In recent years, advances in bioengineering and synthetic biology techniques have been used to create carotenoid diversity in the laboratory. In this chapter, we describe the step-by-step method to perform directed evolution of carotenoid biosynthetic enzymes. We first explain how to establish an efficient Escherichia coli colony-based screening, including a detailed description of plasmid DNA construction design as well as tips and tricks to handle and manipulate cells to produce stable colonies. As an example for the directed evolution experiment, we engineer a bacterial phytoene desaturase CrtI to obtain a C50-phytoene desaturase, which catalyzes formation of a non-natural long-chain carotenoid. The method described in this chapter can be applied to many carotenoid biosynthetic enzymes, whose numbers have been rapidly expanding with recent advances in genomics. The use of directed evolution for carotenoid enzymes will contribute not only to the discovery of novel carotenoids but also to a deeper understanding of the creation and evolution of carotenoid biosynthetic pathways in nature.

<title>Abstract</title>A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism <italic>Saccharomyces cerevisiae</italic>. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system’s utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (&gt;100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.

A variety of positive/negative selection systems have been exploited as genome engineering tools and screening platforms for genetic switches. While numerous positive-selection systems are available, only a handful of negative-selection systems are useful for such applications. We previously reported a powerful negative-selection system using herpes simplex virus thymidine kinase (HsvTK) and the mutagenic nucleoside analog 6-(β-d-2-deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido [4,5-c][1,2] oxazin-7-one (dP). Upon addition of 1000 nM dP, cells expressing HsvTK quickly die, with unprecedented efficacy. However, this selection procedure elevates the spontaneous mutation rate of the host cells by 10-fold due to the mutagenic nature of dP. To decrease the operative concentration of dP required for negative selection, we systematically created the strains of Escherichia coli either by removing or overexpressing genes involved in DNA/RNA metabolism. We found that over-expression of NupC and NupG (nucleoside uptake-related inner membrane transporters), Tsx (outer membrane transporter), NdK (nucleotide kinase) sensitized E. coli cells to dP. Simultaneous overexpression of these three genes (ndk-nupC-tsx) significantly improved the dP-sensitivity of E. coli, lowering the necessary operative concentration of dP for negative selection by 10-fold. This enabled robust and selective elimination of strains harboring chromosomally-encoded hsvtk simply by adding as low as 100 nM dP, which causes only a modest increase in the spontaneous mutation frequency as compared to the cells without hsvtk.

Heterologous production of a useful carotenoid astaxanthin was achieved in a cyanobacterium Synechocystis sp. PCC 6803 with the aid of marine bacterial genes. Astaxanthin and its intermediates emerged at high levels, whereas β-carotene and zeaxanthin disappeared in the strain. Total carotenoid accumulation was nearly two fold compared with wild type. The astaxanthin-producing strain was capable of only growing heterotrophically, which was likely due to the absence of β-carotene. Further enhanced accumulation was pursued by gene overexpression for possible rate-limiting steps in the biosynthesis pathway.

Stringency (low leak) is one of the most important specifications required for genetic circuits and induction systems, but it is challenging to evolve without sacrificing the maximum output level. This problem also comes from the absence of truly tunable negative selection methods. This paper reports that stringently switching variants can sometimes emerge with surprising frequency upon mutations. We randomly mutated the previously generated leaky variants of LuxR, the quorum-sensing transcription activator from Vibrio fischeri, to restore the stringency. We found as much as 10-20% of the entire population exhibited significantly improved signal-to-noise ratios compared with their parents. This indicated that these mutants arose by the loss of folding capability by accumulating destabilizing mutations, not by introducing rare adaptive mutations, thereby becoming AHL-dependent folders. Only four rounds of mutagenesis and ON-state selection resulted in the domination of the entire population by the improved variants with low leak, without direct selection pressure for stringency. With this surprising frequency, conversion into the "ligand-addicted folders" should be one of the prevailing modes of evolving stringency both in the laboratory and in nature, and the workflow described here provides a rapid and versatile method of improving the signal-to-noise ratio of various genetic switches.

Genetic switches provide core components of gene expression induction systems, genetic circuits, and metabolite sensors, including those for high-throughput screening and selection of enzyme functions. However, it is rare that natural transcription factors meet the required specifications for each application. Fortunately, the directed evolution of transcription switches is a straightforward process, given that the two states of genetic switches (on and off) are both selectable, using a wide range of positive and negative selection tools developed in the field of molecular genetics. On/off-state selections based on bactericidal mechanism allow greatly accelerate the entire process. The key to success is finding the selection conditions that minimize false-positive and false-negative clones as with any directed evolution experiment. We introduce a reliable and automatable directed evolution platform to rapidly evolve genetic switches with desired specifications in this chapter. Highlighting the importance and ease of screening for selection conditions, we demonstrate how selection conditions influence the resultant population of the selected pools.

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 μg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).

Carotenoids are structurally diverse pigments with various important biological functions. There has been a large interest in the search for novel carotenoid structures, since only a slight structural changes can result in a drastic difference in their biological functions. Carotenoid-modifying enzymes show remarkable substrate promiscuity, allowing rapid access to a vast set of novel carotenoids by combinatorial biosynthesis. We previously constructed a nonnatural carotenoid biosynthetic pathway in Escherichia coli that can produce C50 carotenoids having a longer chain than their natural C40 counterparts. In this study, a carotenoid 2,2'-hydroxylase (crtG) from Brevundimonas sp. SD212 was coexpressed together with our laboratory-engineered C50-zeaxanthin and C50-astaxanthin biosynthetic pathways. We identified six novel nonnatural C50-xanthophylls, namely, C50-nostoxanthin, C50-caloxanthin, C50-adonixanthin, C50-4-ketonostoxanthin, C50-2-hydroxyastaxanthin, and C50-2,2'-dihydroxyastaxanthin.

A weak acid cation exchange fiber used for large-scale antibody purification was prepared by radiation-induced graft polymerization of acrylic acid (AA) onto a commercially available nylon-6 fiber. The AA-grafted fiber was post-treated by conditioning in NaOH aqueous solution at 298 K followed by immersion in water at 353 K to increase the protein binding capacity of the cation exchange fiber. A 2 g/L lysozyme solution buffered at pH 6.0 flowed through a column charged with a treated or untreated AA-grafted fiber at a space velocity of 20 h(-1). The 10% dynamic binding capacity of the column charged with the former fiber of 200 mg/mL-column was 5.9-fold that of the column charged with the latter fiber, and 2.3-fold that (88 mg/mL-column) of a column charged with CM Sepharose (TM) Fast Flow (GE Healthcare).

Longer-chain carotenoids have interesting physiological and electronic/photonic properties due to their extensive polyene structures. Establishing nonnatural biosynthetic pathways for longer-chain carotenoids in engineerable microorganisms will provide a platform to diversify and explore the potential of these molecules. We have previously reported the biosynthesis of nonnatural C50 carotenoids by engineering a C30-carotenoid backbone synthase (CrtM) from Staphylococcus aureus. In the present work, we conducted a series of experiments to engineer C60 carotenoid pathways. Stepwise introduction of cavity-expanding mutations together with stabilizing mutations progressively shifted the product size specificity of CrtM toward efficient synthases for C60 carotenoids. By coexpressing these CrtM variants with hexaprenyl diphosphate synthase, we observed that C60-phytoene accumulated together with a small amount of C65-phytoene, which is the largest carotenoid biosynthesized to date. Although these carotenoids failed to serve as a substrate for carotene desaturases, the C25-half of the C55-phytoene was accepted by the variant of phytoene desaturase CrtI, leading to accumulation of the largest carotenoid-based pigments. Continuing effort should further expand the scope of carotenoids, which are promising components for various biological (light-harvesting, antioxidant, and communicating) and nonbiological (photovoltaic, photonic, and field-effect transistor) systems.

While the majority of the natural carotenoid pigments are based on 40-carbon (C40) skeleton, some carotenoids from bacteria have larger C50 skeleton, biosynthesized by attaching two isoprene units (C5) to both sides of the C40 carotenoid pigment lycopene. Subsequent cyclization reactions result in the production of C50 carotenoids with diverse and unique skeletal structures. To produce even larger nonnatural novel carotenoids with C50 + C5 + C5 = C60 skeletons, we systematically coexpressed natural C50 carotenoid biosynthetic enzymes (lycopene C5-elongases and C50-cyclases) from various bacterial sources together with the laboratory-engineered nonnatural C50-lycopene pathway in Escherichia coli. Among the tested enzymes, the elongases and cyclases from Micrococcus luteus exhibited significant activity toward C50-lycopene, and yielded the novel carotenoids C60-flavuxanthin and C60-sarcinaxanthin. Moreover, coexpression of M. luteus elongase with Corynebacterium cyclase resulted in the production of C60-sarcinaxanthin, C60-sarprenoxanthin, and C60-decaprenoxanthin.

Substrate tolerance of bacterial cyclases has been demonstrated in various contexts, but little is known about that of plant cyclases. Here, we tested two plant ε-cyclases to convert C50-lycopene, which we previously established by rounds of directed evolution. Unlike bacterial β-cyclases, two-end cyclase from lettuce exhibited complete specificity against this molecule, indicating that this enzyme has some mechanism that exerts size-specificity. Arabidopsis one-end cyclase At-y2 showed detectable activity to C50-lycopene. Interestingly, we found that it functions as a two-end cyclase in a C50 context. Based on this observation, a possible model for substrate discrimination of this enzyme is proposed.

The cellular activities of gymnosperms monoterpene synthases are largely compromised due to their requirement for manganese, which is deficient in microbial cells. Through site-saturation mutagenesis of the residue adjacent to metal-binding glutamate, we found that pinene synthase is highly mutable at this position yet drastically alter their metal binding preference, thereby quickly improving the cellular performance in heterologous hosts.

An efficient method for rare metal recovery from environmental water and urban mines is in high demand. Toward rapid and high-resolution rare metal ion separation, a novel bis(2-ethylhexyl) phosphate (HDEHP)-impregnated graft-type particle as a filler for a chromatography column is proposed. To achieve rapid and high-resolution separation, a convection-flow-aided elution mode is required. The combination of 35 μm non-porous particles and a polymer-brush-rich particle structure minimizes the distance from metal ion binding sites to the convection flow in the column, resulting in minimized diffusional mass transfer resistance and the convection-flow-aided elution mode. The HDEHP-impregnated graft-type non-porous-particle-packed cartridge developed in this study exhibited a higher separation performance for model rare metals, neodymium (III) and dysprosium (III) ions, and a narrower peak at a higher linear velocity, than those of previous HDEHP-impregnated fiber-packed and commercially available Lewatit® VP OC 1026-packed cartridges.

<p>緑茶抽出液からカテキンを除去するために，放射線グラフト重合法を適用して，市販の6-ナイロン繊維を基材にN-ビニルピロリドン（NVP）グラフト重合繊維（NVP繊維）を作製した．NVP密度3.9 mmol/gのNVP繊維へのカテキンの吸着平衡関係はLangmuir型吸着等温式に整理できた．飽和吸着量および吸着平衡定数は，それぞれ0.23および13 L/mmolであった．また，NVP繊維に吸着したカテキンを0.1 M NaOHによって処理することで吸着量を100%再生できた．この後，2から4回目まで吸着と溶出を繰り返しても吸着量はほとんど減少することなく一定のままであった．</p>

To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GESM53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.

A concentric pulse by motile cells of Escherichia coli (E. coli) propagates and the cells aggregate to form self-organized patterns.We summarize experimental and numerical results on the self-organized pattern formation of E. coli to elucidate some aspects of its mechanism. Our presentation includes experiments on E. coli patterns, as well as numerical simulations on the basis of a reaction-diffusionchemotaxis model.We find good agreement for one-dimensional propagating fronts in observation and simulation. However, corresponding results for two-dimensional circular bacterial clusters have still not been obtained.

Hydrous cerium oxide was impregnated onto a sufonic-acid-group-containing fiber (SS fiber) prepared by radiation-induced graft polymerization and subsequent chemical modifications. Cerium ions (Ce³⁺) were adsorbed onto SS fiber before the formation of hydrous cerium oxide (H-CeO) via precipitation by a reaction with a sodium hydroxide (NaOH) solution at various NaOH concentrations in the range from 0.001 to 0.1 mol L^&minus;1. The maximum percentage impregnation of H-CeO was 12 %. In the batch mode, neither SS fiber nor anion-exchange fiber removed any antimony ions, whereas H-CeO fiber captured antimony ions at a high rate.

<p>タンパク質を高容量に吸着するカチオン交換繊維をグラフト重合によって作製するために，モノマー液に溶液とエマルションを使う2種類のグラフト重合法，すなわち放射線前照射溶液および乳化グラフト重合法（以後，溶液および乳化グラフト重合法）を採用し，比較した．市販の6-ナイロン製繊維へグリシジルメタクリレート（GMA）をグラフト重合後，亜硫酸ナトリウムとの反応によってポリGMA鎖のエポキシ基をスルホン酸基へ転化し，カチオン交換繊維を作製した．得られた2種類のカチオン交換繊維を充填したカラムに，リゾチーム溶液（pH 9.0）を空間速度60 h⁻¹で流通させた．GMAグラフト率が81–87%のとき，乳化グラフト重合法によって作製した繊維のカラム体積あたりの10%動的吸着容量は，溶液グラフト重合法によって作製したそれよりも4倍高い値を示した．乳化グラフト重合法によって作製した繊維の周縁部に形成されたグラフト鎖が，タンパク質の高速吸着に寄与すると推察される．</p>

LuxR family transcriptional regulators are the core components of quorum sensing in Gram-negative bacteria and exert their effects through binding to the signaling molecules acyl-homoserine lactones (acyl-HSLs). The function of the LuxR homologs is remarkably plastic, and naturally occurring acyl-HSLs are structurally diverse. To investigate the molecular basis of the functional plasticity of Vibrio fischeri LuxR, we directed the evolution of LuxR toward three different specificities in the laboratory. We found an orthogonal pair of LuxR mutants specific either to 3-oxo-hexanoyl homoserine lactone or to 3-oxo-octanoyl homoserine lactone. Interestingly, the majority of the specificity changes did not arise from modulating the recognition event but rather from changing the efficiency of the transition from the inactive form to the active form upon signal binding. This finding explains how quorum sensing systems can rapidly diverge in nature and in the laboratory and how signal orthogonality and mutual inhibition frequently occur among closely related diverging systems.

Synthetic biologists are in need of genetic switches, or inducible sensor/promoter systems, that can be reliably integrated in multiple contexts. Using a liquid-based selection method, we systematically engineered the choline-inducible transcription factor BetI, yielding various choline-inducible and choline-repressive promoter systems with various input-output characteristics. In addition to having high stringency and a high maximum induction level, they underwent a graded and single-peaked response to choline. Taking advantage of these features, we demonstrated the utility of these systems for controlling the carotenoid biosynthetic pathway and for constructing two-input logic gates. Additionally, we demonstrated the rapidity, throughput, robustness, and cost-effectiveness of our selection method, which facilitates the conversion of natural genetic controlling systems into systems that are designed for various synthetic biology applications.

LuxR is the core component of Vibrio fischeri quorum sensing. It acts as the transcriptional activator by binding to its cognate signaling molecules 3-oxo-hexanoyl-homoserine lactone (3OC6HSL). Although several acyl-HSLs with 3-oxo groups are known to activate LuxR with similar efficiency, acyl-HSLs without 3-oxo groups are very weak inducers. We conducted a round of LuxR directed evolution to acquire LuxR mutants with higher signal sensitivity to octanoyl-homoserine lactone (C8HSL). All of the isolated mutants showed increased signal sensitivity to many other acyl-HSLs, including C8HSL, and some to the LuxR antagonist p-coumaroyl-HSL. The evolution of their ligand sensitivity proceeded through the stabilization of the signal-bound state, thereby elevating the effective concentration of LuxR at the ON-state.

Successful feeding of the substrate geranylpyrophosphate (GPP) to monoterpene synthase is critical to the efficient microbial production of monoterpenes. Overexpression of GPP synthases, metabolic channeling from GPP synthase to terpene synthases, and down-tuning of endogenous competitors have been successfully used to increase the production of monoterpene. Nevertheless, the production of monoterpenes has remained considerably lower than that of hemi-/sesqui-terpenoids. We tested whether it is effective to improve the cellular activity of monoterpene synthases. To this end, we developed a high-throughput screening system to monitor for elevated GPP consumption. Through a single round of mutagenesis and screening, we isolated a pinene synthase variant that outperformed the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibited drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese-deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenylpyrophosphate isomerase, and GPP synthase yielded 140 mg/L pinene in a flask culture.

At Tokyo Electric Power Company (TEPCO) Fukushima Daiichi nuclear power plant (NPP), water contaminated with radionuclides such as Cs-137 and Sr-90 has been stored in tanks and seawater-intake area. We have prepared cobalt-ferrocyanide-impregnated fibers via four steps: the grafting of an epoxy-group-containing monomer, the conversion of the epoxy group into positively charged groups, the binding of ferrocyanide ions ([Fe(CN)(6)](4-)), and the precipitation of cobalt ferrocyanide (Co-2[Fe(CN)(6)]) by contact with cobalt ions. However, the impregnation structure of cobalt ferrocyanide microparticles onto the fiber remains unclear. Here, we describe the impregnation structure from the results of rebinding [Fe(CN)(6)](4-) to the cobalt-ferrocyanide-impregnated fiber. The amount of [Fe(CN)(6)](4-) re-bound onto the fiber was found to decrease with increasing amount of Co-2[Fe(CN)(6)] initially impregnated. This suggests that the microparticles of cobalt ferrocyanide become entangled with the grafted polymer chains via multipoint electrostatic interactions.

塩酸溶液からのパラジウム（Pd）の回収のため，Pdを特異的に捕捉する中性抽出試薬ジオクチルスルフィド（DOS）を，放射線グラフト重合法によって6-ナイロン繊維に付与した疎水性基固定高分子鎖に担持した．ドデカンチオールと6-ナイロン繊維にグラフト重合したポリグリシジルメタクリレート（GMA）鎖中のエポキシ基との反応によって，2.0 mmol/gの疎水性基（C12S基）を繊維に固定した．C12S基は，ドデシル鎖との疎水性相互作用によってDOSを担持する，およびDOSと同様の機構でPdを吸着する2つの機能をもつ．さらに，疎水性基を付与したグラフト鎖上に，0.40–1.1 mmol/gの密度でDOSを担持した．0.40 mmol/gのDOSを担持した繊維の，1 mol/L塩酸中でのPd飽和吸着量は0.70 mmol/gであった．Pd飽和吸着量とDOS担持量との関係から，PdとDOSおよびC12S基との結合モル比が，それぞれ0.50および0.27と算出された．また，DOS担持繊維は，塩酸濃度が1–4 mol/LのPdおよび白金（Pt）混合溶液から，Ptをほとんど吸着せずに，Pdを特異的に吸着できた．

By assembly and evolutionary engineering of T7-phage-based transcriptional switches made from endogenous components of the bet operon on the Escherichia coli chromosome, genetic switches inducible by choline, a safe and inexpensive compound, were constructed. The functional plasticity of the BetI repressor was revealed by rapid and high-frequency identification of functional variants with various properties, including those with high stringency, high maximum expression level, and reversed phenotypes, from a pool of BetI mutants. The plasmid expression of BetI mutants resulted in the choline-inducible (Bet-ON) or choline-repressible (Bet-OFF) switching of genes under the pT7/betO sequence at unprecedentedly high levels, while keeping the minimal leaky expression in uninduced conditions.

Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C50-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C50 backbones produced. We then further extend the C50 pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C50 carotenoids, including ∼ 90% C50-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.

Squalene is a precursor of thousands of bioactive triterpenoids and also has industrial value as a lubricant, health-promoting agent, and/or drop-in biofuel. To establish an efficient Escherichia coli-based system for squalene production, we tested two different squalene synthases and their mutants in combination with precursor pathways. By co-expressing a chimeric mevalonate pathway with human or Thermosynechococcus squalene synthase, E. coli accumulated squalene up to 230 mg/L or 55 mg/g-DCW in flask culture. We also determined that a significant truncation of squalene synthase at the C-terminus retains partial cellular activity. The squalene-producing strain described herein represents a convenient platform for gene discovery and the construction of the pathway toward natural and non-natural hopanoids/steroids.

酸性抽出試薬であるリン酸ビス（2-エチルヘキシル）（HDEHP）を，6-ナイロン繊維上に接ぎ木した高分子鎖に0.43 mol/kg-productの密度で担持した．HDEHP担持繊維充填カラムに負荷したネオジムとジスプロシウムイオンとを，3種類の濃度の塩酸を使った溶出クロマトグラフィーによって分離した．さまざまな空間速度および総負荷量であっても，HDEHP担持繊維充填カラムは，HDEHP担持ビーズ充填カラムと比較して，溶出クロマトグラムにおいて高いピークおよび小さい半値幅を示した．これは，イオンの拡散物質移動距離が短く，カラム体積あたりの表面積が大きいためである．担持されたHDEHPの漏出は検出されなかった．

Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counter-selectable markers limits the throughput of this method. Additionally, the emergence of 'selection escapees' can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating hsvtk gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications.

The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3')-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C30 carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a "dehydrosqualene synthase." All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.

Most natural carotenoids have 40-carbon (C40) backbones, while some bacteria produce carotenoids with C30 backbones. Carotenoid backbone synthases, the enzyme that catalyze the first committed step in carotenoid biosynthesis, are known to be highly specific. Previously, using C30 backbone synthase (diapophytoene synthase, CrtM) from Staphylococcus aureus, we reported two size-shifting mutations, F26A and W38A, which confer C40 synthase activity at the cost of the original C30 synthase activity. In this study, we performed a directed evolution of the C40-specialist variant CrtMF26A in search of mutations that restore the original C30 synthase function. Examination of the resultant mutants, together with the site-directed mutagenesis study identified three new mutations (H12A, D27A and I240F) that affect the size specificity of this enzyme. After re-defining the reading frame, we obtained CrtM variants that are highly active in C30 and C40 carotenoid synthesis.

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C30 carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C30 carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a "squalene route" and a "lycopersene route" for C30 and C40 carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.

Potassium cobalt hexacyanoferrate compounds (KCo-HCFe's) were impregnated onto a 6-nylon fiber by radiation-induced graft polymerization and subsequent chemical modifications. First, dimethylaminoethyl methacrylate was graft-polymerized onto the nylon fiber. Second, hexacyanoferrate ions were bound to graft chains via an anion-exchange interaction. Third, KCo-HCFe's were formed on the nylon fiber via the precipitation reaction of hexacyanoferrate ions with cobalt ions in the presence of potassium chloride. The resulting KCo-HCFe-impregnated fiber had an impregnation percentage of the fiber for KCo-HCFe's of 7%. The cesium concentration in 10. ppm cesium chloride solution with the immersion of this fiber decreased to 0.6. ppm within 60. min at a ratio of liquid volume (10. mL) to fiber mass (0.1. g). The fiber was fabricated into a braid with a length of 100. cm and a diameter of 8. cm for practical use at sites contaminated with cesium. © 2013 Elsevier Ltd.

Ever-increasing repertories of RNA-based switching devices are enabling synthetic biologists to construct compact, self-standing, and easy-to-integrate regulatory circuits. However, it is rather rare that the existing RNA-based expression controllers happen to have the exact specification needed for particular applications from the beginning. Evolutionary design of is powerful strategy for quickly tuning functions/specification of genetic switches. Presented here are the steps required for rapid and efficient enrichment of genetic switches with desired specification using recently developed nucleoside kinase-based dual selection system. Here, the library of genetic switches, created by randomizing either the part or the entire sequence coding switching components, is subjected to OFF (negative) selection and ON (positive) selection in various conditions. The entire selection process is completed only by liquid handling, facilitating the parallel and continuous operations of multiple selection projects. This automation-liable platform for genetic selection of functional switches has potential applications for development of RNA-based biosensors, expression controllers, and their integrated forms (genetic circuits).

Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

6-ナイロン繊維上にグラフトした高分子鎖のドデシルアミノ基上にリン酸ビス（2-エチルヘキシル）（HDEHP）を担持した．この繊維でのネオジムおよびジスプロシウムイオンの分配係数は，ドデカンに溶解したHDEHPのそれとほぼ一致した．疎水性グラフト鎖上に担持されたHDEHPは，有機溶媒中に溶解しているHDEHPのような挙動を示すことがわかった．HDEHP担持繊維を充填したカラムを使って，ネオジムおよびジスプロシウムイオンを，溶出クロマトグラフィーによって分離した．このとき，グラフト鎖上に担持されているHDEHPの液中への漏出は無視できることがわかった．

Aliquat 336 was impregnated onto the polymer chain grafted onto a 2.0-mm-thick porous sheet with a porosity of 75% and a pore size of 1.2 mu m via the graft polymerization of glycidyl methacrylate and the subsequent reaction of the epoxy group with mercaptoundecanoic acid. In a 2:1 ethanol/4M NaOH (v/v) mixture, Aliquat 336 was impregnated at a density of 0.85mmol/g, which was comparable to that of conventional Aliquat-336-impregnated polymeric beads. The dynamic binding capacity for palladium was 0.60mmol/g when 100mg-Pd/L palladium chloride solution was forced to permeate at a space velocity of 3700h(-1.</SUP)

Glycidyl methacrylate (GMA) was graft-polymerized onto a porous sheet (75% porosity and 1.6-mu m average pore size). previously irradiated with an electron beam. The resultant grafted poly-GMA chain can be classified as a polymer brush. extending from the pore surface toward the pore interior and a polymer root invading the polymer matrix. The boundary crossing the pore/matrix interface can be detected in two independent ways: molar conversion of the epoxy group into a sulfonic. group providing a plateau in the molar conversion versus reaction time curve and molar conversion exhibiting a breakthrough point in the swelling ratio versus molar conversion curve. A higher irradiation dose was found to lead to a higher mole percentage of polymer brush in the graft chain. The dose range from 20 to 200 kGy corresponded to the mole percentage range of polymer brush from 6% to 15%.

Dimethylamino-gamma-butyric acid (DMGABA) as an ampholite was reacted with the epoxy group of the poly-glycidyl methacrylate chain grafted onto the pore surface of a porous hollow-fiber polyethylene membrane by radiation-induced graft polymerization. DMGABA was dissolved in a mixture of dioxane and water at various dioxane volume fractions, defined by dividing the dioxane volume by the total volume. The equilibrium binding capacity (EBC) of the DMGABA-immobilized porous hollow-fiber membrane for lysozyme was evaluated in the permeation mode. The EBC was varied from a 1/50-fold monolayer binding capacity to a 10-fold monolayer binding capacity by controlling the composition of the solvent used for DMGABA immobilization and the molar conversion of the epoxy group into the DMGABA group. (c) 2013 Elsevier Ltd. All rights reserved.

地表水中へ放出された放射性セシウムを除去するために，セシウムイオンを特異的に吸着する不溶性フェロシアン化物（KCoFC）を，6-ナイロン繊維にvinylbenzyltrimethylammonium chloride（VBTAC）とN-vinyl-2-pyrrolidone（NVP）を共グラフト重合して得られた高分子鎖に担持した．0–0.5 Mの範囲で塩化ナトリウムを添加した10 mg/L塩化セシウム水溶液を，不溶性フェロシアン化物（KCoFC）担持繊維に流通させた．0.5 M塩化ナトリウムを添加したときのセシウム動的吸着容量は，添加していないときのそれと比較して30倍大きくなった．また，海水中での吸着等温式はLangmuir型の式に整理でき，不溶性フェロシアン化物（KCoFC）担持繊維のセシウム飽和吸着量は20 mg-Cs/g-dryであった．

さまざまな接触方式で海水から放射性セシウムを高速除去できる不溶性フェロシアン化コバルト担持繊維を作製した。まず，アニオン交換基をもつビニルモノマー，ジメチルアミノエチルメタクリレート（DMAEMA）を，γ線を予め照射した 6-ナイロン繊維にグラフト重合した。つぎに，フェロシアン化物イオンを DMAEMA グラフト繊維に吸着させた。さらに，コバルトイオンとフェロシアン化物イオンとの沈殿反応によって繊維上に不溶性フェロシアン化コバルト（K₂Co[Fe(CN)₆］) を担持した。パイロット規模の反応器を使って，ワインド型のセシウム吸着繊維を 1 回で約 100 kg を製造した。繊維に対する海水の重量比を 100 にして，回分の接触方式によって，初期濃度 10 mg-Cs/L が 30 分後に検出限界（0.2 mg-Cs/L）以下に減った。また，吸着等温式は Langmuir 型の式に整理でき，海水中のセシウム濃度 1 mg-Cs/L のときの濃縮係数は 21000 であった。さらに，不溶性フェロシアン化コバルト担持繊維を，空気中，500°C で，シアン化水素を発生させることなく焼却でき，減容可能であった。<br>

Removal of the major urinary protein, cauxin, a carboxylesterase, from cat urine is essential for distinguishing between physiological and abnormal proteinuria by a urine dipstick. We have previously developed a material for removing cauxin by using lens culinaris agglutinin (LCA) lectin which targets the N-linked oligosaccharides present in cauxin. To improve the affinity and specificity toward cauxin, we immobilized 1,1,1-trifluoro-3-(2-sulfanylethylsulfanyl) propane-2-one, an inhibitor of esterases, to a polymer chain grafted on to a porous hollow-fiber membrane by applying radiation-induced graft polymerization. Normal male urine was forced to permeate through the pores rimmed by the ligand-immobilized polymer chain. Cauxin could not be detected in the effluent from the membrane. The residence time of the urine across a membrane thickness of 1 mm was set at 7 s. The respective dynamic and equilibrium binding capacities of the membrane for cauxin were 2 and 3 mg/g. The developed cauxin-affinity membrane material was more effective for diagnosing cat kidney diseases than the LCA lectin tip.

An epoxy-group-containing vinyl monomer, i.e., glycidyl methacrylate, was graft-polymerized onto polyethylene particles with an average diameter of 35 mu m at various reaction temperatures within 278-333 K. The produced epoxy group was converted into a diethylamino group as an anion-exchange group. From the equilibrium binding capacity of the resultant particle-packed bed for bovine serum albumin (BSA) and the pressure loss required for a protein solution to flow through the bed, the formation of graft chains that were sufficiently long to hold BSA in multilayers and allow convective flow among them was predicted. The achieved ideal adsorption characteristics enabled a higher flow rate of the protein solution, which leads to a higher overall adsorption rate of the protein because of convective flow among the graft chains.

Dimethylaminoethyl methacrylate (DMAEMA) was graft-polymerized at a density of 1.7 mmol/g onto a 6-nylon fiber by radiation-induced graft polymerization. Urease was bound to the resultant anion-exchange fiber by electrostatic interaction. After crosslinking with transglutaminase, 80% of the bound urease was immobilized at a density of 41 mg/g of the fiber. Urea in water was quantitatively hydrolyzed during the permeation of a 0.20 mg/L urea solution through the urease-immobilized-fiber-packed bed with a diameter of 5.5 mm and a height of 27 mm in the residence time range of 12-180 s. The activity of immobilized urease did not deteriorate after repeated use of the fiber, i.e., during reaction and storage.

An epoxy-group-containing vinyl monomer, glycidyl methacrylate, was graft-polymerized onto an electron-beam-irradiated porous sheet to form a graft chain. Graft chains can be categorized according to the formation site into the polymer brush extending from the pore surface of a porous sheet and polymer root penetrating into the polymer matrix of the porous sheet. The polymer brush and root of the porous sheet govern its protein adsorptivity and liquid permeability. The mole percentages of the polymer brush and the root were determined from the dependence of the degree of swelling on the molar conversion of epoxy groups into ion-exchange groups with water as the solvent during functionalization. This method enables us to better understand the manner in which polymer chains are grafted onto porous polymers and helps us to design porous polymers for protein purification and enzyme immobilization.

Directed evolution is a well-established strategy to confer novel catalytic functions to the enzymes. Thanks to the relative ease of establishing color screening, carotenogenic enzymes can be rapidly evolved in the laboratory for novel functions. The combinatorial usages of the evolvants result in the creation of diverse set of novel, sometimes unnatural carotenoids. This chapter describes the directed evolution of diapophytoene (C(30) carotenoid) synthase CrtM to function in the foreign C(40) pathway, and the use of the CrtM variants thus obtained for the production of novel backbone structures.

This study investigates improvements in high-resolution elution chromatography of proteins loaded to a bed charged by way of cation-exchange polymer-brush-immobilized particles. A sulfonic acid group as a cation-exchange group was introduced into a polymer brush grafted onto a polyethylene particle with an average diameter of 35 mu m. A bed charged with the resulting cation-exchange polymer-brush-immobilized particles, SS bed, has a sulfonic acid group density of 0.36 mmol/mL-bed. A mixture of alpha-chymotripsinogen A (chy), cytochrome c (cyt), and lysozyme (lys) was loaded onto the SS bed. The subsequent linear gradient elution performance of the proteins adsorbed by the SS bed with 20 mM phosphate buffer (pH 6.0) and 1 M NaCl is compared with those by commercially available cation-exchange-bead-packed beds. The resolutions of the SS bed of both the chy/cyt and cyt/lys pairs at a linear velocity of 600 cm/h exceeded 1.9 at a loading amount of 0.8 mg/mL-bed, which was not achieved using the beds charged with SOURCE (R) 30S, POROS (R) 50HS, or Fractogel (R) EMD SO3- (s). Even at a loading amount of 12 mg/mL-bed, the resolutions of the SS bed for the chy/cyt and cyt/lys pairs at a linear velocity of 300 cm/h were 1.4 and 2.7, respectively.

An octadecylamino-group-introduced polymer chain grafted onto a porous sheet was impregnated with bis(2-ethylhexyl) hydrogen phosphate (HDEHP). A mixture of HDEHP and ethanol of various HDEHP concentrations was used for the impregnation. The porous sheet into which a C18H37NH group was introduced was immersed in HDEHP/ethanol solution before ethanol evaporation. The liquid permeability of a cartridge charged with the HDEHP-impregnated porous sheet in disk form prepared in 50 (v/v)% HDEHP/ethanol solution was 96% that of the starting-porous-disk-packed cartridge. The equilibrium binding capacity of the HDEHP-impregnated porous disk for yttrium ions was 0.32 mol per kg of the disk. In addition, the HDEHP-impregnated-porous-disc-packed cartridge was found to be applicable to the preconcentration of trace amounts of lanthanides in a multielement solution prior to their measurement by inductively coupled plasma mass spectrometry.

The first step toward elucidating the mutagenic effects of chemicals and pathways is to determine the specificity of the mutations generated spontaneously or in response to treatment with mutagens. We constructed a set of plasmid-encoded probes for the specific detection of each type of base substitution mutation. Using these probes, we were able to quickly determine both the mutation rate and the specificity of the mutations caused by different types of mutagens and mutagenic conditions. We also developed a PCR-based method to rapidly and robustly determine the mutation spectrum in response to various mutagenic samples in parallel. This system allows one to not only analyze the mutation specificity of various chemicals, but also to search for novel genetic elements that promote the specific mutation events.

By using electron-beam-induced graft polymerization, an epoxy-group-containing monomer, glycidyl methacrylate (GMA), was appended onto a 6-nylon fiber; subsequently, N-methylglucamine as a chelate-forming moiety was added to the epoxy group. The chelating group density of the resultant chelating fiber was 2.0 mmol/g, which was 74% of that of a commercially available chelating bead containing the same functionality. A 150 mg-B/L boron solution was forced to flow through the chelating-fiber-packed bed at the space velocity range from 10 to 100 h(-1), defined by dividing flow rate by bed volume (0.3 mL). At a space velocity of 20 h(-1), the dynamic binding capacity of the chelating-fiber-packed bed was 2.5-fold higher than that of the chelating-bead-packed bed.

The development of genetic switches and their integrated forms (genetic circuits) with desired specifications/functions is key for success in synthetic biology. Due to the difficulty in rational design, genetic switches and circuits with desirable specifications are mostly obtained by directed evolution. Based on a virus-derived nucleotide kinase as a single-gene dual selector, we constructed a robust, efficient and stringent selection system for genetic switches. This method exhibited unprecedented enrichment efficacy (>30,000-fold) of functional switches from non-functional ones in a single selection cycle. In addition, negative (OFF) selection was exceptionally stringent, allowing the rapid and efficient selection of non-leaky from leaky circuits.

Strongly acidic cation-exchange membranes were prepared by the electron-beam-induced graft polymerization of glycidyl methacrylate onto a high-density polyethylene film with a thickness of 35 μm and the subsequent conversion of the resulting epoxy group into a sulfonic acid group. The resulting cation-exchange membranes with various ion-exchange capacities or sulfonic acid group densities ranging from 1.9 to 2.7 mmol/g were applied to the enrichment of 0.50 mol/L sulfuric acid by electrodialysis. Concentrated sulfuric acids at concentrations of 1.4 to 2.9 mol/L were obtained in the concentrate chamber during the electrodialysis operated at 30 mA/cm² and 298 K, using a pair of this cation-exchange membrane and a commercially available anion-exchange membrane.<br>

A neutral extractant, tri-n-octylphosphine oxide (TOPO), was used to chemically modify a porous sheet, with a final density of 1.0 mmol/g. First, glycidyl methacrylate (GMA) was graft-polymerized onto a porous polyethylene sheet with an average pore diameter, porosity, and thickness of 1.2 mu m, 75%, and 2.0 mm, respectively. Second, an octadecane thiol group was introduced into the poly-GMA graft chain. Third. TOPO was deposited on the graft chain via a hydrophobic interaction. Bismuth chloride solution (BiCl3 in 0.15 M HNO3) was forced through the pores of the TOPO-modified porous sheet. The equilibrium binding capacity for bismuth was 0.19 mmol/g. Bismuth ions bound to TOPO were quantitatively eluted with 11 M HNO3. (C) 2010 Elsevier Ltd. All rights reserved.

We prepared nucleic-acid-base-immobilized porous membranes of a hollow-fiber form with pore size, porosity, and thickness of 0.2 mu m, 70%, and approximately 0.7 mm, respectively. Glycidyl methacrylate was graft-polymerized onto a polyethylene-made porous hollow-fiber membrane, followed by ring-opening of the epoxy group with the amino groups of adenine, guanine, and cytosine. The collection of palladium ions was achievable during the permeation of palladium chloride solution through the adenine-immobilized porous hollow-fiber membrane. The diffusional mass-transfer resistance of palladium ion to immobilized adenine was negligible because palladium ion was transported by permeative flow through the pores. The adenine-immobilized porous membrane with an immobilization density of 0.85 mol/kg of the membrane exhibited the highest molar binding ratio of palladium ion to immobilized adenine of 0.31 in 1 M hydrochloric acid. In addition, a quantitative elution with 4 M hydrochloric acid was experimentally demonstrated. (c) 2007 Published by Elsevier B.V.

A chelating porous sheet for use in solid-phase extraction was prepared by radiation-induced graft polymerization and subsequent chemical modifications. An epoxy-group-containing vinyl monomer was graft-polymerized onto a porous sheet made of polyethylene. The produced epoxy group of the graft chain was converted into an iminodiacetate group. The chelating porous sheet with a density of the iminodiacetate group of 2.1 mol/kg was cut into disks 13 mm in diameter to fit an empty cylindrical cartridge with a capacity of 6 mL. Breakthrough curves using the chelating-porous-disk-packed cartridge overlapped irrespective of the flow rate of the solution ranging up to 1500 mL/h because of negligible diffusional mass-transfer resistance of the copper ions to the iminodiacetate group of the graft chain.

Three kinds of ampholites, i.e., 3-aminopropionic acid (NH2C2H4COOH), (2-aminoethyl)-phosphonic acid (NH2C2H4PO3H2), and 2-aminoethane-1-sulfonic acid (NH2C2H4SO3H), were introduced into an epoxy group-containing polymer brush grafted onto a porous hollow-fiber membrane with a porosity of 70% and pore size of 0.36 mu m. The amphoteric group density of the hollow-fiber ranged from 0.50 to 0.72 mmol/g. Three kinds of proteins, i.e., lactoferrin (Lf), cytochrome c (Cyt c), and lysozyme (Ly), were captured by the amphoteric polymer brush during the permeation of the protein solution across the ampholite-immobilized porous hollow-fiber membrane. Multilayer binding of the protein to the amphoteric polymer brush, with a degree of multilayer binding of 3.3, 8.6, and 15 for Lf, Cyt c, and Ly, respectively, with the (2-aminoethyl)-phosphonic acid-immobilized porous hollow-fiber membrane, was demonstrated with a negligible diffusional mass-transfer resistance of the protein to the ampholite immobilized. The 2-aminoethane-1-sulfonic acid-immobilized porous hollow-fiber membrane exhibited the lowest initial flux of the protein solution, 0.41 m/h at a transmembrane pressure of 0.1 MPa and 298 K, and the highest equilibrium binding capacity of the protein, e.g., 130 mg/g for lysozyme. Extension and shrinkage of the amphoteric polymer brushes were observed during the binding and elution of the proteins.

As an extracurricular activity, 18 students in chemistry major tried to create four Escherichia coli 'robots': (1) an imaging system with swimmy bacteria
(2) a switchable aroma generator
(3) a balloon bacteria with a light-triggered inflator
and (4) an E. coli clock. None of the projects were completed, but a number of BioBricks were generated. They include an aroma synthesiser, a motility controller, and a size and shape controller. By assembling and tuning these BioBricks, we will be able to complete our robot manufacture. We belive these BioBricks would expand the toolbox in bacterial robotics. © 2007 The Institution of Engineering and Technology.

The mutation spectrum, together with mutation frequency, is decisive for the size and nature of genetic diversity. We constructed plasmid-encoded probes for specific detection of each and all of the six base substitutions. Using the set of the probes, we analyzed the mutation spectrumon the plasmids caused by different types of mutagens and mutator enzymes/alleles.

Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products--those that could be made biosynthetically--remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these "evolved" pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed.

Using methods of laboratory evolution to force the C(30) carotenoid synthase CrtM to function as a C(40) synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C(45) and C(50) carotenoid backbones (mono- and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.

To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C(30) carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C(40) synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies.

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C(30) carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C(35) backbone. The products of condensation of farnesyldiphosphate and GDP, C(35) structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C(40) or C(30) pathways accepted and converted the C(35) substrate, thus creating a C(35) carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.

The C30 carotene synthase CrtM from Staphylococcus aureus and the C40 carotene synthase CrtB from Erwinia uredovora were swapped into their respective foreign C40 and C30 biosynthetic pathways (heterologously expressed in Escherichia coli) and evaluated for function. Each displayed negligible ability to synthesize the natural carotenoid product of the other. After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C40 carotenoids. In contrast, we failed to find a single variant of CrtB with detectable C30 activity. Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C40 pathway. These mutants showed significant variation in performance in their original C30 pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity. We discovered that Phe 26 alone determines the specificity of CrtM. The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures.

In this research, we synthesized a novel DNA-polymer conjugate and evaluated its application to an affinity precipitation separation of TATA-box binding protein (TBP), which is a representative general transcription factor. The conjugate was composed of two fractions. One was a double-stranded DNA modified by the grafting of poly(N-isopropylacrylamide) (PNIPAAm), which is known as a thermosensitive vinyl polymer. The other fraction is a native double-stranded DNA containing a specific base sequence (5'-TATAAA-3') called a TATA-box. These two fractions, which have EcoRI termini, were treated with T4 DNA ligase, and the block conjugate was obtained as a precipitate after two wash processes. When the resultant block conjugate was introduced into a sample solution containing TBP (0.26 microM) and bovine serum albumin (BSA) (0.39 microM), a rapid and selective precipitation separation of TBP under homogeneous conditions was achieved by controlling temperature. The purity of TBP in the precipitation fraction was estimated to be above 90%.

In this study we develop a sequence-specific precipitation separation system of oligonucleotide (ODN) using a conjugate between poly(N-isopropylacrylamide) (PNIPAM) and ODN. PNIPAM is known as a thermoresponsive polymer and dehydrates to precipitate above its phase transition temperature in an aqueous milieu. The principal advantage of this separation system using the conjugate is that the hybridization reaction between the conjugate and oligonucleotide is conducted in homogeneous solution. The conjugate was prepared by copolymerization between N-isopropylacrylamide and a vinyl-derivatized (dT)8. The obtained conjugate efficiently precipitated (dA)8 from solution when the solution contained more than 1.5 M NaCl. The conjugate containing 3 nmol of (dT)8 residue was able to precipitate 1.4 nmol of (dA)8, suggesting that the (dT)8 residue of the conjugate formed a triple helix with (dA)8. From an equimolar mixture of (dA)8 and its one point mutant, the conjugate selectively precipitated (dA)8: the highest selectivity was obtained for the isolation of (dA)8 from the mixture consisting of (dA)4dT(dA)3 and (dA)8. When the conjugate was applied for the precipitation of five oligo(dA)s having different chain lengths, the longer oligo(dA)s tended to be precipitated by the conjugate more efficiently than the shorter ones. The conjugate could be used repeatedly for precipitation of (dA)8 without showing any loss in precipitation efficiency. © 2001 John Wiley &amp
Sons, Inc.

Single-chain observation of giant DNAs grafted with poly(N-isopropylacrylamide) (PNIPAAM) was performed using fluorescence microscopy while changing the solution temperature. The PNIPAAM-grafted DNA was prepared through the intercalation of psoralen-terminated PNIPAAM. Individual DNAs grafted with PNIPAAM exhibit a sharp but continuous transition at around 34 °C, from an elongated coil to a collapsed compact state. It is found that the width of the transition is almost the same between the level of individual DNAs and the level of ensemble DNAs. The unique nature of this transition is discussed in comparison with the discrete nature of the folding transition in native double-strand DNAs. With the addition of spermidine, a trivalent amine, the conformation of individual T4DNAs changes in a markedly discrete manner, on the level of individual giant DNAs, whereas in the ensemble, the size of T4 DNAs changes rather mildly
i.e., the transition appears to occur over a temperature range of 30 degrees. Thus, it is confirmed that the cooperative transition on the DNAs grafted with PNIPAAM exhibits a sharper transition than the change in the ensemble average for the discrete phase transition in native DNAs induced by the polycation.

We have synthesized psoralen-terminated poly(N-isopropylacrylamide)(1) and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)(2), both of which show temperature-responsive properties and DNA-binding ability. These polymers were grafted on plasmid double stranded DNA through a photochemical reaction of their terminal psoralen groups. The double stranded DNA grafted with the vinyl polymers turned out to lose the function as templates for RNA synthesis, while it showed characteristic solution property derived from the vinyl polymers. Ligation reaction between the polymer-grafted DNA and native DNA resulted in the AB-type block conjugate with the polymer-grafted DNA as A-segment and native DNA as B-segment. It was found that the block conjugate which have two different functional domains retains two functions independently; B-domain of the block conjugate worked as a template for RNA synthesis, while A-domain responded to temperature and made the conjugate precipitate. In this way, one can construct tailor-made and precisely-regulated structures with multiple functions at his own will by using recombinant DNA techniques.

The conjugate between oligo(dT)16 and thermo-responsive polymer, poly(N-isopropylacrylamide), was prepared for isolation of poly(A)+ RNA from total RNA. The hybridization reaction between the conjugate and poly(A) (average length: 320 base) was equilibrated in 10 min, and all the poly(A) (16 nmol base for 24 nmol base of conjugate) was precipitated when raising the solution temperature to 35 degrees C. The precipitate was dissolved in water, and poly(A) was dissociated from the conjugate by heating to 65 degrees C. This separation system was successfully applied to the isolation of poly(A)+ RNA from total RNA.

Soluble conjugates between double stranded DNA and poly(N-isopropylacrylamide) (polyNIPAAm) was synthesized and applied to the selective separation of DNA fragments: the target fragments were connected to the DNA-polyNIPAAm conjugate with the aid of T4 DNA ligase, followed by temperature-induced precipitation. Target DNA was collected from the resultant precipitate in high purity.

Poly(N-isopropylacrylamide) having a terminal psoralen group was synthesized and covalently bound to plasmid pBR 322 DNA through a photochemical reaction. The resulting conjugate exhibited temperature-responsive precipitation due to the aggregation of poly(N-isopropylacrylamide) chains when heated above 31 °C. This system was used for the one-pot separation of restriction endonuclease EcoRI.

Polyacrylamide hydrogels comprising double-stranded DNA were synthesized and applied as the absorbent for DNA-binding mutagenic molecules. Vinyl groups were immobilized on salmon sperm DNA via photoreaction between DNA and a vinyl derivative of psoralen. The resulting DNA macromonomer was fed into the polymerization system of acrylamide and N,N'-methylenebisacrylamide to give a hybrid hydrogel between DNA and polyacrylamide. Adsorption functions of the DNA hydrogel were studied for a variety of DNA-binding drugs and dyes. Copyright (C) 1998 Elsevier Science B.V.

A temperature-responsive polymer, poly(N-isopropyl acrylamide) terminated with psoralen, has been synthesized. Photo-induced reaction between DNA and psoralen end group on the polymer resulted in the grafting of poly(N-isopropyl acrylamide) on double-stranded DNA. The conjugate was found to form a precipitate at temperatures above 31°C and was easily collected by centrifugation. In this precipitation process, the conjugate collected DNA binders from solution. On the other hand, the binding efficiency of these molecules to the conjugate decreased significantly with an increasing level of DNA modification by the polymer. This blocking effect was utilized for protein imprinting on the DNA. Pre-incubation of the restriction endonuclease EcoRI with DNA results in selective preservation of its binding site from polymer modification. Bioimprinting is a potentially facile method for the preparation of selective collectors for site-specific DNA binders.

We synthesized poly(N-isopropylacrylamide) terminated with psoralen. Photo-induced reaction between DNA and the psoralen end group resulted in the grafting of poly(N-isopropylacrylamide) on double-stranded DNA. On the other hand, binding efficiency of DNA binding proteins such as restriction endonucleases to the DNA decreased significantly with increasing degree of polymer modification: At a certain level of polymer introduction, DNA was revealed to be practically 'jacketed'. Lithography was attempted to this grafting procedure. We found that pre-incubation of a restriction endonuclease EcoRI with DNA resulted in selective preservation of its binding site from polymer modification. The application of this technique would allow us facile methods to construct a precisely regulated nanoscale architecture.

We immobilized double-stranded DNA on poly(N-isopropylacrylamide), which is temperature-sensitive and gives precipitates from an aqueous solution when heated over 31°C. Photochemical conjugation with a vinyl derivative of psoralen was demonstrated to make the DNA polymerizable with a vinyl monomer. The radical copolymerization with N-isopropylacrylamide gave the temperature-responsive conjugate. The conjugate was shown to capture ethidium, a DNA-binding genotoxin, and precipitate with it when heated. About 95% of ethidium was separated from its highly diluted solution (3 ppm).

We describe a vinyl monomer having a psoralen moiety, which can form a photoadduct with double-helical DNA. This monomer (2) which is bound covalently to DNA gives the double-helical DNA a polymerizable function. The covalently bound DNA with vinyl groups can be copolymerized with a comonomer to give rise to a DNA-vinyl polymer conjugate. In contrast to our previously reported psoralen-containing monomer (1) which has a relatively short spacer from ethylenediamine, the present monomer provides a highly efficient conjugation. Therefore, the psoralen-containing monomer 2 should be a useful tool for anchoring double-helical DNA on polymeric materials when applying the DNA as an affinity ligand for bioseparation and bioanalysis.

We have synthesized a vinyl monomer having a psoralen moiety, which can form a photoadduct with double-helical DNA. The monomer 1 was proved to have an ability to crosslink DNA double strands through a photochemical reaction when irradiated by UV light. The resulting DNA having vinyl groups was copolymerized with a comonomer such as acrylamide and N-isopropylacrylamide to give rise to a DNA—vinyl polymer conjugate. A conjugation based on covalent bondings was verified by using gel electrophoresis
the conjugation efficiency was found to be dependent upon concentration of the monomer which had been used in the antecedent photochemical reaction. This monomer will be a useful tool when anchoring double-helical DNA on polymeric materials for separating and sensing DNA-binding substances. © 1994, American Chemical Society. All rights reserved.