<title>ABSTRACT</title>
Owing to carboxylation activity, reversible decarboxylases can use CO2 as a C1-building block to produce useful carboxylic acids. Although many reversible decarboxylases can synthesize aromatic monocarboxylic acids, only a few reversible decarboxylases have been reported to date that catalyze the synthesis of aromatic dicarboxylic acids. In the present study, a reversible 4-hydroxyisophthalic acid decarboxylase was identified in Cystobasidium slooffiae HTK3. Furthermore, recombinant 4-hydroxyisophthalic acid decarboxylase was prepared, characterized, and used for 4-hydroxyisophthalic acid production from 4-hydroxybenzoic acid.

Toward a sustainable synthesis of value-added chemicals, the method of CO2 utilization attracts great interest in chemical process engineering. Biotechnological CO2 fixation is a promising technology
however, efficient methods that can fix carbon dioxide are still limited. Instead, some parts of microbial decarboxylases allow the introduction of carboxy group into phenolic compounds using bicarbonate ion as a C1 building block. Here, we identified a unique decarboxylase from Arthrobacter sp. K8 that acts on resorcinol derivatives. A high-throughput colorimetric decarboxylase assay facilitated gene cloning of orsellinic acid decarboxylase from genomic DNA library of strain K8. Sequence analysis revealed that the orsellinic acid decarboxylase belonged to amidohydrolase 2 family, but shared low amino acid sequence identity with those of related decarboxylases. Enzymatic characterization unveiled that the decarboxylase introduces a carboxy group in a highly regio-selective manner. We applied the decarboxylase to enzymatic carboxylation of resorcinol derivatives. Using Escherichia coli expressing the decarboxylase gene as a whole cell biocatalyst, orsellinic acid, 2,4-dihydroxybenzoic acid, and 4-methoxysalicylic acid were produced in the presence of saturated bicarbonate. These findings could provide new insights into the production of useful phenolic acids from resorcinol derivatives.

Sesquiterpenoids are one of the most diverse groups in natural compounds with various chemical structures and bioactivities. In our previous work, we developed the chemoenzymatic oxygenation method based on the combination of Fe(II)-EDTA and ferric-chelate reductase that could synthesize (−)-rotundone, a key aroma sesquiterpenoid of black pepper. Fe(II)-EDTA catalyzed the oxygenation of sesquiterpene to sesquiterpenoid, and ferric-chelate reductase catalyzed the supply and regeneration of Fe(II)-EDTA in this system. We then investigated the effect of various Fe2+-chelates on the catalytic oxygenation of sesquiterpene and applied this system to the synthesis of odor sesquiterpenoids. We determined Fe(II)-NTA to be an efficient oxygenation catalyst by the screening approach focusing on ligand structures and coordination atoms of Fe2+-chelates. Valuable odor sesquiterpenoids such as (+)-nootkatone, (−)-isolongifolenone, and (−)-β-caryophyllene oxide were oxygenatively synthesized from each precursor sesquiterpene by 66%, 82%, and 67% of the molar conversion rate, respectively. Abbreviations: EDTA: ethylenediaminetetraacetate
NTA: nitrilotriacetate
DTPA: diethylenetriaminepentaacetate
phen: o-phenanthroline
cyclam: 1,4,8,11-tetraazacyclotetradecane
TPA: tris(2-pyridylmethyl)amine
GlcDH: glucose dehydrogenase
HP-β-CD: hydroxypropyl-β-cyclodextrin.

Piceatannol is a valuable natural polyphenol with therapeutic potential in cardiovascular and metabolic disease treatment. In this study, we screened for microorganisms capable of producing piceatannol from resveratrol via regioselective hydroxylation. In the first screening, we isolated microorganisms utilizing resveratrol, phenol, or 4-hydroxyphenylacetic acid as a carbon source for growth. In the second screening, we assayed the isolated microorganisms for hydroxylation of resveratrol. Using this screening procedure, a variety of resveratrol-converting microorganisms were obtained. One Gram-negative bacterium, Ensifer sp. KSH1, and one Gram-positive bacterium, Arthrobacter sp. KSH3, utilized 4-hydroxyphenylacetic acid as a carbon source for growth and efficiently hydroxylated resveratrol to piceatannol without producing any detectable by-products. The hydroxylation activity of strains KSH1 and KSH3 was strongly induced by cultivation with 4-hydroxyphenylacetic acid as a carbon source during stationary growth phase. Using the 4-hydroxyphenylacetic acid–induced cells as a biocatalyst under optimal conditions, production of piceatannol by strains KSH1 and KSH3 reached 3.6 mM (0.88 g/L) and 2.6 mM (0.64 g/L), respectively. We also cloned genes homologous to the monooxygenase gene hpaBC from strains KSH1 and KSH3. Introduction of either hpaBC homolog into Escherichia coli endowed the host with resveratrol-hydroxylating activity.

Amide bond formation serves as a fundamental reaction in chemistry, and is practically useful for the synthesis of peptides, food additives, and polymers. However, current methods for amide bond formation essentially generate wastes and suffer from poor atom economy under harsh conditions. To solve these issues, we demonstrated an alternative synthesis method for diverse tryptophyl-N-alkylamides by the combination of the first adenylation domain of tyrocidine synthetase 1 with primary or secondary amines as nucleophiles. Moreover, the physiological role of this domain is l-phenylalanine adenylation
however, we revealed that it displayed broad substrate flexibility from mono-substituted tryptophan analogues to even d-tryptophan. To the best of our knowledge, this is the first evidence for an adenylating enzyme-mediated direct amide bond formation via a sequential enzymatic activation of amino acids followed by nucleophilic substitution by general amines. These findings facilitate the design of a promising tool for biocatalytic straightforward amide bond formation with less side products.

An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported
however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class III polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906T, coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87 mM of L-Trp-L-Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2 mM L-Trp-L-Pro. When the coupling reaction was applied to whole-cell reactions in E. coli BL21(DE3) pepQ− putA−, ATP was successfully regenerated from AMP by Deipr_1912, and 6.7 mM of L-Trp-L-Pro was produced after 24 h with the supplementation of 10 mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only L-Trp-L-Pro, but also various other L-Xaa-L-Pro (Xaa = Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase.

Hydroxylation via COH bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available L-lysine with L-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two L-lysine 3S-hydroxylases and four L-lysine 4R-hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereo-selective hydroxylation into either C-3 or C-4 positions of L-lysine, yielding (2S, 3S)-3-hydroxylysine and (2S, 4R)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant L-lysine hydroxylase-expressing Escherichia coli cells were used as a biocatalyst for L-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S, 3S)-3-hydroxylysine was produced from 600 mM L-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S, 4R)-4-hydroxylysine was produced from 300 mM L-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies.
IMPORTANCE The present study identified six L-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of L-lysine hydroxylases was relatively narrow, we found that (2S, 3S)-3-hydroxylysine was hydroxylated by 4R-hydroxylase and (2S, 5R)-5-hydroxylysine was hydroxylated by both 3S- and 4R-hydroxylases. Moreover, the L-arginine hydroxylase VioC also hydroxylated L-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of L-lysine into (2S, 3S)-3-hydroxylysine and (2S, 4R)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic L-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.

Vanillin is an important and popular plant flavor, but the amount of this compound available from plant sources is very limited. Biotechnological methods have high potential for vanillin production as an alternative to extraction from plant sources. Here, we report a new approach using immobilized enzymes for the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sep-abeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use. This immobilized Cso2 catalyst allowed 6.8 mg yield of vanillin from isoeugenol through ten reaction cycles at a 1 mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demonstrated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin production. (C) 2016 Elsevier B.V. All rights reserved.

In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy) proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Delta(1)-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of alpha-to beta-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3-and 4-hydroxyprolines.

In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy) proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Delta(1)-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of alpha-to beta-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3-and 4-hydroxyprolines.

L-Amino acid ligase (Lal) catalyzes dipeptide synthesis from unprotected L-amino acids by hydrolysis ATP to ADP. Each Lal displays unique substrate specificity, and many different dipeptides can be synthesized by selecting suitable Lal. We have already successfully synthesized Met-Gly selectively by replacing the Pro85 residues of Lal from Bacillus licheniformis (BL00235). From these results, we deduced that the amino acid residue at position 85 had a key role in enzyme activity, and applied these findings to other Lals. When Pro and Gly were used as substrates, TabS from Pseudomonas syringae, synthesized the salt taste enhancing dipeptide Pro Gly and other three dipeptides (Gly-Pro, Pro-Pro, and Gly-Gly) was hardly synthesized from its substrate specificity. However, the amount of Pro Gly was low. Therefore, to alter the substrate specificity and increase the amount of Pro Gly, we selected amino acid residues that might affect the enzyme activity, Ser85 corresponding to Pro85 of BL00235, and His294 on the results from previous studies and the predicted structure of TabS. These residues were replaced with 20 proteogenic amino acids, and Pro Gly synthesizing reactions were conducted. The S85T and the H294D mutants synthesized more Pro Gly than wild-type. Furthermore, the S85T/H294D double mutant synthesized considerably more Pro Gly than the single mutant did. These results showed that the amino acid position 85 of TabS affect the enzyme activity similarly to BL00235. In addition, replacing the amino acid residue positioning around the N-terminal substrate and constructing the double mutant led to increase the amount of Pro-Gly. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3,5-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9mM (1.8g L-1) in 48h.

Naturally occurring l-hydroxyproline in its four regio- and stereoisomeric forms has been explored as a possible precursor for pharmaceutical agents, yet the selective synthesis of trans-3-hydroxy-l-proline has not been achieved. Our aim was to develop a novel biocatalytic asymmetric method for the synthesis of trans-3-hydroxy-l-proline. So far, we focused on the rhizobial arginine catabolic pathway: arginase and ornithine cyclodeaminase are involved in l-arginine degradation to l-proline via l-ornithine. We hypothesized that trans-3-hydroxy-l-proline should be synthesized if arginase and ornithine cyclodeaminase act on (2S,3S)-3-hydroxyarginine and (2S,3S)-3-hydroxyornithine, respectively. To test this hypothesis, we cloned the genes of l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase and overexpressed them in Escherichia coli, with subsequent enzyme purification. After characterization and optimization of each enzyme, a three-step procedure involving l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase (in this order) was performed using l-arginine as a starting substrate. At the second step of the procedure, putative hydroxyornithine was formed quantitatively by arginase from (2S,3S)-3-hydroxyarginine. Nuclear magnetic resonance and chiral high-performance liquid chromatography analyses revealed that the absolute configuration of this compound was (2S,3S)-3-hydroxyornithine. In the last step of the procedure, trans-3-hydroxy-l-proline was synthesized selectively by ornithine cyclodeaminase from (2S,3S)-3-hydroxyornithine. Thus, we successfully developed a novel synthetic route, comprised of three reactions, to convert l-arginine to trans-3-hydroxy-l-proline. The excellent selectivity makes this procedure simpler and more efficient than conventional chemical synthesis.

We examined 66 aromatics for carboxylation by the gamma-resorcylic acid decarboxylase from Rhizobium radiobacter WU-0108 under reverse reaction conditions. The enzyme carboxylated resorcinol, catechol, 5-methylresorcinol and three hydroxystilbenes (resveratrol, gnetol, and piceatannol) with high yields. Except for catechol, the structures of these substrates include a 1,3-dihydroxybenzene moiety. Other compounds gave no reaction products. The reaction products from resveratrol and gnetol were 2,6-dihydroxy-4-[(E)-2-(4-hydroxyphenyl)ethenyl]benzoic acid and 2,6-dihydroxy-4-[(E)-2-(2,6-dihydroxyphenyl)ethenyl]benzoic acid, respectively, as determined by mass spectrometry and nuclear magnetic resonance analyses. Kinetic analyses of the carboxylation reactions indicated that resveratrol and gnetol are better substrates than resorcinol or catechol. (C) 2015 Elsevier B.V. All rights reserved.

RizA is an L-amino-acid ligase from Bacillus subtilis that participates in the biosynthesis of rhizocticin, an oligopeptide antibiotic. The substrate-free form of RizA has been crystallized and the structure was solved at 2.8 angstrom resolution. The amino-acid-binding site appears to be capable of accommodating multiple amino acids, consistent with previous biochemical studies.

Mycobacteria such as Mycobacterium smegmatis strain mc2155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E.coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol.

We developed a novel process for efficient synthesis of L-threo-3-hydroxyaspartic acid (L-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of L-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize L-THA. By using these recombinant enzymes, L-THA was obtained by L-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. L-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased L-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the L-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, L-THA was successfully obtained from L-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of L-THA.

We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp-hydroxamate-Fe&lt
sup&gt
3+&lt
/sup&gt
complex and the formation of l-Trp-l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.

Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl&lt
inf&gt
2&lt
/inf&gt
to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75mM of this substrate to 4-vinylguaiacol within 2h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52mM (7.8gL&lt
sup&gt
-1&lt
/sup&gt
) in 24h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl2 to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75 mM of this substrate to 4-vinylguaiacol within 2 h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52 mM (7.8 g L-1) in 24 h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the L-Trp-hydroxamate-Fe3+ complex and the formation of L-Trp-L-Pro, where L-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl L-Pro formations was observed. (C) 2015 Elsevier Inc. All rights reserved.

Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl2 to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75 mM of this substrate to 4-vinylguaiacol within 2 h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52 mM (7.8 g L-1) in 24 h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Pro line hydroxylases are representative members of the nonheme Fe2+/alpha-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium rneliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Pro line hydroxylases are representative members of the nonheme Fe2+/alpha-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium rneliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Pro line hydroxylases are representative members of the nonheme Fe2+/alpha-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium rneliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

:HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3',5'-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100 mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9 mM (1.8 g L(-1)) in 48 h.

Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an L-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.

We developed a novel process for efficient synthesis of L-threo-3-hydroxyaspartic acid (L-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693- D246N) were adaptable to the direct hydroxylation of L-aspartic acid
however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize L-THA. By using these recombinant enzymes, L-THA was obtained by L-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. L-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased L-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the L-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, L-THA was successfully obtained from L-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of L-THA.

:We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp-hydroxamate-Fe(3+) complex and the formation of l-Trp-l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.

CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell etal. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and S247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol
the whole cells expressing the S247D mutant completely converted 1mM p-cresol to p-hydroxybenzylalcohol in only 30min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2.

Many dipeptides have unique physiological functions such as antihypertensive effects and taste enhancing effects. In this study, we focused on the taste of dipeptides and conducted screening for dipeptides as salt taste enhancers. Dipeptides were synthesized using TabS, an L-amino acid ligase (Lal) from Pseudomonas syringae NBRC14081. Six kinds of amino acids, which were easily released by the hydrolysis of proteins or peptides, reacted with 20 proteogenic amino acids, and the reaction mixtures were evaluated using sensory evaluation. In the first screening, the reaction mixture or L-Leu-L-Ser, a known salt taste enhancing dipeptide, was added to a salt solution containing ATP and panelists judged the salt taste intensities. In the second screening, the reaction mixture or residual substrate amino acids was added to a salt solution containing ATP and subjected to sensory evaluation. L-Met-Gly was identified as a candidate salt taste enhancer. In addition to sensory evaluation, the salt taste enhancing effect of L-Met-Gly was evaluated using a taste sensor. Taste sensor analysis showed that L-Met-Gly had a salt taste enhancing effect, and the relative sensor response for L-Met-Gly was equal to or higher than that for L-Leu-L-Ser. This is the first report that L-Met-Gly is a salt taste enhancing dipeptide. Furthermore, we propose that the screening method using reaction mixtures of Lal is applicable for the taste evaluation of other dipeptides.

Many dipeptides have unique physiological functions such as antihypertensive effects and taste enhancing effects. In this study, we focused on the taste of dipeptides and conducted screening for dipeptides as salt taste enhancers. Dipeptides were synthesized using TabS, an L-amino acid ligase (Lal) from Pseudomonas syringae NBRC14081. Six kinds of amino acids, which were easily released by the hydrolysis of proteins or peptides, reacted with 20 proteogenic amino acids, and the reaction mixtures were evaluated using sensory evaluation. In the first screening, the reaction mixture or L-Leu-L-Ser, a known salt taste enhancing dipeptide, was added to a salt solution containing ATP and panelists judged the salt taste intensities. In the second screening, the reaction mixture or residual substrate amino acids was added to a salt solution containing ATP and subjected to sensory evaluation. L-Met-Gly was identified as a candidate salt taste enhancer. In addition to sensory evaluation, the salt taste enhancing effect of L-Met-Gly was evaluated using a taste sensor. Taste sensor analysis showed that L-Met-Gly had a salt taste enhancing effect, and the relative sensor response for L-Met-Gly was equal to or higher than that for L-Leu-L-Ser. This is the first report that L-Met-Gly is a salt taste enhancing dipeptide. Furthermore, we propose that the screening method using reaction mixtures of Lal is applicable for the taste evaluation of other dipeptides.

CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell et al. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and 5247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol; the whole cells expressing the S247D mutant completely converted 1 HIM p-cresol to p-hydroxybenzylalcohol in only 30 min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell et al. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and S247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol; the whole cells expressing the S247D mutant completely converted 1 mM p-cresol to p-hydroxybenzylalcohol in only 30 min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2.

Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 gL(-1)) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to L-proline and L-pipecolinic acid, we found that 3,4-dehydro-L-proline, L-azetidine-2-carboxylic acid, cis-3-hydroxy-L-proline, and L-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-L-proline, cis-3-hydroxy-L-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline.

Piceatannol, a valuable biologically active stilbene derivative, was efficiently synthesized from resveratrol. Whole-cell catalysis with HpaBC monooxygenase enabled the regioselective hydroxylation of resveratrol to produce 23 mM (5.2 g L-1) of piceatannol. (C) 2014 Elsevier Ltd. All rights reserved.

Microbial hydroxylases were screened for the capacity to effect direct hydroxylation of proline and pipecolinic acid, based on genomic information. Of the eight candidates screened, 2-oxoglutarate-dependent hydroxylase from Streptosporangium roseum NBRC 3776T and aspartyl/asparaginyl β-hydroxylase from Catenulispora acidiphila NBRC 102108T showed both proline and pipecolinic acid hydroxylation activities. In the case of l-proline hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-l-proline and cis-4-hydroxy-l-proline, and cis-4-hydroxy-l-proline was preferentially produced. In the case of l-pipecolinic acid hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-l-pipecolinic acid and cis-5-hydroxy-l-pipecolinic acid. While the former enzyme preferentially produced cis-3-hydroxy-l-pipecolinic acid, the latter enzyme preferentially produced cis-5-hydroxy-l-pipecolinic acid. © 2013 Elsevier B.V.

4-Hydroxyphenylacetate 3-hydroxylases (HPAHs) of the two-component flavin-dependent monooxygenase family are attractive enzymes that possess the catalytic potential to synthesize valuable ortho-diphenol compounds from simple monophenol compounds. In this study, we investigated the catalytic activity of HPAH from Pseudomonas aeruginosa strain PAO1 toward cinnamic acid derivatives. We prepared Escherichia coli cells expressing the hpaB gene encoding the monooxygenase component and the hpaC gene encoding the oxidoreductase component. E. coli cells expressing HpaBC exhibited no or very low oxidation activity toward cinnamic acid, o-coumaric acid, and m-coumaric acid, whereas they rapidly oxidized p-coumaric acid to caffeic acid. Interestingly, after p-coumaric acid was almost completely consumed, the resulting caffeic acid was further oxidized to 3,4,5-trihydroxycinnamic acid. In addition, HpaBC exhibited oxidation activity toward 3-(4-hydroxyphenyl)propanoic acid, ferulic acid, and coniferaldehyde to produce the corresponding ortho-diphenols. We also investigated a flask-scale production of caffeic acid from p-coumaric acid as the model reaction for HpaBC-catalyzed syntheses of hydroxycinnamic acids. Since the initial concentrations of the substrate p-coumaric acid higher than 40 mM markedly inhibited its HpaBC-catalyzed oxidation, the reaction was carried out by repeatedly adding 20 mM of this substrate to the reaction mixture. Furthermore, by using the HpaBC whole-cell catalyst in the presence of glycerol, our experimental setup achieved the high-yield production of caffeic acid, i.e., 56.6 mM (10.2 g/L) within 24 h. These catalytic activities of HpaBC will provide an easy and environment-friendly synthetic approach to hydroxycinnamic acids.

Microbial hydroxylases were screened for the capacity to effect direct hydroxylation of proline and pipecolinic acid, based on genomic information. Of the eight candidates screened, 2-oxoglutaratedependent hydroxylase from Streptosporangium roseum NBRC 3776T and aspartyl/asparaginyl p-hydroxylase from Catenulispora acidiphila NBRC 102108T showed both proline and pipecolinic acid hydroxylation activities. In the case of L-proline hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-L-proline and cis-4-hydroxy-L-proline, and cis-4-hydroxy-L-proline was preferentially produced. In the case of L-pipecolinic acid hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-L-pipecolinic acid and cis-5-hydroxy-L-pipecolinic acid. While the former enzyme preferentially produced cis-3-hydroxy-L-pipecolinic acid, the latter enzyme preferentially produced cis-5-hydroxy-L-pipecolinic acid. (C) 2013 Elsevier B.V. All rights reserved.

:Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 g L(-1) ) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to L-proline and L-pipecolinic acid, we found that 3,4-dehydro-L-proline, L-azetidine-2-carboxylic acid, cis-3-hydroxy-L-proline, and L-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-L-proline, cis-3-hydroxy-L-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline.

:4-Hydroxyphenylacetate 3-hydroxylases (HPAHs) of the two-component flavin-dependent monooxygenase family are attractive enzymes that possess the catalytic potential to synthesize valuable ortho-diphenol compounds from simple monophenol compounds. In this study, we investigated the catalytic activity of HPAH from Pseudomonas aeruginosa strain PAO1 toward cinnamic acid derivatives. We prepared Escherichia coli cells expressing the hpaB gene encoding the monooxygenase component and the hpaC gene encoding the oxidoreductase component. E. coli cells expressing HpaBC exhibited no or very low oxidation activity toward cinnamic acid, o-coumaric acid, and m-coumaric acid, whereas they rapidly oxidized p-coumaric acid to caffeic acid. Interestingly, after p-coumaric acid was almost completely consumed, the resulting caffeic acid was further oxidized to 3,4,5-trihydroxycinnamic acid. In addition, HpaBC exhibited oxidation activity toward 3-(4-hydroxyphenyl)propanoic acid, ferulic acid, and coniferaldehyde to produce the corresponding ortho-diphenols. We also investigated a flask-scale production of caffeic acid from p-coumaric acid as the model reaction for HpaBC-catalyzed syntheses of hydroxycinnamic acids. Since the initial concentrations of the substrate p-coumaric acid higher than 40 mM markedly inhibited its HpaBC-catalyzed oxidation, the reaction was carried out by repeatedly adding 20 mM of this substrate to the reaction mixture. Furthermore, by using the HpaBC whole-cell catalyst in the presence of glycerol, our experimental setup achieved the high-yield production of caffeic acid, i.e., 56.6 mM (10.2 g/L) within 24 h. These catalytic activities of HpaBC will provide an easy and environment-friendly synthetic approach to hydroxycinnamic acids.

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. L-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal
it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., L-pipecolic acid, hydroxy-L-proline, and Β-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: L-arginyl-L-phenylalanine (antihypertensive effect
yield, 62%), L-leucyl-L-isoleucine (antidepressive effect
yield, 77%), L-glutaminyl-L-tryptophan (precursor of L-glutamyl-L-tryptophan, which has antiangiogenic activity
yield, 54%), L-leucyl-L-serine (enhances saltiness
yield, 83%), and L-glutaminyl-L-threonine (precursor of L-glutamyl-L-threonine, which enhances saltiness
yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis. © 2013, American Society for Microbiology.

Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. L-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., L-pipecolic acid, hydroxy-L-proline, and beta-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: L-arginyl-L-phenylalanine (antihypertensive effect; yield, 62%), L-leucyl-L-isoleucine (antidepressive effect; yield, 77%), L-glutaminyl-L-tryptophan (precursor of L-glutamyl-L-tryptophan, which has antiangiogenic activity; yield, 54%), L-leucyl-L-serine (enhances saltiness; yield, 83%), and L-glutaminyl-L-threonine (precursor of L-glutamyl-L-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis.

The proton motive force (PMF) is bio-energetically important for various cellular reactions to occur. We developed PMF-photogenerating mitochondria in cultured mammalian cells. An archaebacterial rhodopsin, delta-rhodopsin, which is a light-driven proton pump derived from Haloterrigena turkmenica, was expressed in the mitochondria of CHO-K1 cells. The constructed stable CHO-K1 cell lines showed suppression of cell death induced by rotenone, a pesticide that inhibits mitochondrial complex I activity involved in PMF generation through the electron transport chain. Delta-rhodopsin was also introduced into the mitochondria of human neuroblastoma SH-SY5Y cells. The constructed stable SH-SY5Y cell lines showed suppression of dopaminergic neuronal cell death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an inducer of Parkinson's disease models, which acts through inhibition of complex I activity. These results suggest that the light-activated proton pump functioned as a PMF generator in the mitochondria of mammalian cells, and suppressed cell death induced by inhibition of respiratory PMF generation.

Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine
Gln, l-glutamine
and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid
and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ± 30 nmol min-1 mg-1·protein and 136 ± 6 nmol min-1 mg-1·protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa. © 2012 The Society for Biotechnology, Japan.

Dipeptides exhibit unique physiological functions and physical properties, e.g., L-aspartyl-L-phenylalanine-methyl. ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an L-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, L-asparagine; Gln, L-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, L-aspartic add; and Glu, L-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated L-asparaginyl-L-valine (Asn-Val) and L-glutaminyl-glycine (Gln-Gly), but did not deamidate L-valyl-L-asparagine and L-alanyl-L-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 +/- 30 nmol min(-1) mg(-1) protein and 136 +/- 6 nmol min(-1) mg(-1) protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

The mimABCD gene clusters in Mycobacterium smegmatis strain mc 2155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the Rhodococcus-Escherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD. The active expression of mycobacterial binuclear iron monooxygenases (MimABCD) in a heterologous host was dependent on the co-expression of a specific chaperonin-like protein (MimG). SDS/PAGE and western blotting analyses demonstrated that MimG played essential roles in productive folding of MimA and that the resulting soluble MimA protein led to the active expression of MimABCD. © 2012 The Authors Journal compilation.

The mimABCD gene clusters in Mycobacterium smegmatis strain mc2155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the RhodococcusEscherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD.

:5-Hydroxy-l-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified l-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, l-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the λ red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of l-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4α-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration.

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes. © 2013, American Society for Microbiology.

5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells - wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing - were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production
the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively. © 2013 Elsevier B.V.

Microorganisms are capable of producing a wide variety of biopolymers. Homopoly(amino acid)s and homooligo(amino acid)s, which are made up of only a single type of amino acid, are relatively rare; in fact, only two homopoly(amino acid)s have been known to occur in nature: poly(epsilon-L-lysine) (epsilon-PL) and poly(gamma-glutamic acid) (gamma-PGA). Bacterial enzymes that produce homooligo(amino acid)s, such as L-beta-lysine-, L-valine-, L-leucine-, L-isoleucine-, L-methionine-, and L-glutamic acid-oligopeptides and poly(alpha-L-glutamic acid) (alpha-PGA) have recently been identified, as well as epsilon-PL synthetase and gamma-PGA synthetase. This article reviews the current knowledge about these unique enzymes producing homopoly(amino acid)s and homooligo(amino acid)s.

The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP's retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP. © 2012 Springer-Verlag.

5-Hydroxy-L-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified L-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, L-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the λ red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of L-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4α-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration. © 2013 Hara and Kino.

L -Amino-acid ligases (LALs) are enzymes which catalyze the formation of dipeptides by linking two l-amino acids. Although many dipeptides are known and expected to have medical and nutritional benefits, their practical use has been limited owing to their low availability and high expense. LALs are potentially desirable tools for the efficient production of dipeptides; however, the molecular basis of substrate recognition by LAL has not yet been sufficiently elucidated for the design of ideal LALs for the desired dipeptides. This report presents the crystal structure of the LAL BL00235 derived from Bacillus licheniformis NBRC 12200 determined at 1.9 angstrom resolution using the multi-wavelength anomalous dispersion method. The overall structure of BL00235 is fairly similar to that of YwfE, the only LAL with a known structure, but the structure around the catalytic site contains some significant differences. Detailed structural comparison of BL00235 with YwfE sheds some light on the molecular basis of the substrate specificities.

Scope We found that a dipeptide, Arg-Phe (RF), had vasorelaxing activity in mesenteric artery isolated from spontaneously hypertensive rats (SHRs) (EC50 = 580 nM). We then investigated its mechanism of action, and elucidated its physiological functions. Methods and results Vasorelaxing activities of RF-related peptides were tested. The retro-sequence dipeptide FR was inactive, suggesting that the RF sequence is important for a potent vasorelaxing effect. RA and AF were also inactive. RF-nh2 had vasorelaxing activity, implying that the C-terminal amidation of RF is tolerated. Nitric oxide (NO) and prostaglandins (PGs) are known to be vasorelaxing factors; however, the vasorelaxing activity of RF was inhibited by neither NG-nitro-l-arginine methyl ester (l-NAME), an NO synthase inhibitor, nor indomethacin, a COX inhibitor. Interestingly, the activity was blocked by lorglumide, an antagonist of the cholecystokinin (CCK)1 receptor; however, RF had no affinity for CCK receptors, suggesting that RF stimulates CCK release. Orally administered RF decreased blood pressure in SHRs, and this antihypertensive activity was also blocked by a CCK1 antagonist. RF had CCK-like suppressive effects on food intake and gastrointestinal transit. RF increased intracellular Ca2+ flux and CCK release in enteroendocrine STC-1 cells. Conclusion A novel CCK-dependent vasorelaxing RF decreases both blood pressure and food intake.

Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 angstrom from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F1851, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.

The alpha-glucosyl transfer enzyme (XgtA), a novel type alpha-glucosidase produced by Xanthomonas campestris WU-9701, was purified from the cell-free extract and characterized. The molecular weight of XgtA is estimated to be 57 kDa by SOS-PAGE and 60 kDa by gel filtration, indicating that XgtA is a monomeric enzyme. Kinetic properties of XgtA were determined for alpha-glucosyl transfer and maltose-hydrolyzing activities using maltose as the alpha-glucosyl donor, and if necessary, hydroquinone as the acceptor. The V-max value for alpha-glucosyl transfer activity was 1.3 x 10(-2) (mM/s); this value was 3.9-fold as much as that for maltose-hydrolyzing activity. XgtA neither produced maltooligosaccharides nor hydrolyzed sucrose. The gene encoding XgtA that contained a 1614-bp open reading frame was cloned, identified, and highly expressed in Escherichia coli JM109 as the host. Site-directed mutagenesis identified Asp201,Glu270, and Asp331 as the catalytic sites of XgtA, indicating that XgtA belongs to the glycoside hydrolase family 13. (C) 2012 Elsevier B.V. All rights reserved.

:Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.

The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ 54 factor, a gene encoding the σ 54 factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria. © 2011, American Society for Microbiology.

The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the sigma(54) factor, a gene encoding the sigma(54) factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria.

We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H ? transporter) and TF(0)F(1)-ATP synthase (a thermostable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess for various ATP-hydrolyzing bioproductions.

Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher n-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.

Poly-L-alpha-amino acids have various applications because of their biodegradable properties and biocompatibility. Microorganisms contain several enzymes that catalyze the polymerization of L-amino acids in an ATP-dependent manner, but the products from these reactions contain amide linkages at the side residues of amino acids: e.g., poly-gamma-glutamic acid, poly-epsilon-lysine, and cyanophycin. In this study, we found a novel catalytic activity of RimK, a ribosomal protein S6-modifying enzyme derived from Escherichia coli K-12. This enzyme catalyzed poly-alpha-glutamic acid synthesis from unprotected L-glutamic acid (Glu) by hydrolyzing ATP to ADP and phosphate. RimK synthesized poly-alpha-glutamic acid of various lengths; matrix-assisted laser desorption ionization-time of flight-mass spectrometry showed that a 46-mer of Glu (maximum length) was synthesized at pH 9. Interestingly, the lengths of polymers changed with changing pH. RimK also exhibited 86% activity after incubation at 55 degrees C for 15 min, thus showing thermal stability. Furthermore, peptide elongation seemed to be catalyzed at the C terminus in a stepwise manner. Although RimK showed strict substrate specificity toward Glu, it also used, to a small extent, other amino acids as C-terminal substrates and synthesized heteropeptides. In addition, RimK-catalyzed modification of ribosomal protein S6 was confirmed. The number of Glu residues added to the protein varied with pH and was largest at pH 9.5.

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetoneinduced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc2155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc2155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc2155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc2155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria. © 2011, American Society for Microbiology.

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.

Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.

L-Amino acid ligase synthesizes various peptides from unprotected L-amino acids in an ATP-dependent manner. Known L-amino acid ligases catalyze only dipeptide synthesis, but recently we found that RizB of Bacillus subtilis NBRC 3134 catalyzes oligopeptide synthesis. In the present study, we searched for new members of the L-amino acid ligase group that catalyze oligopeptide synthesis. Several hypothetical proteins possessing the ATP-grasp motif were selected by in silico analysis. These recombinant proteins were assayed for L-amino acid ligase activity. We obtained five L-amino acid ligases showing oligopeptide synthesis activities. These proteins showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacterium adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones. We also examined some of their characteristics.

L-Amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We recently identified a new member of L-amino acid ligase, the plu1440 protein, from Photorhabdus luminescens subsp. laumondii TT01 by in silico analysis. This protein was found to synthesize dipeptides containing L-asparagine at the N-terminus, which is a novel substrate specificity. (C) 2009, The Society for Biotechnology, japan. All rights reserved.

A novel metabolic pathway was found in the yeast Trichosporon moniliiforme WU-0401 for salicylate degradation via phenol as the key intermediate. When 20 mM salicylate was used as the sole carbon source for the growth of strain WU-0401, phenol was detected as a distinct metabolite in the culture broth. Analysis of the products derived from salicylate or phenol through reactions with resting cells and a cell-free extract of strain WU-0401 indicated that salicylate is initially decarboxylated to phenol and then oxidized to catechol, followed by aromatic ring cleavage to form cis-cis muconate.

Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon-hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy. CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid, at a rate of 130 mol (mol P450)(-1) min(-1). Furthermore, CYP199A2 also oxidized quinoline-6-carboxylic acid, although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2, C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids.

:L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE protein from Bacillus subtilis 168 was the first reported L-amino acid ligase, and it synthesizes various dipeptides. Thereafter, several L-amino acid ligases were newly obtained by in silico analysis using the ATP-grasp motif. But these L-amino acid ligases synthesize only dipeptide and no longer peptide. A novel L-amino acid ligase capable of catalyzing oligopeptide synthesis is required to increase the variety of peptides. We have previously found a new member of L-amino acid ligase, RizA, from B. subtilis NBRC3134, a microorganism that produces the peptide-antibiotic rhizocticin. We newly found that a gene at approximately 9 kbp upstream of rizA encoded a novel L-amino acid ligase RizB. Recombinant RizB synthesized homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acids, and also synthesized various heteropeptides. RizB is the first reported L-amino acid ligase that catalyzes oligopeptide synthesis. In addition, we obtained L-amino acid ligases showing oligopeptide synthesis activities by in silico analysis using BLAST, which is a set of similarity search programs. These L-amino acid ligases showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacteria adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones.

:L-Amino acid ligase synthesizes various peptides from unprotected L-amino acids in an ATP-dependent manner. Known L-amino acid ligases catalyze only dipeptide synthesis, but recently we found that RizB of Bacillus subtilis NBRC 3134 catalyzes oligopeptide synthesis. In the present study, we searched for new members of the L-amino acid ligase group that catalyze oligopeptide synthesis. Several hypothetical proteins possessing the ATP-grasp motif were selected by in silico analysis. These recombinant proteins were assayed for L-amino acid ligase activity. We obtained five L-amino acid ligases showing oligopeptide synthesis activities. These proteins showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacterium adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones. We also examined some of their characteristics.

:L-amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We recently identified a new member of L-amino acid ligase, the plu1440 protein, from Photorhabdus luminescens subsp. laumondii TT01 by in silico analysis. This protein was found to synthesize dipeptides containing L-asparagine at the N-terminus, which is a novel substrate specificity.

:Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon-hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.

:CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy.CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid,at a rate of 130 mol (mol P450)(-1) min(-1). Furthermore,CYP199A2 also oxidized quinoline-6-carboxylic acid,although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2,C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids.

L-Amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We have purified a new L-amino acid ligase, RizA, which synthesizes dipeptides whose N-terminus is Arg, from Bacillus subtilis NBRC3134, a microorganism that produces a rhizocticin peptide antibiotic. It was suggested that RizA is probably involved in rhizocticin biosynthesis. In this study, we performed sequence analysis of unknown regions around rizA, and newly identified a gene that encodes a protein that possesses an ATP-grasp motif upstream of rizA. This gene was designated rizB, and its recombinant protein was prepared. Recombinant RizB synthesized homo-oligo-mers of branched-chain L-amino acids and L-methionine consisting of two to five amino acids in an ATP-dependent manner. RizB also synthesized various heteropeptides. Further examination showed that RizB might elongate a peptide chain at the N-terminus. This is the first report on an L-amino acid ligase catalyzing oligopeptide synthesis.

:A novel metabolic pathway was found in the yeast Trichosporon moniliiforme WU-0401 for salicylate degradation via phenol as the key intermediate. When 20 mM salicylate was used as the sole carbon source for the growth of strain WU-0401, phenol was detected as a distinct metabolite in the culture broth. Analysis of the products derived from salicylate or phenol through reactions with resting cells and a cell-free extract of strain WU-0401 indicated that salicylate is initially decarboxylated to phenol and then oxidized to catechol, followed by aromatic ring cleavage to form cis-cis muconate.

L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE protein from Bacillus subtilis 168 was the first reported L-amino acid ligase, and it synthesizes various dipeptides. Thereafter, several L-amino acid ligases were newly obtained by in silico analysis using the ATP-grasp motif. But these L-amino acid ligases synthesize only dipeptide and no longer peptide. A novel L-amino acid ligase capable of catalyzing oligopeptide synthesis is required to increase the variety of peptides. We have previously found a new member of L-amino acid ligase, RizA, from B. subtilis NBRC3134, a microorganism that produces the peptide-antibiotic rhizocticin. We newly found that a gene at approximately 9 kbp upstream of rizA encoded a novel L-amino acid ligase RizB. Recombinant RizB synthesized homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acids, and also synthesized various heteropeptides. RizB is the first reported L-amino acid ligase that catalyzes oligopeptide synthesis. In addition, we obtained L-amino acid ligases showing oligopeptide synthesis activities by in silico analysis using BLAST, which is a set of similarity search programs. These L-amino acid ligases showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacteria adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones.

L-Amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We have purified a new L-amino acid ligase, RizA, which synthesizes dipeptides whose N-terminus is Arg, from Bacillus subtilis NBRC3134, a microorganism that produces a rhizocticin peptide antibiotic. It was suggested that RizA is probably involved in rhizocticin biosynthesis. In this study, we performed sequence analysis of unknown regions around rizA, and newly identified a gene that encodes a protein that possesses an ATP-grasp motif upstream of rizA. This gene was designated rizB, and its recombinant protein was prepared. Recombinant RizB synthesized homo-oligomers of branched-chain L-amino acids and L-methionine consisting of two to five amino acids in an ATP-dependent manner. RizB also synthesized various heteropeptides. Further examination showed that RizB might elongate a peptide chain at the N-terminus. This is the first report on an L-amino acid ligase catalyzing oligopeptide synthesis.

An ergot fungus Verticillium kibiense E18 produced two cationic peptides, epsilon-poly-L-lysine (ePL) and poly(L-arginyl-D-histidine) (PRH). The ePL was used as a food preservative, and it was expected that PRH would be used as a novel material, such as cationic and antimicrobial peptide. To enhance PRH production of strain E18, various culture conditions were investigated. Glucose was a suitable carbon source for PRH production, although glycerol was a suitable carbon source for growth. The cultivation temperature significantly influenced both cell growth and PRH production. The optimal temperatures for cell growth and PRH production were 28 and 30 degrees C, respectively. Moreover, strain E18 produced more PRH when an additional 5.0 mu g/L FeSO4.7H(2)O was added to the production medium. Under optimal conditions, strain E18 enhanced PRH production, while suppressing ePL production. The maximum PRH production was 183.9 mg/L, which is approximately 60-fold higher than that of the initial culture condition. (C) 2008 Elsevier B.V. All rights reserved.

In the phaseolotoxin biosynthetic gene cluster of Pseudomonas syringae pv. phaseolicola 1448A, the PSPPH_4299 gene encodes a novel L-amino acid ligase. The PSPPH_4299 protein synthesized various heterodipeptides containing basic amino acids in an ATP-dependent manner, and also synthesized alanyl-homoarginine, part of the phaseolotoxin scaffold.

Cyanophycin synthesis is catalyzed by cyanophycin synthetase (CphA). It was believed that CphA requires L-aspartic acid (Asp), L-arginine (Arg), ATP, Mg2+, and a primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of a low-molecular mass cyanophycin. Despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear, and already-known CphAs were primer-dependent enzymes. In the present study, we found that recombinant CphA from Thermosynechococcus elongatus BP-1 (Tlr2170 protein) catalyzed in vitro cyanophycin synthesis in the absence of a primer. The Tlr2170 protein showed strict substrate specificity toward Asp and Arg. The optimum pH was 9.0, and Mg2+ or Mn2+ was essential for cyanophycin synthesis. KCl enhanced the cyanophycin synthesis activity of the Tlr2170 protein; in contrast, dithiothreitol did not. The Tlr2170 protein appeared to be a 400 +/- 9 kDa homo-tetramer. The Tlr2170 protein showed thermal stability and retained its 80% activity after a 60-min incubation at 50 degrees C. In addition, we examined cyanophycin synthesis at 30 degrees C, 40 degrees C, 50 degrees C, and 60 degrees C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.

L-Amino acid a-ligase (EC 6.3.2.28) catalyzed formation of a-peptide bond in unprotected L-amino acids in an ATP-dependent manner. BL00235 gene in Bacillus licheniformis NBRC12200 coded as a new L-amino acid ligase. BL00235 substrate specificity was strict; only methionine or leucine was acceptable as dipeptide N-terminal residues.

Cellobiose phosphorylase from Clostridium thermocellum catalyzed the beta-anomer-selective synthesis of alkyl glucosides from cellobiose. Synthesis of alkyl beta-glucoside from inexpensive sucrose using cellobiose phosphorylase and sucrose phosphorylase from Pseudomonas saccharophilia was investigated. By combined use of these two phosphorylases, alkyl beta-glucoside was anomer-selectively synthesized from sucrose and alkyl alcohol.

Thirty two thermophilic amino acid aminotransferases (AATs) were expressed in Escherichia coli as soluble and active proteins. Based on their primary structures, the 32 AATs were divided into four phylogenetic groups (classes I, II, IV, and V). The substrate specificities of these AATs were examined, and 12 AATs were found capable of synthesizing ring-substituted phenylglycine derivatives such as hydroxyl-, methoxy-, and fluorophenylglycines. Eleven out of the 12 AATs were enzymes belonging to two phylogenetic groups namely, one subgroup of the class I family and the class IV family. AATs in these two groups may thus be useful for the synthesis of a variety of ring-substituted phenylglycine derivatives.

Characterization of orphan enzymes, for which the catalytic functions and actual substrates are still not elucidated, is a significant challenge in the postgenomic era. Here, we describe a general strategy for exploring the catalytic potentials of orphan monooxygenases based on direct infusion analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Eight cytochromes P450 from Bacillus subtilis were recombinantly expressed in Escherichia coli and subjected to a reconstitution system containing appropriate electron transfer components and many potential substrates. The reaction mixtures were directly analyzed using FT-ICR/MS, and substrates of the putative enzymes were readily identified from the mass spectral data. This allowed identification of previously unreported CYP109B1 substrates and the functional assignment of two putative cytochromes P450, CYP107J1 and CYP134A1. The FT-ICR/MS-based approach can be easily applied to large-scale screening with the aid of the extremely high mass resolution and accuracy.

The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (13613), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail.

The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (13613), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail.

Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two mono-oxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.

Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two mono-oxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50degreesC. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45degreesC. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography-mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45degreesC with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50degreesC. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45degreesC. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography-mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45degreesC with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.

D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.

D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and P-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Splingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 2 18 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE. (C) 2004 Elsevier Inc. All rights reserved.

Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52 degreesC. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e. g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO2), converted DBTO2 to 2'-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30 - 52 degreesC, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50 degreesC and DBT desulfurization (2-HBP production) at 40 degreesC in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.

Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52 degreesC. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e. g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO2), converted DBTO2 to 2'-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30 - 52 degreesC, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50 degreesC and DBT desulfurization (2-HBP production) at 40 degreesC in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and P-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Splingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 2 18 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE. (C) 2004 Elsevier Inc. All rights reserved.

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and P-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Splingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 2 18 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE. (C) 2004 Elsevier Inc. All rights reserved.

With the objective of developing an odorless biodegradation process for dimethyl sulfoxide (DMSO), Hyphomicrobium sp. WU-OM3 was isolated. During the cultivation of strain WU-OM3 cells with 20 mM dimethyl sulfone (DMSO2) as the sole carbon source, DMSO2 was completely consumed within 48 h and sulfate ion accumulated in the culture broth. Methanesulfonate was also detected as an intermediate of DMSO2 degradation. By combining the DMSO-oxidizing microorganism and strain WU-OM3 cells, 0.64 mM (50 mg/l) DMSO was degraded to sulfate ion with 80% molar conversion ratio.

The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50degreesC. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55degreesC. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases. (C) 2003 Elsevier Inc. All rights reserved.

The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50degreesC. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55degreesC. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases. (C) 2003 Elsevier Inc. All rights reserved.

With the objective of developing an odorless biodegradation process for dimethyl sulfoxide (DMSO), Hyphomicrobium sp. WU-OM3 was isolated. During the cultivation of strain WU-OM3 cells with 20 mM dimethyl sulfone (DMSO2) as the sole carbon source, DMSO2 was completely consumed within 48 h and sulfate ion accumulated in the culture broth. Methanesulfonate was also detected as an intermediate of DMSO2 degradation. By combining the DMSO-oxidizing microorganism and strain WU-OM3 cells, 0.64 mM (50 mg/l) DMSO was degraded to sulfate ion with 80% molar conversion ratio.

Dimethyl sulfoxide (DMSO)-degrading ability of Hyphomicrobium denitrificans WU-K217 immobilized by entrapment with calcium-alginate gel was examined. The immobilized cells with the cell density of 0.46 mg-dry weight ml(-1) degraded 0.64 mM (50 mg l(-1)) DMSO within 180 min at a temperature range from 20 to 35 degreesC, similarly to the freely- suspended cells. At 40 degreesC, the immobilized cells completely degraded 0.64 mM DMSO within 240 min, although the freely-suspended cells could not degrade it under the same condition. The half-life of DMSO-degradation activity for the immobilized cells stored at 30 degreesC was 96 h and it was extended to more than 240 h by the storage at 4 degreesC. However, in the case of the freely-suspended cells, the half-life was shorter than 48 h even by the storage at 4 degreesC. These results indicated that the immobilized WU-K217 cells had high thermostability and storage stability compared to those of the freely- suspended cells. Considering these characteristics, we performed the repeated DMSO degradation by reuse of the immobilized cells. As a result, even at the tenth reaction, the immobilized cells maintained 90% of DMSO-degradation rate of that at the first reaction. These results suggested that the immobilized cells of WU-K217 might be applicable to a wastewater-treatment system for the removal of DMSO. (C) 2003 Elsevier Science B.V. All rights reserved.

Dimethyl sulfoxide (DMSO)-degrading ability of Hyphomicrobium denitrificans WU-K217 immobilized by entrapment with calcium-alginate gel was examined. The immobilized cells with the cell density of 0.46 mg-dry weight ml(-1) degraded 0.64 mM (50 mg l(-1)) DMSO within 180 min at a temperature range from 20 to 35 degreesC, similarly to the freely- suspended cells. At 40 degreesC, the immobilized cells completely degraded 0.64 mM DMSO within 240 min, although the freely-suspended cells could not degrade it under the same condition. The half-life of DMSO-degradation activity for the immobilized cells stored at 30 degreesC was 96 h and it was extended to more than 240 h by the storage at 4 degreesC. However, in the case of the freely-suspended cells, the half-life was shorter than 48 h even by the storage at 4 degreesC. These results indicated that the immobilized WU-K217 cells had high thermostability and storage stability compared to those of the freely- suspended cells. Considering these characteristics, we performed the repeated DMSO degradation by reuse of the immobilized cells. As a result, even at the tenth reaction, the immobilized cells maintained 90% of DMSO-degradation rate of that at the first reaction. These results suggested that the immobilized cells of WU-K217 might be applicable to a wastewater-treatment system for the removal of DMSO. (C) 2003 Elsevier Science B.V. All rights reserved.

For one-step enzymatic synthesis of eugenyl alpha-glucoside as a promising pro-drug for a hair restorer and a derivative of spices, selective alpha-glucosylation of eugenol was carried out using the alpha-glucosyl transfer enzyme of Xanthomonas campestris WU-9701. When 130 mumol eugenol and crude enzyme showing 1.0 unit of mu-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) containing 1.2 M maltose as a glucosyl donor at 40degreesC, only one form of eugenyl glucoside was selectively obtained as a product and identified as eugenyl alpha-D-glu-copyranoside (alpha-EG) by C-13-NMR, H-1-NMR, and two-dimensional heteronuclear multiple-bond coherence analyses. In the reaction, no other glucosylated products such as maltotriose or eugenyl maltoside were detected in the reaction mixture. The reaction at 40degreesC for 48 h under the above conditions yielded 68 mumol alpha-EG in 2 ml suspension, and the maximum molar conversion yield based on the amount of eugenol supplied reached 52%.

For one-step enzymatic synthesis of eugenyl alpha-glucoside as a promising pro-drug for a hair restorer and a derivative of spices, selective alpha-glucosylation of eugenol was carried out using the alpha-glucosyl transfer enzyme of Xanthomonas campestris WU-9701. When 130 mumol eugenol and crude enzyme showing 1.0 unit of mu-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) containing 1.2 M maltose as a glucosyl donor at 40degreesC, only one form of eugenyl glucoside was selectively obtained as a product and identified as eugenyl alpha-D-glu-copyranoside (alpha-EG) by C-13-NMR, H-1-NMR, and two-dimensional heteronuclear multiple-bond coherence analyses. In the reaction, no other glucosylated products such as maltotriose or eugenyl maltoside were detected in the reaction mixture. The reaction at 40degreesC for 48 h under the above conditions yielded 68 mumol alpha-EG in 2 ml suspension, and the maximum molar conversion yield based on the amount of eugenol supplied reached 52%.

Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by conventional hydrodesulfurization (HDS) treatment using metallic catalysts. The thermophilic DBT-desulfurizing bacterium Mycobacterium phlei WU-F1 grew in a medium with hydrodesulfurized LGO as the sole source of sulfur, and exhibited high desulfurizing ability toward LGO between 30 and 50degreesC. When WU-F1 was cultivated at 45degreesC with B-LGO (390 ppm S), F-LGO (120 ppm S) or X-LGO (34 ppm S) as the sole source of sulfur, biodesulfurization resulted in around 60-70% reduction of sulfur content for all three types of hydrodesulfurized LGOs. In addition, when resting cells were incubated at 45degreesC with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v) oils, biodesulfurization reduced the sulfur content from 390 to 100 ppm S (B-LGO), from 120 to 42 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO). Gas chromatography analysis with an atomic emission detector revealed that the peaks of alkylated DBTs including 4-methyl-DBT, 4,6-dimethyl-DBT and 3,4,6-trimethyl-DBT significantly decreased after biodesulfurization. Therefore, thermophilic M. phlei WU-F1, which could effectively desulfurize HDS-treated LGOs over a wide temperature range up to 50 C, may be a promising biocatalyst for practical biodesulfurization of diesel oil. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by conventional hydrodesulfurization (HDS) treatment using metallic catalysts. The thermophilic DBT-desulfurizing bacterium Mycobacterium phlei WU-F1 grew in a medium with hydrodesulfurized LGO as the sole source of sulfur, and exhibited high desulfurizing ability toward LGO between 30 and 50degreesC. When WU-F1 was cultivated at 45degreesC with B-LGO (390 ppm S), F-LGO (120 ppm S) or X-LGO (34 ppm S) as the sole source of sulfur, biodesulfurization resulted in around 60-70% reduction of sulfur content for all three types of hydrodesulfurized LGOs. In addition, when resting cells were incubated at 45degreesC with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v) oils, biodesulfurization reduced the sulfur content from 390 to 100 ppm S (B-LGO), from 120 to 42 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO). Gas chromatography analysis with an atomic emission detector revealed that the peaks of alkylated DBTs including 4-methyl-DBT, 4,6-dimethyl-DBT and 3,4,6-trimethyl-DBT significantly decreased after biodesulfurization. Therefore, thermophilic M. phlei WU-F1, which could effectively desulfurize HDS-treated LGOs over a wide temperature range up to 50 C, may be a promising biocatalyst for practical biodesulfurization of diesel oil. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

A dimethyl sulfoxide (DMSO)-degrading yeast strain Cryptococcus humicolus WU-2 was isolated and characterized. When 0.64 mM (50 mg/l) DMSO was added as the sole source of sulfur, DMSO was completely consumed by WU-2 in 48 h and oxidized to dimethyl sulfone with a molar conversion ratio of 83%. WU-2 also oxidized alkyl sulfides such as dimethyl sulfide, ethyl methyl sulfide and diethyl sulfide into the corresponding sulfones, which are odorless compounds.

A dimethyl sulfoxide (DMSO)-degrading yeast strain Cryptococcus humicolus WU-2 was isolated and characterized. When 0.64 mM (50 mg/l) DMSO was added as the sole source of sulfur, DMSO was completely consumed by WU-2 in 48 h and oxidized to dimethyl sulfone with a molar conversion ratio of 83%. WU-2 also oxidized alkyl sulfides such as dimethyl sulfide, ethyl methyl sulfide and diethyl sulfide into the corresponding sulfones, which are odorless compounds.

l-Menthyl alpha-D-glucopyranosyl-(1--&gt;4)-alpha-D-glucopyranosid(alpha-MenG(2)), a novel glycoside of l- menthol, was synthesized enzymatically, and its physicochemical properties were characterized. Production of alpha-MenG(2) from l-menthyl alpha-D-glucopyranoside (alpha-MenG) was attempted since we had already succeeded in the high-yield production of alpha-MenG using a Xanthomonas campestris enzyme (Nakagawa, H., et al., J. Biosci. Bioeng., 89, 138-1441. 2000). Through production tests on enzymes, it was confirmed that cyclodextrin glucanotransferase (CGTase) from Bacillus macerans produced l-menthyl alpha-D-maltooligosides (alpha-MenG(n)), containing alpha-MenG(2), from alpha-MenG and soluble starch. When 10 ml of a 10 mM citrate-10 mM phosphate buffer (pH 6.0) containing 150 mg of alpha-MenG, 3 g of soluble starch and CGTase was shaken at 70degreesC for 24 h, a total of 81.8% alpha-MenG was reacted. The molar conversion yields of alpha-MenG(2) and alpha-MenG(n) with alpha-glucose degrees of polymerization of 3-18, based on the amount of alpha-WenG supplied, reached 16.1% and 65.7%, respectively. For efficient production of alpha-MenG(2), the reaction mixture was treated with a-amylase of Aspergillus oryzae, and alpha-MenG(n) were mainly converted into alpha-MenG: finally, the molar conversion yield of alpha-MenG(2) reached 74.2% based on the amount of alpha-MenG supplied. alpha-MenG(2) was purified and its molecular structure was confirmed by C-13-NMR, H-1-NMR and two-dimensional HMBC (heteronuclear multiple-bond coherence).alpha-MenG(2) and its aqueous solution tasted bitter and a little sweet at first, but in a few minutes, a refreshing flavor and sweetness spread. At 20degreesC the solubility of alpha-MenG(2) in pure water was 29.6 g/100 ml, approximately 1570-fold that of alpha-MenG.

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WUK2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c] [1,2]oxathiin S-oxide, benzo[c] [1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl) ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WUK2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c] [1,2]oxathiin S-oxide, benzo[c] [1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl) ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.

l-Menthyl alpha-D-glucopyranosyl-(1--&gt;4)-alpha-D-glucopyranosid(alpha-MenG(2)), a novel glycoside of l- menthol, was synthesized enzymatically, and its physicochemical properties were characterized. Production of alpha-MenG(2) from l-menthyl alpha-D-glucopyranoside (alpha-MenG) was attempted since we had already succeeded in the high-yield production of alpha-MenG using a Xanthomonas campestris enzyme (Nakagawa, H., et al., J. Biosci. Bioeng., 89, 138-1441. 2000). Through production tests on enzymes, it was confirmed that cyclodextrin glucanotransferase (CGTase) from Bacillus macerans produced l-menthyl alpha-D-maltooligosides (alpha-MenG(n)), containing alpha-MenG(2), from alpha-MenG and soluble starch. When 10 ml of a 10 mM citrate-10 mM phosphate buffer (pH 6.0) containing 150 mg of alpha-MenG, 3 g of soluble starch and CGTase was shaken at 70degreesC for 24 h, a total of 81.8% alpha-MenG was reacted. The molar conversion yields of alpha-MenG(2) and alpha-MenG(n) with alpha-glucose degrees of polymerization of 3-18, based on the amount of alpha-WenG supplied, reached 16.1% and 65.7%, respectively. For efficient production of alpha-MenG(2), the reaction mixture was treated with a-amylase of Aspergillus oryzae, and alpha-MenG(n) were mainly converted into alpha-MenG: finally, the molar conversion yield of alpha-MenG(2) reached 74.2% based on the amount of alpha-MenG supplied. alpha-MenG(2) was purified and its molecular structure was confirmed by C-13-NMR, H-1-NMR and two-dimensional HMBC (heteronuclear multiple-bond coherence).alpha-MenG(2) and its aqueous solution tasted bitter and a little sweet at first, but in a few minutes, a refreshing flavor and sweetness spread. At 20degreesC the solubility of alpha-MenG(2) in pure water was 29.6 g/100 ml, approximately 1570-fold that of alpha-MenG.

With the objective of removing dimethyl sulfoxide (DMSO) contained in wastewater from semiconductor or liquid crystal display factories, biodegradation of DMSO, particularly at a low concentration, was examined. Through the screening of DMSO-degrading microorganisms, Hyphomicrobium denitrificans WU-K217 utilizing DMSO as the sole source of carbon was isolated from soil. DMSO at less than 20 mM was degraded to sulfate ion by WU-K217 with 100% molar conversion ratio based on DMSO added during 60-h cultivation at 30degreesC under aerobic conditions. Even in the presence of 116 mM or 225 mM DMSO, WU-K217 showed growth although the amount of DMSO degraded was only 33 mM or 10 mM, respectively. Similar to the growing cells, the resting cells of WU-K217 degraded DMSO at over a wide range of temperature, 20-40degreesC. The highest DMSO-degradation activity was obtained at 30degreesC, and 0.64 mM (50 mg/l) DMSO was completely degraded to sulfate ion with 100% molar conversion ratio within only 15 min. Furthermore, to examine whether WU-K217 would be useful for the removal of DMSO contained in wastewater exhausted in large amounts, continuous degradation of DMSO was examined. When 0.64 mM DMSO was added to the resting cells periodically at 15-min intervals, DMSO was completely degraded to sulfate ion without any decrease of the degradation activity at least during the twelve times of DMSO addition.

With the objective of removing dimethyl sulfoxide (DMSO) contained in wastewater from semiconductor or liquid crystal display factories, biodegradation of DMSO, particularly at a low concentration, was examined. Through the screening of DMSO-degrading microorganisms, Hyphomicrobium denitrificans WU-K217 utilizing DMSO as the sole source of carbon was isolated from soil. DMSO at less than 20 mM was degraded to sulfate ion by WU-K217 with 100% molar conversion ratio based on DMSO added during 60-h cultivation at 30degreesC under aerobic conditions. Even in the presence of 116 mM or 225 mM DMSO, WU-K217 showed growth although the amount of DMSO degraded was only 33 mM or 10 mM, respectively. Similar to the growing cells, the resting cells of WU-K217 degraded DMSO at over a wide range of temperature, 20-40degreesC. The highest DMSO-degradation activity was obtained at 30degreesC, and 0.64 mM (50 mg/l) DMSO was completely degraded to sulfate ion with 100% molar conversion ratio within only 15 min. Furthermore, to examine whether WU-K217 would be useful for the removal of DMSO contained in wastewater exhausted in large amounts, continuous degradation of DMSO was examined. When 0.64 mM DMSO was added to the resting cells periodically at 15-min intervals, DMSO was completely degraded to sulfate ion without any decrease of the degradation activity at least during the twelve times of DMSO addition.

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.(660) 3.3) were shaken in 50 mM K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells.

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.(660) 3.3) were shaken in 50 mM K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells.

alpha-Arbutin, a useful cosmetic ingredient, was selectively synthesized by alpha-anomer-selective glucosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU-9701 as a biocatalyst. When 45 mM hydroquinone and 120 mg of lyophilized cells showing 11 nkat of alpha-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 7.5) containing 1.2 M maltose at 40degreesC, only one form of hydroquinone glucoside was selectively obtained as a product and identified as hydroquinone 1-O-alpha-D-glucopyranoside (alpha-arbutin) by C-13-NMR, H-1-NMR and two-dimensional HMBC analysis. Although hydroquinone has two phenolic -OH groups at the para position in its structure, only one -OH group, but not both -OHs, was glucosylated and no other glucosylated products such as maltotriose were detected in the reaction mixture. The reaction at 40degreesC for 36 h under optimum conditions yielded 42 mM alpha-arbutin, and the maximum molar conversion yield based on the amount of hydroquinone supplied reached 93%.

alpha-Arbutin, a useful cosmetic ingredient, was selectively synthesized by alpha-anomer-selective glucosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU-9701 as a biocatalyst. When 45 mM hydroquinone and 120 mg of lyophilized cells showing 11 nkat of alpha-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 7.5) containing 1.2 M maltose at 40degreesC, only one form of hydroquinone glucoside was selectively obtained as a product and identified as hydroquinone 1-O-alpha-D-glucopyranoside (alpha-arbutin) by C-13-NMR, H-1-NMR and two-dimensional HMBC analysis. Although hydroquinone has two phenolic -OH groups at the para position in its structure, only one -OH group, but not both -OHs, was glucosylated and no other glucosylated products such as maltotriose were detected in the reaction mixture. The reaction at 40degreesC for 36 h under optimum conditions yielded 42 mM alpha-arbutin, and the maximum molar conversion yield based on the amount of hydroquinone supplied reached 93%.

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50 degreesC), could also grow at 50degreesC in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50 degreesC, the resting cells of WU-F1 degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2'-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Therefore, M. phlei WU-F1 can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.

The complementary DNA (cDNA) and chromosomal DNA (icdA) encoding the NADP(+)-specific isocitrate dehydrogenase (EC 1.1.1.42) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Two cDNA clones (cDNA-1, 2.0 kb; cDNA-2, 1.5 kb) were obtained and sequenced, and an ORF of 1494 base pairs (bp) encoding a protein of 498 amino acids (aa) was identified in cDNA-1. The predicted amino acid sequence showed 73% and 67% sequence identities with those of the mitochondrial NADP(+)-ICDHs from Saccharomyces cerevisiae and pig, respectively. The sequence analysis of cDNA-1 and -2 revealed that the cDNA-2 lacks a 500-bp fragment from cDNA-1 which contains a mitochondrial targeting motif. A peroxisomal targeting motif at the C-terminus was found on the aa sequences of cDNA-1 and cDNA-2, but the cDNA-2 product seemed to be localized in the cytoplasm since the peroxisomes were not found in the mycelia of WU-2223L cultivated under the conditions of citric acid production. The expression of both cDNAs in Escherichia coli DEK2004, an isocitrate dehydrogenase-deficient mutant, revealed that both cDNAs complemented the glutamate-requiring phenotype, and that the transformants retained NADP(+)-ICDH activities. Therefore, it was clarified that both of the cDNA-l and -2 products are fully functional. The chromosomal DNA, icdA, was cloned to correspond to CDNA-1, and its nucleotide sequence revealed that it contains seven introns. Southern hybridization using cDNA-1 and cDNA-2 indicated that there is only one copy of icdA on the chromosomes of A. niger WU-2223L. Northern hybridization analysis as for total RNA of WU-2223L revealed that two mRNAs of different sizes, 2.0 kb and 1.5 kb, were hybridized to the ORF of cDNA-1 used as a probe. Therefore, it was found that approximately 1500-nt and 2000-nt mRNAs were transcribed from only one icdA chromosomal gene in A. niger. Such a transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.

The complementary DNA (cDNA) and chromosomal DNA (icdA) encoding the NADP(+)-specific isocitrate dehydrogenase (EC 1.1.1.42) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Two cDNA clones (cDNA-1, 2.0 kb; cDNA-2, 1.5 kb) were obtained and sequenced, and an ORF of 1494 base pairs (bp) encoding a protein of 498 amino acids (aa) was identified in cDNA-1. The predicted amino acid sequence showed 73% and 67% sequence identities with those of the mitochondrial NADP(+)-ICDHs from Saccharomyces cerevisiae and pig, respectively. The sequence analysis of cDNA-1 and -2 revealed that the cDNA-2 lacks a 500-bp fragment from cDNA-1 which contains a mitochondrial targeting motif. A peroxisomal targeting motif at the C-terminus was found on the aa sequences of cDNA-1 and cDNA-2, but the cDNA-2 product seemed to be localized in the cytoplasm since the peroxisomes were not found in the mycelia of WU-2223L cultivated under the conditions of citric acid production. The expression of both cDNAs in Escherichia coli DEK2004, an isocitrate dehydrogenase-deficient mutant, revealed that both cDNAs complemented the glutamate-requiring phenotype, and that the transformants retained NADP(+)-ICDH activities. Therefore, it was clarified that both of the cDNA-l and -2 products are fully functional. The chromosomal DNA, icdA, was cloned to correspond to CDNA-1, and its nucleotide sequence revealed that it contains seven introns. Southern hybridization using cDNA-1 and cDNA-2 indicated that there is only one copy of icdA on the chromosomes of A. niger WU-2223L. Northern hybridization analysis as for total RNA of WU-2223L revealed that two mRNAs of different sizes, 2.0 kb and 1.5 kb, were hybridized to the ORF of cDNA-1 used as a probe. Therefore, it was found that approximately 1500-nt and 2000-nt mRNAs were transcribed from only one icdA chromosomal gene in A. niger. Such a transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50 degreesC), could also grow at 50degreesC in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50 degreesC, the resting cells of WU-F1 degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2'-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Therefore, M. phlei WU-F1 can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.

Dibenzothiophene (DBT) derivatives can be detected in diesel oil following hydrodesulfurization treatment, and they are widely recognized as target compounds for more efficient desulfurization. The moderately thermophilic bacterium Mycobacterium phlei WU-F1 was isolated for its ability to grow at 50 degreesC in a medium with DBT as the sole source of sulfur. At 50 degreesC, resting cells of WU-F1 degraded 0.81 mM DBT within only 90 min to produce 2-hydroxybiphenyl as a desulfurized metabolite through the selective cleavage of carbon-sulfur bonds,, and also degraded 0.81 mM of derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT within 8 h. In addition, the resting cells exhibited high DBT-desulfurizing ability over a wide temperature range from 20 to 50 degreesC. Because M. phlei WU-F1 possesses higher desulfurizing ability toward DBT and the derivatives over a wider temperature range than any other microorganisms previously reported, it may have useful practical applications for biodesulfurization. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Dibenzothiophene (DBT) derivatives can be detected in diesel oil following hydrodesulfurization treatment, and they are widely recognized as target compounds for more efficient desulfurization. The moderately thermophilic bacterium Mycobacterium phlei WU-F1 was isolated for its ability to grow at 50 degreesC in a medium with DBT as the sole source of sulfur. At 50 degreesC, resting cells of WU-F1 degraded 0.81 mM DBT within only 90 min to produce 2-hydroxybiphenyl as a desulfurized metabolite through the selective cleavage of carbon-sulfur bonds,, and also degraded 0.81 mM of derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT within 8 h. In addition, the resting cells exhibited high DBT-desulfurizing ability over a wide temperature range from 20 to 50 degreesC. Because M. phlei WU-F1 possesses higher desulfurizing ability toward DBT and the derivatives over a wider temperature range than any other microorganisms previously reported, it may have useful practical applications for biodesulfurization. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Heterocyclic organosulfur compounds such as dibenzothiophene (DBT) in petroleum cannot be completely removed by hydrodesulfurization using chemical catalysts. A moderately thermophilic bacterium Bacillus subtilis WU-S2B, which could desulfurize DBT at 50 degreesC through the selective cleavage of carbon-sulfur (C-S) bonds, was newly isolated. At 50 degreesC, growing tells of WU-S2B could degrade 0.54 mM DBT within 120 h to produce 2-hydroxybiphenyl, and the resting cells could also degrade 0.81 mM DBT within 12 h, The DBT-desulfurizing ability of WU-S2B is high over a wide temperature range from 30 to 50 degreesC, and highest at 50 degreesC for both the growing and resting cells, and this is an extremely advantageous property for the practical biodesulfurization. In addition, WU-S2B could also desulfurize DBT derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT, Therefore, S. subtilis WU-S2B is considered to have more beneficial properties than other desulfurizing bacteria such as Rhodococcus strains previously reported, particularly from the viewpoint of its capacity for thermophilic desulfurization through the C-S bond cleavage.

Heterocyclic organosulfur compounds such as dibenzothiophene (DBT) in petroleum cannot be completely removed by hydrodesulfurization using chemical catalysts. A moderately thermophilic bacterium Bacillus subtilis WU-S2B, which could desulfurize DBT at 50 degreesC through the selective cleavage of carbon-sulfur (C-S) bonds, was newly isolated. At 50 degreesC, growing tells of WU-S2B could degrade 0.54 mM DBT within 120 h to produce 2-hydroxybiphenyl, and the resting cells could also degrade 0.81 mM DBT within 12 h, The DBT-desulfurizing ability of WU-S2B is high over a wide temperature range from 30 to 50 degreesC, and highest at 50 degreesC for both the growing and resting cells, and this is an extremely advantageous property for the practical biodesulfurization. In addition, WU-S2B could also desulfurize DBT derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT, Therefore, S. subtilis WU-S2B is considered to have more beneficial properties than other desulfurizing bacteria such as Rhodococcus strains previously reported, particularly from the viewpoint of its capacity for thermophilic desulfurization through the C-S bond cleavage.

alpha -Anomer-selective glucosylation of(+)-catechin was carried out using the crude enzyme, showing a-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 mi of 10 mM citrate-Na2HPO4 buffer (pH 6.5) containing 1.2 M maltose at 45 degreesC, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3'-O-alpha -D-glucopyranoside (alpha -C-G) by C-13-NMR, H-1-NMR and two-dimensional HMBC analysis. The reaction at 45 degreesC for 36 h under the optimum conditions gave 12 mM alpha -C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20 degreesC, the solubility in pure water of alpha -C-G, of 450 mg/ml, was approximately 100 fold higher than that of(+)-catechin, of 4.6 mg/ml. Since alpha -C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, alpha -C-G may be a desirable additive for foods, particularly sweet foods.

alpha -Anomer-selective glucosylation of(+)-catechin was carried out using the crude enzyme, showing a-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 mi of 10 mM citrate-Na2HPO4 buffer (pH 6.5) containing 1.2 M maltose at 45 degreesC, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3'-O-alpha -D-glucopyranoside (alpha -C-G) by C-13-NMR, H-1-NMR and two-dimensional HMBC analysis. The reaction at 45 degreesC for 36 h under the optimum conditions gave 12 mM alpha -C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20 degreesC, the solubility in pure water of alpha -C-G, of 450 mg/ml, was approximately 100 fold higher than that of(+)-catechin, of 4.6 mg/ml. Since alpha -C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, alpha -C-G may be a desirable additive for foods, particularly sweet foods.

In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX), In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 muM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A, niger.

In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX), In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 muM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A, niger.

l-Menthyl alpha-D-glucopyranoside (alpha-MenG) is a desirable derivative of l-menthol with useful properties for the production of new flavors and novel food additives, Bacteria were screened for alpha-anomer-selective glucosglation activity toward l-menthol, resulting in the isolation of two strains, Xanthomonas campestris WU-9701 and Stenotrophomonas maltophilia WU-9702, from independent soil samples, Since the safety of X, campestris for use in the food industry is well established, WU-9701 was selected as the more suitable strain for further study, When 50 mg X. campestris WU-9701 lyophilized cells as a biocatalyst were incubated with 1.0 M maltose and 100 mg l-menthol in 10 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) at 40 degrees C, alpha-MenG was accumulated, mainly in a crystalline form, through the anomer-selective synthesis reaction without any by-product formation. Under the optimal conditions, 202 mg alpha-MenG was obtained over 48 h with a highest conversion yield of 99.1% based on the supplied l-menthol. Crude alpha-MenG formed through tills "crystal accumulation reaction" was easily collected from the reaction mixture by separation on filter paper. Plank-like crystals of purified alpha-MenG were subsequently obtained by recrystallization in ethyl acetate solution.

l-Menthyl alpha-D-glucopyranoside (alpha-MenG) is a desirable derivative of l-menthol with useful properties for the production of new flavors and novel food additives, Bacteria were screened for alpha-anomer-selective glucosglation activity toward l-menthol, resulting in the isolation of two strains, Xanthomonas campestris WU-9701 and Stenotrophomonas maltophilia WU-9702, from independent soil samples, Since the safety of X, campestris for use in the food industry is well established, WU-9701 was selected as the more suitable strain for further study, When 50 mg X. campestris WU-9701 lyophilized cells as a biocatalyst were incubated with 1.0 M maltose and 100 mg l-menthol in 10 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) at 40 degrees C, alpha-MenG was accumulated, mainly in a crystalline form, through the anomer-selective synthesis reaction without any by-product formation. Under the optimal conditions, 202 mg alpha-MenG was obtained over 48 h with a highest conversion yield of 99.1% based on the supplied l-menthol. Crude alpha-MenG formed through tills "crystal accumulation reaction" was easily collected from the reaction mixture by separation on filter paper. Plank-like crystals of purified alpha-MenG were subsequently obtained by recrystallization in ethyl acetate solution.

An efficient production strain for L-threonine fermentation was derived from Escherichia coli by multiple rounds of mutation programs that aimed at deregulation of the L-threonine biosynthetic pathway and blocking of L-threonine degradation pathways. When the optimum amount of DL-methionine was added, this strain KY10935, an L-methionine auxotroph, gave 100 g/liter L-threonine after 77 h cultivation. In this strain, key enzymes in the L-threonine biosynthetic pathway were highly derepressed, but some were inhibited by lower concentrations of L-threonine than the accumulated level. Such incomplete deregulation of the pathway was accounted for by the intracellular concentration of L-threonine being lower than the extracellular level. In an assessment of L-threonine transport in terms of phenotypic growth responses to the amino acid, L-threonine-auxotrophic mutants with a lesion in the L-threonine operon were derived from strain KY10935 by selection for auxotrophy for dipeptide L-alanyl-L-threonine or glycyl-L-threonine, the transport systems of which were different from those of L-threonine. All three independent mutants isolated needed an extraordinarily high concentration (10mg/ml) of L-threonine, but grew in the presence of a low concentration (10 mu g/ml) of either dipeptide, indicating that strain KY10935 had impaired L-threonine uptake. These results suggested that the strain had an unusual mechanism of L-threonine hyperproduction: the inability to take up L-threonine that had accumulated extracellularly decreased the steady-state level of intracellular L-threonine, freeing the remaining regulatory steps of feedback inhibition.

An efficient production strain for L-threonine fermentation was derived from Escherichia coli by multiple rounds of mutation programs that aimed at deregulation of the L-threonine biosynthetic pathway and blocking of L-threonine degradation pathways. When the optimum amount of DL-methionine was added, this strain KY10935, an L-methionine auxotroph, gave 100 g/liter L-threonine after 77 h cultivation. In this strain, key enzymes in the L-threonine biosynthetic pathway were highly derepressed, but some were inhibited by lower concentrations of L-threonine than the accumulated level. Such incomplete deregulation of the pathway was accounted for by the intracellular concentration of L-threonine being lower than the extracellular level. In an assessment of L-threonine transport in terms of phenotypic growth responses to the amino acid, L-threonine-auxotrophic mutants with a lesion in the L-threonine operon were derived from strain KY10935 by selection for auxotrophy for dipeptide L-alanyl-L-threonine or glycyl-L-threonine, the transport systems of which were different from those of L-threonine. All three independent mutants isolated needed an extraordinarily high concentration (10mg/ml) of L-threonine, but grew in the presence of a low concentration (10 mu g/ml) of either dipeptide, indicating that strain KY10935 had impaired L-threonine uptake. These results suggested that the strain had an unusual mechanism of L-threonine hyperproduction: the inability to take up L-threonine that had accumulated extracellularly decreased the steady-state level of intracellular L-threonine, freeing the remaining regulatory steps of feedback inhibition.

Introduction of plasmid pKW99, which coexpresses the deregulated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tryptophan-biosynthetic enzymes, into tryptophan-producing Corynebacterium glutamicum KY10894 resulted in a marked increase (54%) in yield of tryptophan production (43 g/liter), but incurred two problems. One was a decline in sugar consumption at the late stage of fermentation, and the other the loss of the plasmid in the absence of selective pressure. The retarded sugar assimilation was found to be attributed to the death of cells that arose from the detrimental action of indole, the last intermediate in the tryptophan pathway, accumulated as a by-product. A chain of these events simultaneously disappeared when serine, the other substrate of the final reaction by tryptophan synthase, was added. These results indicated that a limiting supply of serine was the cause of the decline in the sugar consumption. Thus, to increase carbon flux into serine, the gene for 3-phosphoglycerate dehydrogenase (PGD), the first enzyme in the serine pathway, was cloned from wild-type C.glutamicum ATCC 31833 and joined onto pKW99 to generate pKW9901. Strain KY10894 transformed with pKW9901 favorably consumed sugar through fermentation with accumulating little indole. Furthermore, on the basis of the observation that serine in the medium was consumed rapidly by the recombinant cells, we developed a unique plasmid stabilization system composed of KY9218 (a PGD-deficient serine-requiring strain of KY10894) and pKW9901: In its combination, cells lacking the plasmid should not proliferate in the fermentation medium which does not contain serine. Even if selective pressure was not applied, the modified strain KY9218 with pKW9901 stably maintained the plasmid during fermentation and produced 50 g/liter of tryptophan in a 61% increased yield relative to strain KY10894.

Introduction of plasmid pKW99, which coexpresses the deregulated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tryptophan-biosynthetic enzymes, into tryptophan-producing Corynebacterium glutamicum KY10894 resulted in a marked increase (54%) in yield of tryptophan production (43 g/liter), but incurred two problems. One was a decline in sugar consumption at the late stage of fermentation, and the other the loss of the plasmid in the absence of selective pressure. The retarded sugar assimilation was found to be attributed to the death of cells that arose from the detrimental action of indole, the last intermediate in the tryptophan pathway, accumulated as a by-product. A chain of these events simultaneously disappeared when serine, the other substrate of the final reaction by tryptophan synthase, was added. These results indicated that a limiting supply of serine was the cause of the decline in the sugar consumption. Thus, to increase carbon flux into serine, the gene for 3-phosphoglycerate dehydrogenase (PGD), the first enzyme in the serine pathway, was cloned from wild-type C.glutamicum ATCC 31833 and joined onto pKW99 to generate pKW9901. Strain KY10894 transformed with pKW9901 favorably consumed sugar through fermentation with accumulating little indole. Furthermore, on the basis of the observation that serine in the medium was consumed rapidly by the recombinant cells, we developed a unique plasmid stabilization system composed of KY9218 (a PGD-deficient serine-requiring strain of KY10894) and pKW9901: In its combination, cells lacking the plasmid should not proliferate in the fermentation medium which does not contain serine. Even if selective pressure was not applied, the modified strain KY9218 with pKW9901 stably maintained the plasmid during fermentation and produced 50 g/liter of tryptophan in a 61% increased yield relative to strain KY10894.

Intracellular pH and electron motive force of Thiobacillus thiooxidans were measured. Intracelular pH was maintained between 5.5-5.8,and was not effected by environmental pH. Electron motive force value was highest at pH 2.0,mainly because of the contribution of ΔpH (difference of intracellular pH from environmental pH) rether than membrane potential. Total intracellular adenine nucleotides (ATP+ADP) were most abundant in the exponential growth phase. In death phase, the value decreased to one percent of the that in the exponential phase. Therefore it was recognized that adenine nucleotides are important in cell growth. The energy charge of cells was almost constant over the range pH 2-8,but decreased at pH 1.0,as indicated by the fact that cell growth slowed down below pH 1.5.

Biochemical removal of rust from iron surfaces has been investigated. By immersing a rusted iron plate in the culture medium of an iron-oxidizing bacterium, Thiobacillus ferrooxidans, iron adjacent to the rust was dissolved and the rust was peeled off. Since the amount of dissolved iron per unit iron plate surface area correlated with the concentration of ferric iron in the culture medium, the formation of ferric iron is probably involved in dissolving the iron as is the case for bacterial leaching. In the present study, rust removal in a "continuous" system in which the culture medium was circulated from the fermentor to the rust removal vessel and back again to the fermentor, has also been investigated. Although growth inhibition was observed with the formation of ferric iron precipitates during the operation in this system, it was possible to prevent this precipitation by lowering the pH of the medium during the mixed cultivation of T. ferrooxidans and a sulfur-oxidizing bacterium, T. thiooxidans. © 1981 Springer-Verlag.

The growth and the activities of energy-acquiring enzymes of Thiobacillus ferrooxidans, one of the more common iron-oxidizing bacteira having the capacity to oxidize elemental sulfur as well as ferrous iron, were investigated with mixed-substrate medium. Highlights of this study are as follows : 1. Addition of elemental sulfur resulted in delays of the iron oxidation as well as the cell growth.2. On the mixed-substrate medium ferrous iron was preferentially utilized.3. Elemental sulfur was well oxidized after complete oxidation of the ferrous iron, but a gradual oxidation of sulfur had occurred during the iron oxidation.4. Ferrous iron migth induce the synthesis of iron oxidase and repress thiosulfate-oxidizing enzyme while it didn't inhibit rhodanese or AMP-independent sulfite oxidase.5. Diauxic growth ont he mixed-substrate medium might be caused by the conversion of energy metabolism.6. A main pathway was proposed in the elemental sulfur metabolism of T. ferrooxidans : S^0→SO_3^<2->→SO_4^<2->