担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers Media {SA}  

<jats:p>We recently disclosed that the biosynthesis of antiviral γ-poly-D-2,4-diaminobutyric acid (poly-D-Dab) in <jats:italic>Streptoalloteichus hindustanus</jats:italic> involves an unprecedented cofactor independent stereoinversion of Dab catalyzed by PddB, which shows weak homology to diaminopimelate epimerase (DapF). Enzymological properties and mechanistic details of this enzyme, however, had remained to be elucidated. Here, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we fully illustrate the first Dab-specific PLP-independent racemase PddB and further provide an insight into its evolution. The activity of the recombinant PddB was shown to be optimal around pH 8.5, and its other fundamental properties resembled those of typical PLP-independent racemases/epimerases. The enzyme catalyzed Dab specific stereoinversion with a calculated equilibrium constant of nearly unity, demonstrating that the reaction catalyzed by PddB is indeed racemization. Its activity was inhibited upon incubation with sulfhydryl reagents, and the site-directed substitution of two putative catalytic Cys residues led to the abolishment of the activity. These observations provided critical evidence that PddB employs the thiolate-thiol pair to catalyze interconversion of Dab isomers. Despite the low levels of sequence similarity, a phylogenetic analysis of PddB indicated its particular relevance to DapF among PLP-independent racemases/epimerases. Secondary structure prediction and 3D structural modeling of PddB revealed its remarkable conformational analogy to DapF, which in turn allowed us to predict amino acid residues potentially responsible for the discrimination of structural difference between diaminopimelate and its specific substrate, Dab. Further, PddB homologs which seemed to be narrowly distributed only in actinobacterial kingdom were constantly encoded adjacent to the putative poly-D-Dab synthetase gene. These observations strongly suggested that PddB could have evolved from the primary metabolic DapF in order to organize the biosynthesis pathway for the particular secondary metabolite, poly-D-Dab. The present study is on the first molecular characterization of PLP-independent Dab racemase and provides insights that could contribute to further discovery of unprecedented PLP-independent racemases.</jats:p>

DOI： 10.3389/fmicb.2021.686023

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担当区分：最終著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media {LLC}  

Polymers of basic amino acids function as polycationic compounds under physiological conditions and exhibit intriguing biological properties, such as antimicrobial and antiviral activities, immunopotentiating ability, and DNA-binding activity. Poly(ε-L-lysine) (ε-PL) produced by some strains of Streptomyces spp. is a cationic homopolymer of L-lysine linking between ε-amino and α-carboxylic acid functional groups and has been used as a food preservative based on its biocompatibility and biodegradability. An ε-PL-producing strain of Streptomyces sp. USE-33 was found to secrete a novel polycationic substance into its culture broth along with ε-PL. High-performance liquid chromatography analyses and one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) experiments, accompanied by NMR titration studies, revealed that the secreted substance was poly[β-(L-diaminopropionyl-L-diaminopropionic acid)], PAP, characterized by an isopeptide backbone linking between the β-amino and α-carboxylic acid groups of L-α,β-diaminopropionic acid (L-Dpr) with pendent L-Dpr residues. PAP had a molecular weight of 500 to 1400, and copolymers composed of the two amino acids L-Dpr and L-lysine were not detected in the producer strain USE-33. The strain coproduced high levels of the two poly(amino acid)s in the presence of glycerol, citrate, and ammonium sulfate at pH 4.0 in a two-stage cultivation procedure. PAP exhibited strong inhibitory activities against several yeasts and weaker activities against bacteria than ε-PL. PAP may share a number of biological functions with ε-PL, and the use of PAP along with ε-PL has potential as a specific and advanced material for technical applications in various fields.Key points• Novel cationic poly(amino acid) was found in an ε-PL-producing Streptomyces species.• The l-α,β-diaminopropionic acid polymer was characterized by a comb-like structure.• The novel poly(amino acid), PAP, exhibited antibacterial and antifungal activities.

DOI： 10.1007/s00253-021-11257-3

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Among the four bioactive cationic homo-poly(amino acids) discovered in nature, two are mirror-image isomers of poly(2,4-diaminobutyric acid) (poly-Dab) whose biosynthesis has long been unexplained. Their structural analogy plausibly suggested that they could share a common biosynthetic pathway utilizing ε-poly(l-lysine) synthetase-like enzymology but with an unprecedented process for enantiomeric inversion of polymer building blocks. To investigate this possibility, we comparatively explored the biosynthesis of poly-l-Dab and its mirror-image isomer poly-d-Dab in Streptomyces celluloflavus USE31 and Streptoalloteichus hindustanus NBRC15115, respectively, through genome mining, genetic inactivation, and heterologous expression combined with biochemical assays. While they shared the same biosynthetic pathway, the poly-d-Dab biosynthetic gene cluster additionally harbored the racemase gene. The critical finding that poly-d-Dab synthetase, in contrast to the synthetase generating the l-isomer, selectively activated d-Dab through adenylation conclusively demonstrated that free diffusible d-Dab preactivationally generated by the racemase is directly activated to be incorporated into the polymer. Our study thus represents the first demonstration of the stereoselective biosynthesis of a nonribosomal peptide governed by adenylation activity for a d-amino acid other than alanine. In silico sequence comparison between poly-Dab synthetases allowed us to identify amino acid residues potentially responsible for the discrimination of Dab enantiomers. Our results will provide significant insight not only for the future discovery of novel bioactive cationic poly(amino acids) but also for the creation of designer nonribosomal peptides with d-configuration.

DOI： 10.1021/acschembio.0c00321

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

ε-Poly-l-lysine (ε-PL) produced as a secondary metabolite of Streptomyces albulus has long been used as a natural food preservative in a number of countries, including Japan, the United States, South Korea, and China. To date, numerous studies employing classical biotechnological approaches have been carried out to improve its productivity. Here we report a modern and rational genetic approach to enhancing metabolic flux toward ε-PL biosynthesis. Based on in silico genome analyses, we revealed that S. albulus NBRC14147 produces five antifungal polyene antibiotics-tetramycin A and B, tetrin A and B, and a trace amount of nystatin A1-concomitantly with antimicrobial ε-PL. Targeted inactivation of the biosynthetic gene cluster for tetramycins and tetrins in a nystatin A1 production-deficient mutant completely abolished the production of polyene macrolides, which in turn led to an approximately 20% improvement in ε-PL production that closely correlated with the polyene defects. The biosynthetic flux for ε-PL was thus successfully enhanced by inactivation of the concomitant secondary metabolite biosynthetic pathways. Since this elimination of concomitantly produced metabolites also allows for simpler purification after fermentation production of ε-PL, the rational strain engineering strategy we show here will improve its industrial production.

DOI： 10.1016/j.jbiosc.2019.12.002

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/MRA.01515-18

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記述言語：英語  

Heterologous expression of natural product biosynthetic gene clusters (BGCs) is a robust approach not only to decipher biosynthetic logic behind natural product (NP) biosynthesis, but also to discover new chemicals from uncharacterized BGCs. This approach largely relies on techniques used for cloning large BGCs into suitable expression vectors. Recently, several whole-pathway direct cloning approaches, including full-length RecE-mediated recombination in Escherichia coli, Cas9-assisted in vitro assembly, and transformation-associated recombination (TAR) in Saccharomyces cerevisiae, have been developed to accelerate BGC isolation. In this chapter, we summarize a protocol for TAR cloning large NP BGCs, detailing the process of choosing TAR plasmids, designing pathway-specific TAR vectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressing BGCs in various host strains. We believe that the established platforms can accelerate the process of discovering new NPs, understanding NP biosynthetic logic, and engineering biosynthetic pathways.

DOI： 10.1016/bs.mie.2019.02.026

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Publishing Group  

In the ongoing effort to unlock the chemical potential of marine bacteria, genetic engineering of biosynthetic gene clusters (BGCs) is increasingly used to awake or improve expression of biosynthetic genes that may lead to discovery of novel bioactive natural products. Previously, we reported the successful capture, engineering and heterologous expression of an orphan BGC from the marine actinomycete Saccharomonospora sp. CNQ-490, which resulted in the isolation of the novel lipopeptide antibiotic taromycin A. Herein we report the isolation and structure elucidation of taromycin B, the second most abundant product of the taromycin biosynthetic series, and show that taromycins A and B exhibit complex chromatographic properties indicative of interconverting conformations. Taromycins A and B display potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium clinical isolates, suggestive that the taromycin molecular scaffold is a promising starting point for further derivatization to produce compounds with promising antibiotic characteristics.

DOI： 10.1038/ja.2017.146

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/anie.201508576

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/AEM.00269-15

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning "plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

DOI： 10.1038/srep09383

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/AEM.01201-14

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention because of their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominases revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds.

DOI： 10.1038/nchembio.1564

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

Recent developments in next-generation sequencing technologies have brought recognition of microbial genomes as a rich resource for novel natural product discovery. However, owing to the scarcity of efficient procedures to connect genes to molecules, only a small fraction of secondary metabolomes have been investigated to date. Transformation-associated recombination (TAR) cloning takes advantage of the natural in vivo homologous recombination of Saccharomyces cerevisiae to directly capture large genomic loci. Here we report a TAR-based genetic platform that allows us to directly clone, refactor, and heterologously express a silent biosynthetic pathway to yield a new antibiotic. With this method, which involves regulatory gene remodeling, we successfully expressed a 67-kb nonribosomal peptide synthetase biosynthetic gene cluster from the marine actinomycete Saccharomonospora sp. CNQ-490 and produced the dichlorinated lipopeptide antibiotic taromycin A in the model expression host Streptomyces coelicolor. The taromycin gene cluster (tar) is highly similar to the clinically approved antibiotic daptomycin from Streptomyces roseosporus, but has notable structural differences in three amino acid residues and the lipid side chain. With the activation of the tar gene cluster and production of taromycin A, this study highlights a unique "plug-and-play" approach to efficiently gaining access to orphan pathways that may open avenues for novel natural product discoveries and drug development.

DOI： 10.1073/pnas.1319584111

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jbiosc.2012.11.020

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Cyanosporasides are marine bacterial natural products containing a chlorinated cyclopenta[a]indene core of suspected enediyne polyketide biosynthetic origin. Herein, we report the isolation and characterization of novel cyanosporasides C-F (3-6) from the marine actinomycetes Salinispora pacifica CNS-143 and Streptomyces sp. CNT-179, highlighted by the unprecedented C-2' N-acetylcysteamine functionalized hexose group of 6. Cloning, sequencing, and mutagenesis of homologous similar to 50 kb cyanosporaside biosynthetic gene clusters from both bacteria afforded the first genetic evidence supporting cyanosporaside's enediyne, and thereby p-benzyne biradical, biosynthetic origin and revealed the molecular basis for nitrile and glycosyl functionalization. This study provides new opportunities for bioengineering of enediyne derivatives and expands the structural diversity afforded by enediyne gene clusters.

DOI： 10.1021/ja311065v

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

Axially chiral biaryl compounds are frequently encountered in nature where they exhibit diverse biological properties. Many are biphenols that have C-C or C-O linkages installed by cytochrome P450 oxygenases that control the regio- and stereoselectivity of the intermolecular coupling reaction. In contrast, bipyrrole-coupling enzymology has not been observed. Marinopyrroles, produced by a marine-derived streptomycete, are the first 1,3'-bipyrrole natural products. On the basis of marinopyrrole's unusual bipyrrole structure, we explored its atropo-selective biosynthesis in Streptomyces sp. CNQ-418 in order to elucidate the N,C-bipyrrole homocoupling enzymology. Through a series of genetic experiments involving the discovery and heterologous expression of marinopyrrole biosynthesis genes, we report that two flavin-dependent halogenases catalyze the unprecedented homocoupling reaction.

DOI： 10.1021/ja305670f

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jbiosc.2011.01.020

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

epsilon-Poly-L-lysine (epsilon-PL) is produced by Streptomyces albulus NBRC14147 as a secondary metabolite and can be detected only when the fermentation broth has an acidic pH during the stationary growth phase. Since strain NBRC14147 produces epsilon-PL-degrading enzymes, the original chain length of the epsilon-PL polymer product synthesized by epsilon-PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the epsilon-PL-degrading enzyme (PldII), which plays a central role in epsilon-PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an epsilon-PL of unchanged polymer chain length, demonstrating that the length is not determined by epsilon-PL-degrading enzymes but rather by Pls itself and that the 25 to 35 L-lysine residues of epsilon-PL represent the original chain length of the polymer product synthesized by Pls in vivo. Transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during epsilon-PL fermentation, rather than the inhibition of the epsilon-PL-degrading enzyme, are necessary for the accumulation of intracellular ATP.

DOI： 10.1128/AEM.00853-10

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担当区分：筆頭著者   掲載種別：論文集(書籍)内論文   出版者・発行元：Springer Berlin Heidelberg  

DOI： 10.1007/978-3-642-12453-2_4

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

epsilon-Poly-L-lysine (epsilon-PL) consists of 25-35 L-lysine residues in isopeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of epsilon-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an epsilon-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an epsilon-PL-producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the nonribosomal peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase-like catalytic activity for peptide bond formation.

DOI： 10.1038/nchembio.125

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We found the occurrence of NAD(P)(+)-dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, and purified to homogeneity. About 50% of the enzyme activity remained even after heating at 50degreesC for 65 min and the highest activity was observed in the range of 55-60degreesC. The enzyme was thermostable and thermophilic, although it was derived from a psychrophile. The circular dichroism at 222 nm of the enzyme showed a peak at 32 degreesC. This temperature was closely similar to the transition temperature in the Arrhenius plots. The stereo specificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R. The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues. The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02523-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL SCIENCE LTD  

We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be approximate to 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD(+) as a coenzyme, and catalyzed the oxidative deamination of L-valine, L-isoleucine, L-leucine and the reductive amination of alpha -ketoisovalerate, alpha -ketovalerate, alpha -ketoisocaproate, and alpha -ketocaproate. The reaction proceeds through an iso-ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 degreesC was estimated to be 2.4 min. The k(cat)/K-m (mu mu (-1).s(-1)) values for L-valine and NAD(+) at 20 degreesC were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.

DOI： 10.1046/j.1432-1327.2001.02353.x

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記述言語：日本語   出版者・発行元：(公社)日本生物工学会  

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記述言語：日本語   出版者・発行元：関西大学先端科学技術推進機構  

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記述言語：日本語   出版者・発行元：関西大学先端科学技術推進機構  

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記述言語：日本語   出版者・発行元：共立出版  

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記述言語：日本語   出版者・発行元：野口研究所  

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記述言語：日本語   出版者・発行元：天然有機化合物討論会実行委員会  

Only two different amino-acid "homopolymers" comprised of a single type of amino acid are known in nature: γ-poly-glutamic acid (γ-PGA) and ε-poly-L-lysine (ε-PL). ε-PL consisting of 25-35 L-lysine residues in isopeptide linkages has shown promise in medical and industrial applications. Elucidating the biosynthetic mechanism of ε-PL should open new avenues for creating novel classes of biopolymers. Here we report on the purification of an ε-PL synthase (Pls; 130kDa) and the cloning of its gene from an ε-PL-producing strain of Streptowyces albulus. Pls was found to be a membrane protein with six transmembrane domains. It was shown to be a single-module nonribosomal peptide synthase (NRPS) with traditional adenylation (A-domain) and thiolation domains (T-domain) in its amino (N)-terminal region. It had no traditional condensation or thioesterase domain; instead, it had three tandem carboxy (C)-terminal acyltransferase domains that catalysed L-lysine polymerization iteratively, yielding chains of diverse length (3-mer to 17-mer) in vitro. Furthermore, these domains catalysed the condensation of free L-lysine monomers (or ε-PL oligomers) as acceptors with enzyme-bound L-lysine as a donor; hence, L-lysine activated by the A-domain and T-domain was used only as an extender unit. A database search showed that Pls homologues were widely distributed among microorganisms and that amino-acid homopolymers, in addition to ε-PL and γ-PGA, might occur in nature.

DOI： 10.24496/tennenyuki.50.0_155

CiNii Books

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記述言語：日本語   出版者・発行元：日本生物工学会  

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J-GLOBAL

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記述言語：日本語   出版者・発行元：日本生物工学会  

CiNii Books

J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.74.5-6_323

CiNii Books

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記述言語：日本語  

Only two different amino-acid "homopolymers" comprised of a single type of amino acid are known in nature: γ-poly-glutamic acid (γ-PGA) and ε-poly-L-lysine (ε-PL). ε-PL consisting of 25-35 L-lysine residues in isopeptide linkages has shown promise in medical and industrial applications. Elucidating the biosynthetic mechanism of ε-PL should open new avenues for creating novel classes of biopolymers. Here we report on the purification of an ε-PL synthase (Pls; 130kDa) and the cloning of its gene from an ε-PL-producing strain of Streptowyces albulus. Pls was found to be a membrane protein with six transmembrane domains. It was shown to be a single-module nonribosomal peptide synthase (NRPS) with traditional adenylation (A-domain) and thiolation domains (T-domain) in its amino (N)-terminal region. It had no traditional condensation or thioesterase domain; instead, it had three tandem carboxy (C)-terminal acyltransferase domains that catalysed L-lysine polymerization iteratively, yielding chains of diverse length (3-mer to 17-mer) in vitro. Furthermore, these domains catalysed the condensation of free L-lysine monomers (or ε-PL oligomers) as acceptors with enzyme-bound L-lysine as a donor; hence, L-lysine activated by the A-domain and T-domain was used only as an extender unit. A database search showed that Pls homologues were widely distributed among microorganisms and that amino-acid homopolymers, in addition to ε-PL and γ-PGA, might occur in nature.

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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記述言語：日本語  

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出願人：福井県, チッソ株式会社

出願番号：特願2007-112078  出願日：2007年4月

公開番号：特開2008-263868  公開日：2008年11月

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