Language：English   Publishing type：Research paper (scientific journal)  

Adenylation enzymes activate amino acid substrates to aminoacyl adenylates and generally transfer this moiety onto the thiol group of the phosphopantetheine arm of a carrier protein for the selective incorporation of aminoacyl building blocks in natural product biosynthesis. In contrast to the canonical thioester-forming adenylation enzymes, the amide-forming adenylation enzyme VinM transfers an l-alanyl group onto the amino group of the aminoacyl unit attached to the phosphopantetheine arm of the carrier protein VinL to generate dipeptidyl-VinL in vicenistatin biosynthesis. It is unclear how VinM distinguishes aminoacyl-VinL from VinL for amide bond formation. Herein we describe structural and biochemical analyses of VinM. We determined the crystal structure of VinM in complex with VinL using a designed pantetheine-type cross-linking probe. The VinM-VinL complex structure in combination with site-directed mutagenesis analysis revealed that the interactions with both the phosphopantetheine arm and VinL are critical for the amide-forming activity of VinM.

DOI： 10.1021/acschembio.3c00517

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Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular type I polyketide synthases (PKSs) and catalyze the decarboxylation of the (alkyl-)malonyl unit bound to the acyl carrier protein (ACP) in the loading module for the construction of the PKS starter unit. Previously, we performed a structural and functional analysis of the GfsA KSQ domain involved in the biosynthesis of macrolide antibiotic FD-891. We furthermore revealed the recognition mechanism for the malonic acid thioester moiety of the malonyl-GfsA loading module ACP (ACPL) as a substrate. However, the exact recognition mechanism for the GfsA ACPL moiety remains unclear. Here, we present a structural basis for the interactions between the GfsA KSQ domain and GfsA ACPL. We determined the crystal structure of the GfsA KSQ-acyltransferase (AT) didomain in complex with ACPL (ACPL=KSQAT complex) by using a pantetheine crosslinking probe. We identified the key amino acid residues involved in the KSQ domain-ACPL interactions and confirmed the importance of these residues by mutational analysis. The binding mode of ACPL to the GfsA KSQ domain is similar to that of ACP to the ketosynthase domain in modular type I PKSs. Furthermore, comparing the ACPL=KSQAT complex structure with other full-length PKS module structures provides important insights into the overall architectures and conformational dynamics of the type I PKS modules.

DOI： 10.1021/acschembio.3c00151

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Lyngbyapeptin B is a hybrid polyketide-nonribosomal peptide isolated from particular marine cyanobacteria. In this report, we carried out genome sequence analysis of a producer cyanobacterium Moorena bouillonii to understand the biosynthetic mechanisms that generate the unique structural features of lyngbyapeptin B, including the (E)-3-methoxy-2-butenoyl starter unit and the C-terminal thiazole moiety. We identified a putative lyngbyapeptin B biosynthetic (lynB) gene cluster comprising nine open reading frames that include two polyketide synthases (PKSs: LynB1 and LynB2), four nonribosomal peptide synthetases (NRPSs: LynB3, LynB4, LynB5, and LynB6), a putative nonheme diiron oxygenase (LynB7), a type II thioesterase (LynB8), and a hypothetical protein (LynB9). In vitro enzymatic analysis of LynB2 with methyltransferase (MT) and acyl carrier protein (ACP) domains revealed that the LynB2 MT domain (LynB2-MT) catalyzes O-methylation of the acetoacetyl-LynB2 ACP domain (LynB2-ACP) to yield (E)-3-methoxy-2-butenoyl-LynB2-ACP. In addition, in vitro enzymatic analysis of LynB7 revealed that LynB7 catalyzes the oxidative decarboxylation of (4R)-2-methyl-2-thiazoline-4-carboxylic acid to yield 2-methylthiazole in the presence of Fe2+ and molecular oxygen. This result indicates that LynB7 is responsible for the last post-NRPS modification to give the C-terminal thiazole moiety in lyngbyapeptin B biosynthesis. Overall, we identified and characterized a new marine cyanobacterial hybrid PKS-NRPS biosynthetic gene cluster for lyngbyapeptin B production, revealing two unique enzymatic logics.

DOI： 10.1021/acschembio.3c00011

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Streptomyces graminofaciens A-8890 produces two macrolide antibiotics, FD-891 and virustomycin A, both of which show significant biological activities. In this study, we identified the virustomycin A biosynthetic gene cluster, which encodes type I polyketide synthases (PKSs), ethylmalonyl-CoA biosynthetic enzymes, methoxymalony-acyl carrier protein biosynthetic enzymes, and post-PKS modification enzymes. Next, we demonstrated that the acyltransferase domain can be exchanged between the Vsm PKSs and the PKSs involved in FD-891 biosynthesis (Gfs PKSs), without encountering supply problems of the unique extender units. We exchanged the malonyltransferase domain in the loading module of Gfs PKS with the ethylmalonyltransferase domain and the methoxymalonyltransferase domain of Vsm PKSs. Consequently, the expected two-carbon elongated analog 26-ethyl-FD-891 was successfully produced with a titer comparable to FD-891 production by the wild type; however, exchange with the methoxymalonyltransferase domain did not produce any FD-891 analogs. Furthermore, 26-ethyl-FD-891 showed potent cytotoxic activity against HeLa cells, like natural FD-891.

DOI： 10.1002/cbic.202200670

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Adenylation domains (A-domains) are responsible for the selective incorporation of carboxylic acid substrates in the biosynthesis of nonribosomal peptides and related natural products. The A-domain transfers an acyl substrate onto its cognate carrier protein (CP). The proper interactions between an A-domain and the cognate CP are important for functional substrate transfer. To stabilize the transient interactions sufficiently for structural analysis of A-domain-CP complex, vinylsulfonamide adenosine inhibitors have been traditionally used as molecular probes. Recently, we have developed an alternative strategy using a synthetic pantetheine-type probe that enables site-specific cross-linking between an A-domain and a CP. In this chapter, we describe the laboratory protocols for this cross-linking reaction.

DOI： 10.1007/978-1-0716-3214-7_10

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Acyltransferase (AT) recognizes its cognate acyl carrier protein (ACP) for functional transfer of an acyl unit in polyketide biosynthesis. However, structural characterization of AT-ACP complexes is limited because of the weak and transient interactions between them. In the biosynthesis of macrolactam polyketide vicenistatin, the trans-acting loading AT VinK transfers a dipeptidyl unit from the stand-alone ACP VinL to the ACP domain (VinP1ACPL) of the loading module of modular polyketide synthase VinP1. Although the previously determined structure of the VinK-VinL complex clearly illustrates the VinL recognition mechanism of VinK, how VinK recognizes VinP1ACPL remains unclear. Here, the crystal structure of a covalent VinK-VinP1ACPL complex formed with a pantetheine-type cross-linking probe is reported at 3.0 Å resolution. The structure of the VinK-VinP1ACPL complex provides detailed insights into the transient interactions between VinK and VinP1ACPL. The importance of residues in the binding interface was confirmed by site-directed mutational analyses. The binding interface between VinK and VinP1ACPL is similar to that between VinK and VinL, although some of the interface residues are different. However, the ACP orientation and interaction mode observed in the VinK-VinP1ACPL complex are different from those observed in other AT-ACP complexes such as the disorazole trans-AT-ACP complex and cis-AT-ACP complexes of modular polyketide synthases. Thus, AT-ACP binding interface interactions are different in each type of AT-ACP pair.

DOI： 10.1021/acs.biochem.2c00645

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The radical S-adenosyl-l-methionine (SAM) methylase Orf29 catalyzes the C-methylation of SAM in the biosynthesis of 1-amino-2-methylcyclopropanecarboxylic acid. Here, we determined that the methylation product is (4″R)-4″-methyl-SAM. Furthermore, we found that the 5'-deoxyadenosyl radical generated by Orf29 abstracts the pro-R hydrogen atom from the C-4″ position of SAM to generate the radical intermediate, which reacts with methylcobalamin to give (4″R)-4″-methyl-SAM. Consequently, the Orf29-catalyzed C-methylation was confirmed to proceed with retention of configuration.

DOI： 10.1021/acs.orglett.2c03555

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DOI： 10.1016/j.cbpa.2022.102212

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Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular polyketide synthases (PKSs) and are proposed to catalyze the decarboxylation of a malonyl or methylmalonyl unit for the construction of the PKS starter unit. KSQ domains have high sequence similarity to ketosynthase (KS) domains, which catalyze transacylation and decarboxylative condensation in polyketide and fatty acid biosynthesis, except that the catalytic Cys residue of KS domains is replaced by Gln in KSQ domains. Here, we present biochemical analyses of GfsA KSQ and CmiP4 KSQ, which are involved in the biosynthesis of FD-891 and cremimycin, respectively. In vitro analysis showed that these KSQ domains catalyze the decarboxylation of malonyl and methylmalonyl units. Furthermore, we determined the crystal structure of GfsA KSQ in complex with a malonyl thioester substrate analogue, which enabled identification of key amino acid residues involved in the decarboxylation reaction. The importance of these residues was confirmed by mutational analysis. On the basis of these findings, we propose a mechanism of the decarboxylation reaction catalyzed by GfsA KSQ. GfsA KSQ initiates decarboxylation by fixing the substrate in a suitable conformation for decarboxylation. The formation of enolate upon decarboxylation is assisted by two conserved threonine residues. Comparison of the structure of GfsA KSQ with those of KS domains suggests that the Gln residue in the active site of the KSQ domain mimics the acylated Cys residue in the active site of KS domains.

DOI： 10.1021/acschembio.1c00856

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Publishing type：Part of collection (book)   Publisher：Elsevier  

DOI： 10.1016/bs.mie.2021.11.025

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Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acs.biochem.1c00536

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Acyltransferases are responsible for the selection and loading of acyl units onto carrier proteins in polyketide and fatty-acid biosynthesis. Despite the importance of protein-protein interactions between the acyltransferase and the carrier protein, structural information on acyltransferase-carrier protein interactions is limited because of the transient interactions between them. In the biosynthesis of the polyketide vicenistatin, the acyltransferase VinK recognizes the carrier protein VinL for the transfer of a dipeptidyl unit. The crystal structure of a VinK-VinL covalent complex formed with a 1,2-bismaleimidoethane cross-linking reagent has been determined previously. Here, the crystal structure of a VinK-VinL covalent complex formed with a pantetheine cross-linking probe is reported at 1.95 Å resolution. In the structure of the VinK-VinL-probe complex, the pantetheine probe that is attached to VinL is covalently connected to the side chain of the mutated Cys106 of VinK. The interaction interface between VinK and VinL is essentially the same in the two VinK-VinL complex structures, although the position of the pantetheine linker slightly differs. This structural observation suggests that interface interactions are not affected by the cross-linking strategy used.

DOI： 10.1107/S2053230X21008761

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Hitachimycin is a macrolactam antibiotic with an (S)-β-phenylalanine (β-Phe) at the starter position of its polyketide skeleton. (S)-β-Phe is formed from l-α-phenylalanine by the phenylananine-2,3-aminomutase HitA in the hitachimycin biosynthetic pathway. In this study, we produced new hitachimycin analogs via mutasynthesis by feeding various (S)-β-Phe analogs to a ΔhitA strain. We obtained six hitachimycin analogs with F at the ortho, meta, or para position and Cl, Br, or a CH3 group at the meta position of the phenyl moiety, as well as two hitachimycin analogs with thienyl substitutions. Furthermore, we carried out a biochemical and structural analysis of HitB, a β-amino acid-selective adenylation enzyme that introduces (S)-β-Phe into the hitachimycin biosynthetic pathway. The KM values of the incorporated (S)-β-Phe analogs and natural (S)-β-Phe were similar. However, the KM values of unincorporated (S)-β-Phe analogs with Br and a CH3 group at the ortho or para position of the phenyl moiety were high, indicating that HitB functions as a gatekeeper to select macrolactam starter units during mutasynthesis. The crystal structure of HitB in complex with (S)-β-3-Br-phenylalanine sulfamoyladenosine (β-m-Br-Phe-SA) revealed that the bulky meta-Br group is accommodated by the conformational flexibility around Phe328, whose side chain is close to the meta position. The aromatic group of β-m-Br-Phe-SA is surrounded by hydrophobic and aromatic residues, which appears to confer the conformational flexibility that enables HitB to accommodate the meta-substituted (S)-β-Phe. The new hitachimycin analogs exhibited different levels of biological activity in HeLa cells and multidrug-sensitive budding yeast, suggesting that they may target different molecules.

DOI： 10.1021/acschembio.1c00003

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2-Deoxy-scyllo-inosose (2DOI, [2S,3R,4S,5R]-2,3,4,5-tetrahydroxycyclohexan-1-one) is a biosynthetic intermediate of 2-deoxystreptamine-containing aminoglycoside antibiotics, including butirosin, kanamycin, and neomycin. In producer microorganisms, 2DOI is constructed from d-glucose 6-phosphate (G6P) by 2-deoxy-scyllo-inosose synthase (DOIS) with the oxidized form of nicotinamide adenine dinucleotide (NAD+). 2DOI is also known as a sustainable biomaterial for production of aromatic compounds and a chiral cyclohexane synthon. In this study, a one-pot enzymatic synthesis of 2DOI from d-glucose and polyphosphate was investigated. First, 3 polyphosphate glucokinases (PPGKs) were examined to produce G6P from d-glucose and polyphosphate. A PPGK derived from Corynebacterium glutamicum (cgPPGK) was found to be suitable for G6P production under ordinary enzymatic conditions. Next, 7 DOISs were examined for the one-pot enzymatic reaction. As a result, cgPPGK and BtrC, the latter of which is a DOIS derived from the butirosin producer Bacillus circulans, achieved nearly full conversion of d-glucose to 2DOI in the presence of polyphosphate.

DOI： 10.1093/bbb/zbaa025

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Kanamycin A is the major 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotic produced by Streptomyces kanamyceticus. The 2DOS moiety is linked with 6-amino-6-deoxy-d-glucose (6ADG) at O-4 and 3-amino-3-deoxy-d-glucose at O-6. Because the 6ADG moiety is derived from d-glucosamine (GlcN), deamination at C-2 and introduction of C-6-NH2 are required in the biosynthesis. A dehydrogenase, KanQ, and an aminotransferase, KanB, are presumed to be responsible for the introduction of C-6-NH2 , although the substrates have not been identified. Here, we examined the substrate specificity of KanQ to better understand the biosynthetic pathway. It was found that KanQ oxidized kanamycin C more efficiently than the 3''-deamino derivative. Furthermore, the substrate specificity of an oxygenase, KanJ, that is responsible for deamination at C-2 of the GlcN moiety was examined, and the crystal structure of KanJ was determined. It was found that C-6-NH2 is important for substrate recognition by KanJ. Thus, the modification of the GlcN moiety occurs after pseudo-trisaccharide formation, followed by the introduction of C-6-NH2 by KanQ/KanB and deamination at C-2 by KanJ.

DOI： 10.1002/cbic.202000839

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DOI： 10.1021/acschembio.0c00403

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Many pharmacologically important peptides are bacterial or fungal in origin and contain nonproteinogenic amino acid (NPA) building blocks. Recently, it was reported that, in bacteria, a cyclopropane-containing NPA 1-aminocyclopropanecarboxylic acid (ACC) is produced from the L-methionine moiety of S-adenosyl-L-methionine (SAM) by non-canonical ACC-forming enzymes. On the other hand, it has been suggested that a monomethylated ACC analogue, 2-methyl-ACC (MeACC), is derived from L-valine. Therefore, we have investigated the MeACC biosynthesis by identifying a gene cluster containing bacterial MeACC synthase genes. In this gene cluster, we identified two genes, orf29 and orf30, which encode a cobalamin (B12)-dependent radical SAM methyltransferase and a bacterial ACC synthase, respectively, and were found to be involved in the MeACC biosynthesis. In vitro analysis using their recombinant enzymes (rOrf29 and rOrf30) further revealed that the ACC structure of MeACC was derived from the L-methionine moiety of SAM, rather than L-valine. In addition, rOrf29 was found to catalyze the C-methylation of the L-methionine moiety of SAM. The resulting methylated derivative of SAM was then converted into MeACC by rOrf30. Thus, we demonstrate that C-methylation of SAM occurs prior to cyclopropanation in the biosynthesis of a bacterial MeACC (norcoronamic acid).

DOI： 10.3390/biom10050775

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Kanosamine (3-amino-3-deoxy-d-glucose) is a characteristic sugar unit found in kanamycins, a group of aminoglycoside antibiotics. The kanosamine moiety originates from d-glucose in kanamycin biosynthesis. However, the timing of the replacement of the 3-OH group of the d-glucose-derived biosynthetic intermediate with the amino group is elusive. Comparison of biosynthetic gene clusters for related aminoglycoside antibiotics suggests that the nicotinamide adenine dinucleotide (NAD+)-dependent dehydrogenase KanD2 and the pyridoxal 5'-phosphate (PLP)-dependent aminotransferase KanS2 are responsible for the introduction of the amino group at the C3 position of kanosamine. In this study, we demonstrated that KanD2 and KanS2 convert kanamycin A, B, and C to the corresponding 3″-deamino-3″-hydroxykanamycins (3″-hks) in the presence of PLP, 2-oxoglutarate, and NADH via a reverse reaction in the pathway. Furthermore, we observed that all of the 3″-hks are oxidized by KanD2 with NAD+, but d-glucose, UDP-d-glucose, d-glucose 6-phosphate, and d-glucose 1-phosphate are not. Crystal structure analysis of KanD2 complexed with 3″-hkB and NADH illustrated the selective recognition of pseudotrisaccharides, especially the d-glucose moiety with 2-deoxystreptamine, by a combination of hydrogen bonds and CH-π interactions. Overall, it was clarified that the kanosamine moiety of kanamycins is constructed after the glucosylation of the pseudodisaccharide biosynthetic intermediates in kanamycin biosynthesis.

DOI： 10.1021/acs.biochem.0c00204

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The myo-inositol-1-phosphate synthase (MIPS) ortholog Ari2, which is encoded in the aristeromycin biosynthetic gene cluster, catalyzes the formation of five-membered cyclitol phosphate using d-fructose 6-phosphate (F6P) as a substrate. To understand the stereochemistry during the Ari2 reaction in vivo, we carried out feeding experiments with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]glucose in the aristeromycin-producing strain Streptomyces citricolor. We observed retention of the 2H atom of (6S)-d-[6-2H1]glucose and no incorporation of the 2H atom from (6R)-d-[6-2H1]glucose in aristeromycin. This indicates that Ari2 abstracts the pro-R proton at C6 of F6P after oxidation of C5-OH by nicotinamide adenine dinucleotide (NAD+) to generate the enolate intermediate, which then attacks the C2 ketone to form the C-C bond via aldol-type condensation. The reaction of Ari2 with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]F6P in vitro exhibited identical stereochemistry compared with that observed during the feeding experiments. Furthermore, analysis of the crystal structure of Ari2, including NAD+ as a ligand, revealed the active site of Ari2 to be similar to that of MIPS of Mycobacterium tuberculosis, supporting the similarity of the reaction mechanisms of Ari2 and MIPS.

DOI： 10.1021/acs.biochem.9b00981

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DOI： 10.1021/acs.biochem.9b00444

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Publisher：Wiley  

DOI： 10.1002/ange.201900318

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Adenylation (A) domains act as the gatekeepers of non-ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A-domains. We show that a single asparagine-to-glycine mutation in an aryl acid A-domain leads to an enzyme that tolerates a wide range of non-native aryl acids. The engineered catalyst is capable of activating non-native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild-type enzyme was observed toward these substrates. Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.

DOI： 10.1002/anie.201900318

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Language：English   Publisher：Springer Nature America, Inc  

DOI： 10.1007/s10295-018-2084-7

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DOI： 10.1039/c8np00022k

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DOI： 10.1021/acs.biochem.8b00614

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DOI： 10.1021/acs.biochem.8b00690

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DOI： 10.1021/jacs.8b04162

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DOI： 10.1080/09168451.2017.1411777

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DOI： 10.1002/cbic.201700429

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DOI： 10.1021/acschembio.7b00419

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DOI： 10.1021/acs.biochem.7b00472

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DOI： 10.1002/prot.25284

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DOI： 10.1038/s41598-017-03308-5

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DOI： 10.1074/jbc.M117.792549

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DOI： 10.1016/j.cbpa.2016.08.030

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DOI： 10.1002/cbic.201600348

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DOI： 10.1038/ja.2016.40

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DOI： 10.1080/09168451.2015.1132155

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DOI： 10.1080/09168451.2015.1132152

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DOI： 10.1038/ja.2015.148

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DOI： 10.1002/tcr.201500210

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DOI： 10.1073/pnas.1520042113

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DOI： 10.1002/cbic.201500533

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DOI： 10.1002/cbic.201500426

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DOI： 10.1002/cbic.201500040

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DOI： 10.1002/cbic.201402612

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DOI： 10.1074/jbc.M114.602326

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DOI： 10.1021/ja507759f

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DOI： 10.1039/c4np00007b

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DOI： 10.1016/j.febslet.2014.01.060

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DOI： 10.1021/ja511223g

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DOI： 10.1271/bbb.130498

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DOI： 10.1002/cbic.201300370

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DOI： 10.1002/cbic.201300153

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DOI： 10.1038/ja.2013.76

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Other Link： http://orcid.org/0000-0002-4788-0063

Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Incednine is a 24 membered macrolactam glycoside isolated from Streptomyces sp. ML694-90F3 as an inhibitor of anti-apoptotic function of Bcl-2/Bcl-xL oncoproteins. In the present study, to understand the biosynthesis of the unique amino acid starter unit of macrolactam aglycon (incednam), a series of incorporation study and enzymatic analyses of the biosynthetic enzymes encoded in the corresponding gene cluster were conducted. Feeding experiments with [1-^<13>C_1] acetate, [1,2-^<13>C_2] acetate, [1-^<13>C_1] propionate, and [^<13>C_3]glycerol revealed that incednam is constructed with five malonyl-CoA, four methylmalonyl-CoA and one methoxymalonyl-CoA (ACP) as the extender units of polyketide synthases (PKSs). Interestingly, one intact acetate was also incorporated into the C1-C2 position of 3-aminobutyrate moiety, suggesting that the starter unit would be derived from L-glutamic acid rather than L-lysine and acetoacetyl-CoA. In fact, intact incorporation of L-[^<13>C_5,^<15>N_1]glutamic acid was observed. Further, to elucidate the missing biosynthetic link between L-glutamic acid and the starter unit, possible deuterium labeled amino acids were synthesized and fed to the producer strain. As a result, the free form of 3-[3-^2H]aminobutyric acid and β-[2,2,4,4-^2H_4]glutamic acid were efficiently incorporated to the starter unit, indicating that these amino acids are direct precursors of the starter unit of incednam. These results also implied that the unprecedented β-decarboxylation of β-glutamic acid to give 3-aminobutyric acid would be involved in the starter unit biosynthesis. As the next, to fish out the expected unique biosynthetic gene cluster of incednine, we used unique deoxysugar biosynthetic genes and methoxymalonyl-ACP biosynthetic gene as probes. Consequently, the incednine biosynthetic gene (idn) cluster, which is localized to a 138 kb contiguous DNA from Streptomyces sp. ML694-90F3, was identified. The idn gene cluster contains six modular type I PKS genes (idnPl-P6), deoxysugar biosynthetic genes (idnS1-S9), possible starter biosynthetic genes (idnLM1-LM7) and several modification enzymatic genes. To understand the unique starter unit biosynthesis, we attempted to characterize several recombinant enzymes, which were expressed in heterologous hosts. One of the most striking results was that IdnLM3 was found to catalyze β-decarboxylation of β-glutamic acid to give 3-aminobutyric acid. In the presentation, we will discuss the unique starter biosynthetic pathway based on the characterized enzymatic functions of the incednine biosynthetic enzymes.

DOI： 10.24496/tennenyuki.54.0_67

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DOI： 10.1021/ol302052c

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DOI： 10.1039/c2md00305h

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DOI： 10.1055/s-0031-1290385

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DOI： 10.1002/anie.201108122

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DOI： 10.1021/ja208927r

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DOI： 10.1016/j.tet.2011.08.073

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DOI： 10.1002/cbic.201100418

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Vicenistatin is a 20-membered macrolactam antibiotic isolated from Streptomyces halstedii HC34. Its hybrid structure consisting of a unique amino acid (3-amino-2-methylpropionate, AmP) and polyketide is characteristic and thus prompted us to investigate the biosynthetic pathway, especially concerning the construction of the unique amino acid and its combination with the polyketide pathway. Our previous incorporation study indicated that the AmP moiety of vicenistatin is derived from L-glutamate (Glu) via 3-methylaspartate (MeAsp). Also, since the free form of AmP was not incorporated into the moiety, MeAsp seemed to be modified as CoA or ACP thioester before decarboxylation. Further, bioinfomatic analysis of the vicenistatin biosynthetic (yin) gene cluster revealed that eight unique proteins (VinH, I, J, K, L, M, N and 0) seemed to be responsible for the unique amino acid biosynthesis accompanying four PKSs and seven deoxysugar biosynthetic enzymes. In this report, we characterized the eight unique proteins, which were all involved in the AmP biosynthesis and the connection of the generated AmP with the polyketide pathway expectedly. At first, inactivation of the vinl gene for glutamate mutase E subunit resulted in the loss of vicenistain production, while the disruptant recovered it when MeAsp was added into the culture. This result suggested that glutamate mutase S subuint VinH and E subuint VinI generate MeAsp from Glu cooperatively. To investigate the other six proteins (VinJ, K, L, M, N and 0), we prepared the recombinant enzymes, which were successfully expressed in Escherichia coli. Two ATP-dependent ligases VinM and VinN were predicted to act as freestanding adenylation domains that catalyze two half reactions: The first is ATP-dependent activation of carboxyl group of amino acid and the second half-reaction is the transfer of the aminoacyl moiety of the adenylate intermediate to ACP (VinL) or its equivalent. Based on typical photometric assay by detection of released pyrophosphate, VinM was found to recognize small size of amino acids such as alanine, glycine and serine. On the other hands, VinN specifically recognized MeAsp. Futher, when the holo form of VinL was added into the VinN reaction mixture with ATP and MeAsp, the expected MeAsp-VinL formation was clearly detected by LC-ESI-MS analysis. Subsequently, the formed MeAsp-VinL was decarboxylated to be AmP-VinL by a PLP-dependent decarboxylase VinO. The generated AmP-VinL was then reacted with VinM, ATP and alanine to examine the additional attachment of aminoacyl group onto AmP-VinL to prevent a presumable thermodynamically favorable six-membered lactam formation during the chain elongation by PKS. As a result, a dipeptidyl ACP, Ala-AmP-VinL, was efficiently formed by VinM. The Ala-AmP-VinL was then used as a substrate for an acyltransferase VinK, which was speculated to catalyze the acyl transfer reaction between VinL and VinPl-ACP domain at the loading module of PKS. As we expected, the dipeptide transfer reaction occurred efficiently to give Ala-AmP-VinP 1 -ACP, which should be a starter molecule of vicenilactam PKS. Finally, to investigate the function of VinJ, a peptidase, we chemically synthesized the ethyl ester of N-alanyl-secovicenilactam as a substrate analog for the elongated polyketide intermediate. As we expected, VinJ catalyzed the hydrolysis of the analog to afford secovicenilactam ethyl ester, which was then macrocyclized by VinP4-TE to give vicnilactam to complete the biosynthesis. In this study, we clarified the unique starter biosynthesis of vicenistatin. It should be noteworthy that these amino acid carrying enzymes are highly conserved in the other macrolactam biosynthetic gene cluster, indicating that the clarified dipeptide strategy to carry amino acids appears to be a common way in the macrolactam biosynthesis. Thus, our finding shed a light on the rational design of engineered biosynthesis to change starter unit of macrolactam by swapping of the genes.

DOI： 10.24496/tennenyuki.53.0_103

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DOI： 10.1038/ja.2010.145

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DOI： 10.1002/cbic.201000214

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DOI： 10.1002/cbic.201090064

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DOI： 10.1038/ja.2009.107

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DOI： 10.1038/ja.2009.88

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Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Neomycins belong to a family of clinically important 2-deoxystreptamine (2DOS) containing aminoglycoside antibiotics including kanamycin, gentamicin, tobtamycin, and butirosin. In 2005, biosynthetic genes for neomycin were identified from the producer strain Streptomyces fradiae and the functions of those gene products have been extensively investigated with recombinant proteins. Among seven conserved enzymes encoded in the neomycin, butirosin, kanamycin, gentamicin, and tobramycin biosynthetic gene clusters, NeoC, NeoS and NeoE were already reorted to be responsible for the construction of 2DOS from D-glucose 6-phosphate (G6P). In the present study, a putative glycosyltransferase NeoM was characterized as UDP-GlcNAc:2DOS GlcNAc transferase. Comparative analysis of aminoglycoside biosynthetic genes revealed that four conserved ribostamycin-related enzymes couls be responsible for the ribosylation of neamine. As a result of enzymatic analysis with recombinant proteins derived from the butirosin producer Bacillus circulans, we verified that BtrL (NeoL homolog) catalyzes phosphoribosylation of ribostamycin, and BtrP (NeoP homolog) catalyzes following dephospholylation giving ribostamycin. Two conserved neomycin-related enzymes seemed to be involved in the last glycosylation in neomycin biosynthesis. One of the enzymes NeoF was then found to catalyze the GlcNAc transfer reation onto ribostamysin. The NeoF reaction product was further deacetylated by repetitive functional N-Ac-paromamine deacetylase NeoD, followed by dehydrogenation (NeoQ) and transamination (NeoB) leading to neomycin C. In addtion to previous results from our and British groups, all biosynthetic enzymes to synthesize neomycin C from G6P have been finally characterized.

DOI： 10.24496/tennenyuki.51.0_67

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DOI： 10.1038/ja.2009.76

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DOI： 10.1016/S0076-6879(09)04620-5

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DOI： 10.1039/b817312e

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DOI： 10.1021/bi800509x

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.tet.2008.05.024

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DOI： 10.1002/cbic.200700717

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DOI： 10.1021/ja072481t

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DOI： 10.1038/ja.2007.63

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DOI： 10.1016/j.bmc.2007.04.040

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DOI： 10.1016/j.bmc.2006.09.042

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DOI： 10.24496/intnaturalprod.2006.0__P-188_

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DOI： 10.1038/ja.2006.51

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DOI： 10.1007/s00776-005-0983-8

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DOI： 10.1016/j.tetlet.2006.01.008

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DOI： 10.1038/ja.2005.104

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DOI： 10.1038/ja.2005.47

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DOI： 10.1021/ja0445948

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DOI： 10.1021/ja044921b

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DOI： 10.1007/s00776-004-0828-x

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2-Deoxy-scyllo-inosose (DOI) synthase is the key enzyme in the biosynthesis of aminocyclitol antibiotics and catalyzes cyclization of D-glucose-6-phosphate (G-6-P) into the 6-membered carbocycle DOI. DOI synthase from Bacillus circulans was isolated as a heterodimeric protein comprising from a 20kDa and a 40kDa subunits. While the 40kDa subunit (BtrC) is known to be responsible for catalysis, the function of the 20kDa subunit (BtrC2) has yet to be revealed. In this study, we first carried out identification of the gene encoding BtrC2 from B. circulans by reverse genetics. The amino acid sequence of BtrC2 deduced from btrC2 closely resembled to that of YaaE (function unknwon) of Bacillus subtilis. Instead, BtrC2 appeared to have sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Supplementation of NH_4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. These observations suggest that, in addition to the secondary metabolism, BtrC2 is mainly involved in the primary metabolism in B. circulans. In order to study the reaction mechanism and substrate-enzyme interaction of BtrC, a carbacyclic analog (C-6-P) was designed as a inhibitor. The synthesized C-6-P was found to have strong inhibitory activity with inhibition constant K_1, of 12μM. Furthermore, the C-6-P analog showed time- and concentration-dependent inactivation of BtrC (K_1=224μM, k_<inact>=3.22×10^<-3>s^<-1>). Subsequent LC/ESI-MS analysis of the enzyme after reaction with C-6-P showed the molecular mass of 40768, which was ca. 160mass unit larger than that of native BtrC (40608). These results clearly indicated that C-6-P was a mechanism-based irreversible inhibitor by a covalent bond formation. In order to precisely determine the amino acid residue binding to C-6-P, BtrC-C-6-P complex was digested by chymotrypsin, and the resulting peptide mixture was analyzed by LC/MS. From comparison of the peptides derived from native BtrC, C-6-P was found to bind to a 139-146 peptide fragment, SIKQAVNL, and further, Lys-141 was determined as a binding site of C-6-P by LC/MS/MS analysis. Lys-141 appears to recognize the phosphate group of the substrate G-6-P and play an important role in the DOI synthase reaction.

DOI： 10.24496/tennenyuki.44.0_247

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DOI： 10.1021/ja000023d

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DOI： 10.1016/S0040-4039(00)00064-2

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DOI： 10.1271/bbb.62.2396

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2-Deoxystreptamine 1 is a common central aglycon in the major group of clinically important aminocyclitol antibiotics. The initial step of 2-deoxystreptamine biosynthesis is the intramolecular C-C bond formation between C-1 and C-6 of D-glucose-6-phosphate 3 to form 2-deoxy-scyllo-inosose 2 by 2-deoxy-scyllo-inosose synthase. An aldol-type cyclization involving transient oxidation of C-4 with NAD^+ and subsequent elimination of inorganic phosphate has been proposed. In this study, enzyme purification was carried out from centrifuged supernatant of the disrupted Bacillus circulans cells by ammonium sulfate fractionation, followed by DEAE-, Dye-ligand-, Q-Sepharose FF-, Mono Q- (x2) and gel filtration chromatography to afford two major spieces. The molecular weight of these enzymes were estimated to be about 45,000. Cross-over experiments with doubly labeled D-[4-^2H, 3-^<18>O] glucose-6-phosphate 1 1 were performed with the partially purified enzymes. The deuterium label at C-4 of the substrate was retained at C-6 of the product without scrambling the doubly-labeled isotope, and kinetic isotope effect (k_H/k_D ≒2.4-2.7) were observed in the enzyme reaction. These results strongly suggest that "2-deoxy-scyllo-inosose synthase" is a single enzyme, which catalyzes multistep reaction involving oxidation-reduction with NAD^+ cofactor and aldol-type carbocycle formation. Further, reaction with deoxy-G-6-P was investigated to clarify whether or not the other hydroxyl groups of G-6-P are participated in the reaction. Both 2-deoxy- and 3-deoxy-G-6-P were converted to some extent into their corresponding cyclitols. This indicates that the C-2 and C-3 hydroxyl groups are involved in the substrates recognition, but are not necessary for triggering the reaction.

DOI： 10.24496/tennenyuki.38.0_25

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DOI： 10.1021/ja511223g

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DOI： 10.1002/cbic.201000214

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DOI： 10.1016/j.jbiotec.2008.07.1953

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Venue：Frankfurt, Germany  

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Venue：Mannheim, Germany  

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Venue：立教大学  

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Venue：ホテル阪急エキスポパーク  

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Venue：Gyeongju, Korea  

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Venue：東京農業大学  

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Venue：Sanda  

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Venue：Hyogo  

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Venue：Oregon State University, Corvallis, OR  

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Venue：秋保リゾートホテルクレセント  

天然物化学談話会 奨励賞受賞記念講演

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Venue：Kyoto  

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Venue：国立日高青少年自然の家  

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Venue：Honolulu, HI (USA)  

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Venue：微生物化学研究センター  

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Venue：神奈川大学  

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Venue：広島  

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Venue：福岡  

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Venue：名古屋  

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Venue：仙台  

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Venue：東京  

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Venue：静岡  

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Venue：名古屋  

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Venue：Hangzhou, China  

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Venue：Ch_terais_ Gateaux Kingdom Sapporo  

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Venue：La Jolla, CA  

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Venue：Chulabhorn Convention Center, Thailand  

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Venue：The Hong Kong University of Science and Technology (HKUST), The University of Hong Kong (HKU), The Chinese University of Hong Kong (CUHK)  

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Venue：Ch_terais_ Gateaux Kingdom Sapporo  

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Venue：Yugawara  

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Venue：Saarland University, Germany  

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Venue：University of Nottingham, UK  

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Venue：Baltimore, U.S.A  

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Venue：Sapporo  

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Venue：Durham University, United Kingdom  

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Venue：Honolulu, HI (USA)  

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Venue：Awaji shima  

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Venue：Daejeon, Korea  

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Venue：Sapporo  

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Venue：Chulabhorn Research Institute, Mahidol University, Srinakharinwirot University, Chulalongkorn University, Kasetsart University  

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Venue：Sapporo  

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Venue：Jeju, Korea  

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Venue：University of Warwick, UK  

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Venue：慶應大学  

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Venue：北海道大学  

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Venue：北海道大学、札幌  

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Venue：Clearwater Beach, FL  

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Venue：Shanghai  

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Venue：草津セミナーハウス  

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Venue：Wuhan, China  

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Venue：東北大学川内萩ホール  

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Venue：Shanghai Jiao Tong University  

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Venue：サントリー研究センター  

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Venue：高知  

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Venue：University of Strasbourg, France  

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Venue：Frankfurt, Germany  

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Venue：Honolulu, HI (USA)  

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Venue：National Cheng Kung University, National Tsing Hua University, Genomics Research Center, Academia Sinica  

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Venue：Honolulu, HI (USA)  

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Venue：Eastin Hotel Petaling Jaya, Malaysia  

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