記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Angewandte Chemie - International Edition  

Pseurotins comprise a family of structurally related Aspergillal natural products having interesting bioactivity. However, little is known about the biosynthetic steps involved in the formation of their complex chemical features. Systematic deletion of the pseurotin biosynthetic genes in A. fumigatus and invivo and invitro characterization of the tailoring enzymes to determine the biosynthetic intermediates, and the gene products responsible for the formation of each intermediate, are described. Thus, the main biosynthetic steps leading to the formation of pseurotinA from the predominant precursor, azaspirene, were elucidated. The study revealed the combinatorial nature of the biosynthesis of the pseurotin family of compounds and the intermediates. Most interestingly, we report the first identification of an epoxidase C-methyltransferase bifunctional fusion protein PsoF which appears to methylate the nascent polyketide backbone carbon atom in trans. A maze: Pseurotins are a family of structurally related bioactive natural products from Aspergilli. Through genetic and biochemical studies, the biosynthetic pathway for the formation of azaspirene, synerazol, and pseurotin A/D have been elucidated, and reveal the combinatorial nature of their biosyntheses. PsoF was identified as bifunctional epoxidase methyltransferase enzyme, thus providing the first example of a trans-acting polyketide C-methyltransferase. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/anie.201404804

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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：Royal Society of Chemistry  

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DOI： 10.1039/d2ob00328g

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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：In Vivo  

Background/Aim: Among colorectal cancer-associated intestinal microbiota, colibactin-producing (clb+) bacteria are attracting attention. We aimed to clarify the interaction between clb+ Escherichia coli and normal colorectal epithelial cells in vivo and in vitro. Materials and Methods: Five-week-old female Balb/c mice were divided in an untreated group, a group treated with clb+ E. coli isolated from a Japanese patient with colorectal cancer (E. coli-50), and a group treated with non colibactin-producing E. coli (E. coli-50/ΔclbP). Mice were sacrificed at 18 weeks of treatment. Results: Treatment with clb+ E. coli increased positivity for H2A histone family member X phosphorylated at Ser-139 (γH2AX) in epithelial cells of the luminal surface of the mouse rectum but this did not occur in the E. coli-50/ΔclbP and untreated groups. In an in vitro setting, the ratio of apoptotic cells was increased and cell counts were reduced by treatment with clb+ E. coli more than in untreated cells and normal rat colorectal epithelial cells. Conclusion: E. coli-50 induced DNA damage in the mouse rectum, possibly by direct interaction between clb+ E. coli and normal colorectal epithelial cells. Our findings imply that regulation of clb+ E. coli infection may be a useful strategy for colorectal cancer control.

DOI： 10.21873/invivo.12746

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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：ChemBioChem  

Biosynthetic genes are not only responsible for the formation of bioactive substances but also suited for other applications including gene therapy. To test the feasibility of human cells producing antibiotics in situ when provided with a heterologous biosynthetic gene, we focused on cytochrome P450, the class of enzymes important in conferring bioactivity to natural product precursors. We selected Fma-P450 that plays a central role in the fumagillin antimicrobial biosynthesis in Aspergillus fumigatus to examine fungal metabolite production by HeLa cells that express fma-P450 heterologously. Here we show that HeLa cells harboring fma-P450 can biosynthesize 5-hydroxyl-β-trans-bergamoten and cytotoxic 5-epi-demethoxyfumagillol when supplemented with the nontoxic precursor β-trans-bergamotene. While the production level was insufficient to effect cell death, we demonstrate that programming human cells to autogenerate antibiotics by introducing a heterologous biosynthetic gene is feasible.

DOI： 10.1002/cbic.202100645

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記述言語：日本語   出版者・発行元：日本健康支援学会  

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

Escherichia coli containing polyketide synthase in the gut microbiota (pks+ E coli) produce a polyketide-peptide genotoxin, colibactin, and are suspected to play a role in the development of colorectal neoplasia. To clarify the role of pks+ E coli in the early stage of tumorigenesis, we investigated whether the pks status of E coli was associated with the prevalence of colorectal neoplasia. This cross-sectional analysis of data from a prospective cohort in Izu Oshima, Japan included asymptomatic residents aged 40-79 years who underwent screening colonoscopy and provided a stool sample. We identified 543 participants with colorectal neoplasia (22 colorectal cancer and 521 adenoma) as cases and 425 participants with normal colon as controls. The pks status of E coli was assayed using stool DNA and specific primers that detected pks+ E coli. The proportion of pks+ E coli was 32.6% among cases and 30.8% among controls. Compared with those with pks− E coli, the odds ratio (OR) (95% confidence interval) for participants with pks+ E coli was 1.04 (0.77-1.41) after adjusting for potential confounders. No statistically significant associations were observed regardless of tumor site or number of colorectal adenoma lesions. However, stratified analyses revealed increased ORs among participants who consumed cereals over the median intake or vegetables under the median intake. Overall, we found no statistically significant association between pks+ E coli and the prevalence of colorectal adenoma lesions among this Japanese cohort. However, positive associations were suggested under certain intake levels of cereals or vegetables.

DOI： 10.1111/cas.15196

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記述言語：日本語   出版者・発行元：東京 : 北隆館  

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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：BMC Microbiology  

Background: The Escherichia coli strain that is known to produce the genotoxic secondary metabolite colibactin is linked to colorectal oncogenesis. Therefore, understanding the properties of such colibactin-positive E. coli and the molecular mechanism of oncogenesis by colibactin may provide us with opportunities for early diagnosis or prevention of colorectal oncogenesis. While there have been major advances in the characterization of colibactin-positive E. coli and the toxin it produces, the infection route of the clb + strain remains poorly characterized. Results: We examined infants and their treatments during and post-birth periods to examine potential transmission of colibactin-positive E. coli to infants. Here, analysis of fecal samples of infants over the first month of birth for the presence of a colibactin biosynthetic gene revealed that the bacterium may be transmitted from mother to infant through intimate contacts, such as natural childbirth and breastfeeding, but not through food intake. Conclusions: Our finding suggests that transmission of colibactin-positive E. coli appears to be occurring at the very early stage of life of the newborn and hints at the possibility of developing early preventive measures against colorectal cancer.

DOI： 10.1186/s12866-021-02292-1

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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：BMC Microbiology  

Background: Colibactin-producing Escherichia coli containing polyketide synthase (pks+E. coli) has been shown to be involved in colorectal cancer (CRC) development through gut microbiota analysis in animal models. Stool status has been associated with potentially adverse gut microbiome profiles from fecal analysis in adults. We examined the association between stool patterns and the prevalence of pks+E. coli isolated from microbiota in fecal samples of 224 healthy Japanese individuals. Results: Stool patterns were determined through factorial analysis using a previously validated questionnaire that included stool frequency, volume, color, shape, and odor. Factor scores were classified into tertiles. The prevalence of pks+E. coli was determined by using specific primers for pks+E. coli in fecal samples. Plasma and fecal fatty acids were measured via gas chromatography-mass spectrometry. The prevalence of pks+E. coli was 26.8%. Three stool patterns identified by factorial analysis accounted for 70.1% of all patterns seen (factor 1: lower frequency, darker color, and harder shape; factor 2: higher volume and softer shape; and factor 3: darker color and stronger odor). Multivariable-adjusted odds ratios (95% confidence intervals) of the prevalence of pks+E. coli for the highest versus the lowest third of the factor 1 score was 3.16 (1.38 to 7.24; P for trend = 0.006). This stool pattern exhibited a significant positive correlation with fecal isobutyrate, isovalerate, valerate, and hexanoate but showed a significant negative correlation with plasma eicosenoic acid and α-linoleic acid, as well as fecal propionate and succinate. No other stool patterns were significant. Conclusions: These results suggest that stool patterns may be useful in the evaluation of the presence of tumorigenic bacteria and fecal fatty acids through self-monitoring of stool status without the requirement for specialist technology or skill. Furthermore, it may provide valuable insight about effective strategies for the early discovery of CRC.

DOI： 10.1186/s12866-021-02255-6

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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：Journal of the American Chemical Society  

Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activity-based fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.

DOI： 10.1021/jacs.1c01495

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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：Chemical Research in Toxicology  

Monocyclic aromatic amines, o-toluidine (o-Tol) and its structural analog o-anisidine (o-Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD), a p-semidine-type homodimer of o-Tol, was detected and identified in an in vitro reaction of o-Tol with S9 mix and in vivo urinary samples of o-Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o-Ans is converted to active metabolites to induce DNA damage in a similar manner as o-Tol. In this study, we report that a novel o-Ans metabolite, 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding (p-semidine form), was for the first time identified in o-Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o-Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o-Ans was coincubated with o-Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o-Ans, o-Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

DOI： 10.1021/acs.chemrestox.0c00536

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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：Journal of Natural Medicines  

Natural products, which can be isolated from living organisms worldwide, have played a pivotal role in drug discovery since ancient times. However, it has become more challenging to identify a structurally novel molecule with promising biological activity for pharmaceutical development, mainly due to the limited methodologies for their acquisition. In this review, we summarize our recent studies that activate the biosynthetic potential of filamentous fungi by genetic engineering to harness the metabolic flow for the efficient production of unprecedented natural products. The recent revolution in genome sequencing technology enables the accumulation of vast amounts of information on biosynthetic genes, the blueprint of the molecular construction. Utilizing the established heterologous expression system, activation of the pathway-specific transcription factor coupled with a knockout strategy, and manipulating the global regulatory gene, the biosynthetic genes were exploited to activate biosynthetic pathways and decipher the encoded enzyme functions. We show that this methodology was beneficial for acquiring fungal treasures for drug discovery. These studies also enabled the investigation of the molecular function of natural products in fungal development.

DOI： 10.1007/s11418-020-01466-x

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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 Catalysis  

We have previously reported the identification of CghA, a proposed Diels–Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA–product complex at a resolution of 2.0 Å. Our result provides the second structural determination of eukaryotic Diels–Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exo adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels–Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications. [Figure not available: see fulltext.].

DOI： 10.1038/s41929-021-00577-2

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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：国立感染症研究所 Japanese Journal of Infectious Diseases 編集委員会  

We investigated the relationship between colibactin-producing (clb+) Escherichia coli and colorectal adenocarcinoma. In total, 729 E. coli colonies were isolated from tumor and surrounding non-tumor regions in resected specimens from 34 Japanese patients; 450 colonies were from the tumor regions and 279 from the non-tumor regions. clb+ bacteria were found in tumor regions of 11 patients (11/34, 32.4%) and they were also detected in the non-tumor regions of 7 out of these 11 patients (7/34, 20.6%). The prevalence of clb+ isolates was 72.7% (327/450) and 44.1% (123/279) in tumor and non-tumor regions, respectively. All the recovered clb+ isolates belonged to the phylogenetic group B2 and were the most predominant type in tumor regions. Hemolytic (α-hemolysin-positive, hlyA+) and non-hemolytic (α-hemolysin-negative, hlyA-) clb+ isolates were obtained from patient #19; however, the prevalence of hlyA+ clb+ isolates was significantly higher in tumor regions (35/43, 81.4%) than in non-tumor regions (3/19, 15.8%). Moreover, a significantly higher production of N-myristoyl-D-asparagine, a by-product of colibactin biosynthesis, was observed in hlyA+ clb+ isolates than in hlyA- clb+ isolates. Our results suggest that hlyA+ clb+ E. coli may have a selective advantage in colorectal colonization and, consequently, might play a role in carcinogenesis. The presence of hlyA+ clb+ bacteria in healthy individuals is a potential risk marker of colorectal cancer.

DOI： 10.7883/yoken.JJID.2020.066

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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：Journal of Antibiotics  

Basidiomycetes are known to biosynthesize many biologically interesting compounds, including terpenoids. However, they are notoriously difficult to manipulate. Previously, we identified the gene cluster encoding enzymes responsible for the biosynthesis of lagopodins, cuparene-type sesquiterpenoid quinone natural products in Coprinopsis cinerea. In this study, we attempted to increase the productivity of lagopodin B (1) and related pathway products by overexpressing the terpene cyclase gene cop6 in C. cinerea to determine the details of the complex lagopodin and hitoyol biosynthetic pathway. Random integration of the cop6 into the genome of the ku70-deficient C. cinerea strain resulted in an ~2.4-fold increase in the production of 1. However, integration of cop6 into a highly transcribed position within the chromosome we designated as an expression boost area (EBA) resulted in an ~14-fold greater production of 1. Furthermore, the EBA-integration strain allowed us to isolate a previously undetected product 2, which we determined to be the known compound, hydroxylagopodin B. This finding expanded our understanding of the lagopodin–hitoyol biosynthetic pathway and allowed us to hypothesize a possible mechanism for the biosynthesis of a related homodimeric compound, lagopodin C. Our results demonstrate the potential of targeting EBA to integrate key biosynthetic genes into the genome for enhancing the production of difficult-to-obtain compounds for studying the biosynthesis of complex secondary metabolites in basidiomycetes and other complex eukaryotic organisms.

DOI： 10.1038/s41429-020-0355-9

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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：Scientific Reports  

The relative contribution of diet to colorectal cancer (CRC) incidence is higher than that for other cancers. Animal models have revealed that Escherichia coli containing polyketide synthase (pks+E. coli) in the gut participates in CRC development. The purpose of this cross-sectional study was to examine the relationship between dietary intake and the prevalence of pks+E. coli isolated from the microbiota in faecal samples of 223 healthy Japanese individuals. Dietary intake was assessed using a previously validated brief-type self-administered diet history questionnaire. The prevalence of pks+E. coli was evaluated using faecal samples collected from participants and specific primers that detected pks+E. coli. The prevalence of pks+E. coli was 26.9%. After adjusting for baseline confounders, the prevalence of pks+E. coli was negatively associated with the intake of green tea (odds ratio [OR], 0.59 [95% confidence interval (CI) 0.30–0.88] per 100 g/1,000 kcal increment) and manganese (OR, 0.43 [95% CI 0.22–0.85] per 1 mg/1,000 kcal increment) and was positively associated with male sex (OR, 2.27 [95% CI 1.05–4.91]). While futher studies are needed to validate these findings, these results provide insight into potential dietary interventions for the prevention of CRC.

DOI： 10.1038/s41598-020-72245-7

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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：Scientific Reports  

When the microfloral composition deteriorates, it triggers low-level chronic inflammation associated with several lifestyle-related diseases including obesity and diabetic mellitus. Fecal volatile organic compounds (VOCs) have been found to differ in gastrointestinal diseases as well as intestinal infection. In this study, to evaluate a potential association between the pathogenesis of lifestyle-related diseases and VOCs in the intestinal tract, fecal VOCs from obese/diabetic KK-Ay mice (KK) or controls (C57BL/6J mice; BL) fed a normal or high fat diet (NFD or HFD) were investigated using headspace sampler-GC-EI-MS. Principal component analysis (PCA) of fecal VOC profiles clearly separated the experimental groups depending on the mouse lineage (KK vs BL) and the diet type (NFD vs HFD). 16 s rRNA sequencing revealed that the PCA distribution of VOCs was in parallel with the microfloral composition. We identified that some volatile metabolites including n-alkanals (nonanal and octanal), acetone and phenol were significantly increased in the HFD and/or KK groups. Additionally, these volatile metabolites induced proinflammatory activity in the RAW264 murine macrophage cell line indicating these bioactive metabolites might trigger low-level chronic inflammation. These results suggest that proinflammatory VOCs detected in HFD-fed and/or diabetic model mice might be novel noninvasive diagnosis biomarkers for diabetes.

DOI： 10.1038/s41598-020-62541-7

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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：BMC  

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DOI： 10.1186/s41021-020-00149-z

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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  

DOI： 10.24496/tennenyuki.62.0_181-186

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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  

DOI： 10.24496/tennenyuki.62.0_391-396

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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：Biochemistry  

Biosynthesis of certain fungal polyketide-peptide synthetases involves C-methyltransferase activity that adds one or more S-adenosyl-l-methionine-derived methyl groups to the carbon framework. The previously reported PsoF-MT, the stand-alone C-methyltransferase (MT) from the pseurotin biosynthetic pathway that exists as a domain within a trifunctional didomain enzyme PsoF, was characterized crystallographically and kinetically using mutants with substrate analogs to understand how a trans-acting C-MT works and compare it to known polyketide synthase-associated C-MTs. This study identified key active-site residues involved in catalysis and substrate recognition, which led us to propose the mechanism of C-methylation and substrate specificity determinants in PsoF-MT.

DOI： 10.1021/acs.biochem.9b00702

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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：Organic Letters  

While high-colibactin-producing Escherichia coli is thought to be associated with colorectal oncogenesis, this study is complicated part due to an inability to isolate colibactin adequately. Here, we created fluorescent probes activated by ClbP, the colibactin-maturing peptidase, to identify high-colibactin-producing strains. Our probe served as a valuable clinical diagnostic tool that allowed simple high-throughput diagnostic screening of clinical samples. Furthermore, the probe also allowed identification of high-colibactin producers that would help advance our understanding of colibactin biosynthesis.

DOI： 10.1021/acs.orglett.9b01345

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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：Organic and Biomolecular Chemistry  

Use of the ku70-deficient strain of Coprinopsis cinerea enabled confirmation within the native context of the central role the sesquiterpene synthase Cop6 plays in lagopodin biosynthesis. Furthermore, yeast in vivo bioconversion and in vitro assays of two cytochrome P450 monooxygenases Cox1 and Cox2 allowed elucidation of the network of oxidation steps that build structural complexity onto the α-cuprenene framework during the biosynthesis of lagopodins. Three new compounds were identified as intermediates formed by the redox enzymes.

DOI： 10.1039/c8ob02814a

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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：一般社団法人　日本毒性学会  

Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotox-ic potency of the colibactin-producing (clb+) E. coli strains that carry the polyketide synthases “pks” gene cluster (pks+) and an isogenic clb- mutant in which the colibactin-producing ability is impaired. Measurement of phosphorylated histone H2AX indicated that DNA double strand breaks were induced in mammalian CHO AA8 cells infected with the clb+ E. coli strains. Induction of DNA damage response (SOS response) by crude extract of the clb+ strains was 1.7 times higher than that of the clb- E. coli in an umu assay with a Salmonella typhimurium TA1535/pSK1002 tester strain. Micronucleus test with CHO AA8 cells revealed that infection with the clb+ strains induced genotoxicity, i.e., the frequencies of micronucle-ated cells infected with clb+ strain were 4-6 times higher than with the clb- strain. Since the intestinal flora are affected by dietary habits that are strongly associated with ethnicity, these data may contribute to both risk evaluation and prevention of colorectal cancer in the Japanese population.

DOI： 10.2131/jts.44.871

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：公益社団法人 有機合成化学協会  

Colibactin, a secondary metabolite produced by a commensal Escherichia coli harboring pks island, exhibits a genotoxicity to mammalian cells and progresses to colorectal cancer. Regardless of potential risk for the promotion of cancer by colibactin, which shows neither a biological mechanism of action nor determination of chemical structure. Analysis of a genetic mutant of colibactin-producing E. coli enabled us to provide biosynthetic intermediates and shunt products associated with a partial structure of colibactin. This review covers the recent findings of the biosynthesis and implication of chemical structure of colibactin.

DOI： 10.5059/yukigoseikyokaishi.76.490

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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  

シクロペナーゼはcyclopenin (1) をviridicatin (2) へと変換する糸状菌酵素として1967年にLucknerによって存在が提唱された酵素である (図１) 1。その正体は未解明のままであったが、近年我々はpenigequinoloneの生合成に関与するヘモシアニン類似タンパク質PngLがシクロペナーゼであることを見出し本討論会で報告している 2, 3 。しかし、ヘモシアニンに似た酵素が二次代謝に関わっている例は他になく、どのようなメカニズムで骨格変化を伴う反応を触媒しているか不明であった。今回、このシクロペナーゼの反応機構について各種実験と計算化学によって検証したのでこれを報告する。

シクロペナーゼの生化学的解析
シクロペナーゼの生化学的な解析のため、PngLおよびAspergillus nidulansが有するホモログAsqIをそれぞれ大腸菌に合成させることを試みた。両遺伝子ともに大腸菌での発現に成功し、その菌体破砕液を用いて酵素活性を調べたところ、PngLは4’-methoxycyclopenin (MCPN, 3) のみを、AsqIは1と3の両方を基質として対応する2および4’-methoxyviridicatin (MVDT, 4) へと変換した。続いて酵素反応の詳細な情報を得るべく両酵素の精製を試みた。PngLは各種発現ベクターの検討を行っても溶解性に乏しかったため少量の獲得にとどまった。一方、AsqIは6xHisタグを利用したアフィニティ精製後、ゲル濾過クロマトグラフィーに付すことで酵素活性を保った状態で必要量を精製することができた。
シクロペナーゼであるAsqIはヘモシアニンに類似したアミノ酸配列を有している。ヘモシアニンとは甲殻類や軟体動物の酸素運搬タンパク質であり、金属結合

DOI： 10.24496/tennenyuki.60.0_37-42

CiNii Research

記述言語：日本語   出版者・発行元：公益社団法人 日本薬学会  

冬虫夏草という語を初めて目にし「虫なのか草なのか，一体これは何者だろう？」と興味を抱く人は少なくない．昆虫の亡骸からキノコが生えた奇妙な姿形や，その正体が糸状菌(いわゆるカビ)である事実に驚かされたのは筆者だけではないだろう．冬虫夏草の一種であるサナギタケ<i>Cordyceps</i> <i>militaris</i>は，平板培地においてもキノコを形成するため人工栽培法が確立され，国内でも健康食品として市販されている．その成分の1つがコルジセピン(cordycepin：COR)であり，抗がん，免疫賦活，精力増強など，様々な薬理活性を示すことが知られている．CORはアデノシンの3́位デオキシ体であり，アデノシンとの類似性が故にポリメラーゼによる伸長途中のRNA鎖に取り込まれる．ところが，3́位水酸基が欠如しているため更なる伸長反応が起こらず，細胞増殖が阻害される．一方，CORは生体内のアデニンデアミナーゼ(adenine deaminase：ADA)によって容易に薬物代謝(脱アミノ化)され，活性の減弱化した3́-デオキシイノシン（3́-deoxyinosine：3́dI）を生成する．そのため，かつて実施された白血病治療薬としての臨床試験においては，ADA阻害薬であるペントスタチン(pentostatin：PTN)との併用療法が検討されていた．今回，未解明であったCOR生合成遺伝子が同定され，その独特な生合成戦略が明らかにされたので紹介する．<br>なお，本稿は下記の文献に基づいて，その研究成果を紹介するものである.<br>1) Tuli H. S. <i>et</i> <i>al</i>., <i>Life</i> <i>Sci</i>., <b>93</b>, 863-869(2013)．<br>2) Xia Y. <i>et</i> <i>al</i>., <i>Cell</i> <i>Chem</i>. <i>Biol</i>., <b>24</b>, 1479-1489(2017)．<br>3) Wu P. <i>et</i> <i>al</i>., <i>Cell</i> <i>Chem</i>. <i>Biol</i>., <b>24</b>, 171-181(2017)．

DOI： 10.14894/faruawpsj.54.5_458

CiNii Research

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Fungal Genetics and Biology  

Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of pex1 disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that pex1 is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous C. cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

DOI： 10.1016/j.fgb.2017.10.002

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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：Chemical Record  

During the last decade, we have revealed biosynthetic pathways responsible for the formation of important and chemically complex natural products isolated from various organisms through genetic manipulation. Detailed in vivo and in vitro characterizations enabled elucidation of unexpected mechanisms of secondary metabolite biosynthesis. This personal account focuses on our recent efforts in identifying the genes responsible for the biosynthesis of spirotryprostatin, aspoquinolone, Sch 210972, pyranonigrin, fumagillin and pseurotin. We exploit heterologous reconstitution of biosynthetic pathways of interest in our study. In particular, extensive involvement of oxidation reactions is discussed. Heterologous hosts employed here are Saccharomyces cerevisiae, Aspergillus nidulans and A. niger that can also be used to prepare biosynthetic intermediates and product analogs by engineering the biosynthetic pathways using the knowledge obtained by detailed characterizations of the enzymes. (998 char.).

DOI： 10.1002/tcr.201700015

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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：Organic Letters  

To identify natural products and their associated biosynthetic genes from underutilized, difficult-to-manipulate microbes, chemical screening and bioinformatic analysis were employed to identify secondary metabolites and a potentially associated biosynthetic gene cluster. Subsequently, a heterologous expression system was used to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. This approach successfully identified the curvupallide and spirostaphylotrichin biosynthetic pathways in endophytic fungus Curvularia pallescens and the short-chain pyranonigrin biosynthetic pathway in Aspergillus Niger.

DOI： 10.1021/acs.orglett.7b00559

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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：Angewandte Chemie - International Edition  

The regioselective functionalization of non-activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono- and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O-methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH2-dependent enzyme is uniquely capable of the stepwise gem-dichlorination of a non-activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.

DOI： 10.1002/anie.201604516

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記述言語：英語   出版者・発行元：Molecules  

Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

DOI： 10.3390/molecules21081078

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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：Journal of Antibiotics  

To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an α-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

DOI： 10.1038/ja.2016.54

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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  

<p>1. 研究背景</p><p>　がん化した細胞が増殖し、サイズの大きながん組織になるには血液からの酸素と栄養の供給が必要となり、そのためにがんは血管新生を誘導する。続いて新生した血管を通じて浸潤や転移が誘発され、がんの悪性化が加速される。故に、血管新生を阻害するがん治療戦略が考案され、いくつかの分子標的薬が成功を収めてきた。糸状菌Aspergillus fumigatusは化学構造の全く異なる2種類の血管新生阻害剤、fumagillin (1)およびpseurotin類を生産する。これまでに1の合成誘導体TNP-470について抗がん剤としての臨床開発研究が行われていたが、その副作用や薬物動態の悪さのために現在では開発が中止されている。我々は、依然として医薬品リードとして重要な1について、生合成遺伝子および生合成経路が未解明であったこと、遺伝子工学的手法を用いて非天然型誘導体を創出することで新たな医薬品候補を世に提供できると考え、その生合成研究に着手した。</p><p>2. Fumagillin-pseurotin複合型生合成遺伝子クラスターの発見 </p><p> 　その化学構造より、1はテルペン骨格とポリケタイド骨格から構成されていると推測された。そこでA. fumigatusゲノム中よりテルペン環化酵素、ポリケタイド合成酵素を含む遺伝子クラスターを探索したところ、第8番染色体上にこれらの条件を満たすクラスターが確認された。続いて本領域に含まれるテルペン環化酵素の遺伝子破壊株Dfma-TC株を構築した。その代謝産物をLC-MSにて解析したところ、1の生産消失が認められた。以上の実験から、1の生合成遺伝子クラスターを同定することに成功した。興味深いことに、fma-TCは全く別の二次代謝産物pseurotin A (2)生合成に必須な遺伝子psoA¹のすぐ近傍の約2.5 kb離れた位置に存在していた。</p><p>図1. Fumagillin-pseurotin生合成遺伝子クラスター</p><p>　続いて、1の生合成経路解明を目指した。1の生合成遺伝子クラスターに存在する約15種の酵素遺伝子について、それぞれの遺伝子破壊株を作製した。その結果、8種の遺伝子破壊株において1の生産が失われ、これらの遺伝子が1の生合成に必須であることが示唆された。一方、別の2種の遺伝子破壊株(DpsoF, DpsoG)では、1の生産に変化は認められなかったものの、予想外なことに2の生産が消失していた。さらに転写因子遺伝子fapR破壊株においては、1と2が共に生産されていなかった。以上の結果から、染色体上の本領域において1と2の生合成に必須な遺伝子が互いに交じり合って存在していること、加えて、一つの転写因子FapRにより各化合物の生合成が統一的に制御されていることが示唆された。そこで、当初2の生合成遺伝子として推定されていた計6種の遺伝子(psoAを含む)¹についても同様に遺伝子破壊を行い、うち4種の遺伝子が2の生合成に必須であることを実験的に確認した。以上、全く別の化学構造を持つ1および2が、約20種の酵素遺伝子から構成される一つの巨大クラスターによって生合成されると決定した²(図1)。</p><p>3. fumagillin生合成経路の解明</p><p>2013年、上述した我々の研究とは独立してTangらは1の生合成遺伝子を同定し、テルペン環化酵素Fma-TCが1のテルペン骨格の前駆体で</p><p>(View PDFfor the rest of the abstract.)</p>

DOI： 10.24496/tennenyuki.58.0_oral34

CiNii Research

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

Successful activation of the pyranonigrin biosynthetic gene cluster and gene knockout in Aspergillus Niger plus in vivo and in vitro assays led to isolation of six new products, including a spiro cyclobutane-containing dimeric compound, which served as the basis for the proposed comprehensive pyranonigrin biosynthetic pathway. Two redox enzymes are key to forming the characteristic fused γ-pyrone core, and a protease homologue performs the exo-methylene formation.

DOI： 10.1021/acs.orglett.5b02435

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記述言語：英語   出版者・発行元：Nature Chemical Biology  

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.

DOI： 10.1038/nchembio.1890

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記述言語：日本語   出版者・発行元：[東京] : 病態代謝研究会  

CiNii Research

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

Biochemical studies of purified and dissected fungal polyketide synthase and nonribosomal peptide synthetase (PKS-NRPS) hybrid enzymes involved in biosynthesis of pseurotin and aspyridone indicate that one α-methylation step during polyketide synthesis is a prerequisite and a key checkpoint for chain transfer between PKS and NRPS modules. In the absence of the resulting γ-methyl feature, the completed polyketide intermediate is offloaded as an α-pyrone instead of being aminoacylated by the NRPS domain. These examples illustrate that precisely timed tailoring domain activities play critical roles in the overall programming of the iterative PKS (and NRPS) functions.

DOI： 10.1021/ol503179v

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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：Journal of the American Chemical Society  

Polyene macrolactams are a class of microbial metabolites, many of which show potent biological activities with unidentified modes of action. Here we report that 8-deoxyheronamide C, a new 20-membered polyene macrolactam from a marine-derived actinomycete Streptomyces sp., is a unique membrane binder. 8-Deoxyheronamide C showed a characteristic sensitivity profile against fission yeast sterol mutant cells, indicating that the metabolite targets cell membranes. We detected tight physical interaction between heronamides including 8-deoxyheronamide C and heronamide C and saturated hydrocarbon chains in lipid membranes using surface plasmon resonance experiments. We further show that heronamides induced abnormal cell wall morphology in fission yeast probably by perturbing the structure of membrane microdomains. This work will accelerate the biological and medical investigation of polyene macrolactams. © 2014 American Chemical Society.

DOI： 10.1021/ja500128u

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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：Journal of the American Chemical Society  

Fumagillin (1), a meroterpenoid from Aspergillus fumigatus, is known for its antiangiogenic activity due to binding to human methionine aminopeptidase 2. 1 has a highly oxygenated structure containing a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene β-trans-bergamotene. The chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted-gene deletion experiments. Here, we report the identification and characterization of three oxygenases from the fumagillin biosynthetic pathway, including a multifunctional cytochrome P450 monooxygenase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates. © 2014 American Chemical Society.

DOI： 10.1021/ja500881e

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記述言語：英語   出版者・発行元：AMER CHEMICAL SOC  

DOI： 10.1038/nchembio.1366

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記述言語：日本語   出版者・発行元：[東京] : 病態代謝研究会  

CiNii Research

担当区分：筆頭著者   記述言語：英語  

DOI： 10.1038/NCHEMBIO.1366

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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：Journal of the American Chemical Society  

Postgenomic analysis revealed that many microorganisms carry numerous secondary metabolite biosynthetic genes on their genome. However, activities of those putative genes are not clearly reflected in the metabolic profile of the microorganisms, especially in fungi. A recent genome mining effort is promising in discovering new natural products. However, many fungi and other organisms are not amenable to molecular genetics manipulations, making the study difficult. Here we report successful engineering of Chaetomium globosum, a known producer of various valuable natural products, that allows its genetic manipulation via targeted homologous recombination. This strain permitted us to abolish transcriptional regulators associated with epigenetic silencing of secondary metabolite biosynthetic pathways, leading to the identification of the products generated by different gene clusters and isolation of novel secondary metabolites. We were able to identify six gene clusters that are responsible for the biosynthesis of 11 natural products previously known to be produced by C. globosum, including one cytochalasan and six azaphilone-type compounds. In addition, we isolated two new compounds, mollipilin A and B, that were only recently identified in a related Chaetomium species. Furthermore, our investigation into the mechanism of biosynthesis of those natural products in C. globosum also led to the discovery of a secondary metabolite, aureonitol, that acts like a transcriptional regulator for the biosynthesis of other secondary metabolites. Similar approaches should facilitate exploration of the untapped potential of fungal biosynthetic capability and identification of various unique biological functions that those secondary metabolites possess. © 2013 American Chemical Society.

DOI： 10.1021/ja405128k

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記述言語：英語   出版者・発行元：Current Opinion in Chemical Biology  

Echinomycin is an antitumor antibiotic secondary metabolite isolated from streptomycetes, whose core structure is biosynthesized by nonribosomal peptide synthetase (NRPS). The echinomycin biosynthetic pathway was successfully reconstituted in Escherichia coli. NRPS often contains a thioesterase domain at its C terminus for cyclorelease of the elongating peptide chain. Those thioesterase domains were shown to exhibit significant substrate tolerance. More recently, an oxidoreductase Ecm17, which forms the disulfide bridge in triostin A, was characterized. Surprisingly, an unrelated disulfide-forming enzyme GliT for gliotoxin biosynthesis was also able to catalyze the same reaction, providing another example of broad substrate specificity in secondary metabolite biosynthetic enzymes. Those promiscuous catalysts can be a valuable tool in generating diversity in natural products analogs we can produce heterologously. © 2013 Elsevier Ltd.

DOI： 10.1016/j.cbpa.2013.06.022

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記述言語：英語   出版者・発行元：Natural Product Reports  

Covering: up to November 2012 In this article, we review recent successful efforts to engineer biosynthesis of several important fungal natural products through heterologous expression of relevant biosynthetic genes in Saccharomyces cerevisiae. We also describe an innovative method of rapidly cloning fungal polyketide synthase or nonribosomal peptide synthetase genes, which can be 5-20 kb or longer, from a pool of total RNA obtained from the fungus of interest using the technique we termed the "overlap extension PCR-yeast homologous recombination (ExRec)" method. The process concomitantly incorporates the cloned genes into yeast expression vectors for biosynthesis of corresponding polyketide and nonribosomal peptide compounds in our engineered S. cerevisiae strain, allowing detailed chemical characterizations to identify the activities of those previously uncharacterized biosynthetic megaenzymes. Studies reviewed here highlight yeast as a useful and versatile host not only for production of various natural products and mechanistic investigation of biosynthetic enzymes, but also for mining of uncharacterized fungal genomes for novel secondary metabolite biosynthetic pathways. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3np70037b

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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  

<p>1. 研究背景</p><p>　新しい化学構造、生物活性を有する天然物を獲得することは、学術的にも産業的にも重要である。しかしながら、現在では容易に獲得できる天然物は既に取り尽くされたといっても過言ではない状況にあり、実際に多数の大手製薬企業が天然物をリード化合物とした創薬研究を縮小廃止している。その原因として、新規天然物の獲得には多大な時間と労力が費やされること、獲得効率が悪いこと、生産性が低いなどの理由が挙げられ、これらの問題点を解決した「次世代型の天然物獲得法」が望まれている。</p><p>　ところで、次世代シーケンサーによるDNA配列解読技術の革新により、現在では様々な微生物のゲノム配列を短時間、低コストにて解読可能になった。Cyclosporine A、lovastatin などといった数多くの医薬品を輩出してきた糸状菌についても数多くの種のゲノムが解読されてきた¹。その結果、たった一種の糸状菌のゲノム中に数十種類もの天然物生合成遺伝子が含まれていることが明らかにされた。ところが、実験室内における一般的な培養条件において、実際に代謝産物として単離、同定される天然物の数はそれよりも遥かに少数である。近年のトランスクリプトーム解析により、これら天然物生合成遺伝子の多くが不活性化状態、すなわち休眠状態であることが示された²。つまり、我々は微生物のもつ天然物生産能力のうち、氷山の一角のみを利用していたに過ぎないのかも知れない。一方で、このような生合成遺伝子を人為的に制御し、発現させることができれば、これまでに発見されてこなかった天然物の獲得が期待できる。本研究では、糸状菌に対して遺伝子操作を施すことにより、本来は休眠状態であった天然物生合成経路を覚醒させることで、新規天然物の獲得を目指した。</p><p> </p><p>2. 黒麹菌Aspergillus nigerにおける休眠型生合成遺伝子の覚醒</p><p> i. 休眠型生合成遺伝子のin silico解析</p><p> A. nigerは工業的に有機酸の製造に用いられている麹菌の一種であり、既にそのゲノム解読が完了している¹。そのゲノム中には33個のポリケタイド合成酵素(PKS)、15個の非リボゾーム性ペプチド合成酵素(NRPS)および9つのPKS-NRPS hybrid型酵素遺伝子(HPN)を有する³。我々は、コンピューター上にてこれら天然物生合成遺伝子クラスターを詳細に解析し、新規天然物生合成を担う遺伝子クラスターを推測した。具体的には、まず機能未知の生合成遺伝子クラスターを抽出し、Basic Local Alignment Search Tool (BLAST)検索等により各構成遺伝子の機能を予測した。これらの情報と、既知天然物の生合成経路の文献情報から、一部の生合成遺伝子クラスターについてはその生合成産物が予測可能であった。一方、我々の研究対象はその生合成産物が予測不可能な生合成遺伝子クラスターであり、本クラスターはこれまでに発見されていない天然物（新規化合物）、あるいは生合成経路が明らかになっていない天然物を生合成する可能性を秘めていた。続いて選抜した数十種の生合成遺伝子クラスターについて、様々な培養条件にて得たmRNAをもとに、半定量RT-PCR法によりその転写量の解析を行った。その結果、い</p><p>(View PDFfor the rest of the abstract.)</p>

DOI： 10.24496/tennenyuki.55.0_oral35

CiNii Research

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  

<p>生体膜は特定の環境の内外を仕切る構造体であると同時に、シグナル伝達や物質輸送などを制御する機能的なオルガネラとしても働いている。近年、その主要な構成成分である脂質分子が、生体膜において機能的な微小環境を構築・制御している可能性に注目が集まっている。例えば、スフィンゴ脂質とステロールが豊富な膜ドメインは脂質ラフトとよばれ、シグナル伝達を効果的に行うために必要であるとされている¹。しかし、脂質分子による生体膜微小環境の形成・維持機構や生理機能の実態は、依然として掴めていないのが現状である。この原因の一つに、脂質が遺伝子に直接コードされる分子ではないことが挙げられる。すなわち、タンパク質の機能解析に有効な遺伝学の方法論を十分に活用できないのである。そこで、脂質に直接はたらきかける化合物を用いた化学遺伝学的なアプローチが期待される。特に、脂質との相互作用を起点に可逆的な機能変調を引き起こすような化合物は、その作用機序解析を通じて脂質分子の機能に直接迫れる可能性がある。ところが、脂質を認識することが知られている化合物は激しい膜傷害性を有する場合が多く、生体膜の解析ツールには適していない。このような背景のもと我々は、生体膜脂質の機能解明を目標に、脂質分子と相互作用する新しい天然化合物の取得と作用機序解析を行っている。本発表では、海洋由来放線菌より得られたポリエンマクロラクタムheronamide 類の単離・構造決定および生物活性について報告する。</p><p>1. 脂質認識物質の探索とheronamide類の単離</p><p>　Ergosterol 生合成経路に変異を持つ酵母細胞は、脂質と相互作用する化合物に対する感受性が野生株と比べて低下する。例えばergosterolを標的とするamphotericin B (AmB)の分裂酵母に対する生育阻害活性は、erg2遺伝子破壊株では野生株の1/4程度である。この野生株と変異株の感受性の差を指標に、約2,000 の微生物培養液抽出物をスクリーニングしたところ、海洋由来放線菌Streptomyces sp. NSU893 の培養液が目的とする選択性を示すことを見出した。そこで、本培養液 (5 L)から菌体を濾取しCHCl₃/MeOH (1:1)で抽出後、各種クロマトグラフィーにより活性成分の精製を行い、新規化合物1 (14.5 mg)を得た。化合物1 は、同じく海洋由来放線菌から報告されたポリエンマクロラクタムheronamides A-C の類縁化合物であった (図1)²。実際、我々の放線菌培養液からもheronamide C (2, 16.1 mg) およびA (3, 11.6 mg) が得られた。</p><p>2. Heronamide類の構造解析</p><p>化合物1の平面構造は、各種スペクトル解析からheronamide C (2)の8 位デオキシ体8-deoxyheronamide C (8-dHC)と決定した (図1)。Heronamide類とよく似た構造を持つBE-14106 (図6)の生合成経路を参考にすると、8-dHC (1)はheronamide C (2)の生合成前駆体であり、シトクロムP450により酸化を受けることで化合物2へと変換されると考えられる³。また、heronamide C (2)は酸化と環化反応によりheronamide A (3)へと変換されると推測されている²。以上より、化合物1-3の8, 9, 19位の立体化学は保存されていると予想し、8-dHC (1)の立体化学を化合物2, 3との比較解析から決定することにした。しかしスペクトル解析の過程</p><p>(View PDFfor the rest of the abstract.)</p>

DOI： 10.24496/tennenyuki.55.0_oral20

CiNii Research

掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/chin.201247204

記述言語：日本語   出版者・発行元：天然有機化合物討論会  

The article describes the detailed mechanism of the formation of the spiro-carbon moiety in an anti-mitotic fungal nonribosomal peptide (NRP), spirotryprostatin A, mediated by a key enzyme, FAD-dependent monooxygenase (FMO). The presence of spiro-carbon moieties is prevalent among natural products, yet the detailed mechanism of their biosynthesis is poorly characterized. Those spiro-centers remain to be a significant challenge to synthetic chemistry as well. Thus, we sought to investigate how a sterically constrained chiral spiro-carbon could be synthesized enzymatically. We focused on spirotryprostatins because of their useful biological activity and complex chemical structure involving a spiro-carbon moiety. Since similar structural motif is also found in other indole alkaloid natural products, we felt that our findings here will have a broad impact on our understanding of the mechanism of complex natural product biosynthesis. What is frequently the major obstacle in conducting detailed mechanistic studies of enzymes involved in complex natural product biosynthesis is the lack of adequate supply of substrates and reference materials. This was true for studying the spiro-carbon formation in spirotryprostatins. To resolve this problem, we successfully carried out metabolic engineering of yeast for heterologous production of tryprostatins (substrates for spirotryprostatins formation) and other compounds involved in the biosynthesis of tryprostatins and spirotryprostatins using our engineered strain of Saccharomyces cerevisiae, SCKW5. Using the compounds obtained from yeast, we characterized the activity of an FMO, Afua_12060, involved in the fumiquinazoline biosynthesis and confirmed that Afua_12060 was responsible for an poxidation-driven semipinacol-type rearrangement of tryprostatin A that set up the compound for the ensuing formation of spirotryprostatin A. Comparison of our finding with the proposed biosynthetic mechanism of related fumiquinazoline and notoamide natural products revealed that FMO-catalyzed epoxidation of unactivated indole plays a key role in the formation of various complex chemical structures (namely spiro-carbon and imidazoindolone ring systems). However, what was most intriguing about our finding was the discovery of an unusual crosstalk between two biosynthetic pathways (fumiquinazoline and fumitremorgin biosynthetic pathways) that resulted in the formation of new compounds with interesting chemical features (spirotryprostatins).

DOI： 10.24496/tennenyuki.54.0_79

CiNii Books

担当区分：筆頭著者   記述言語：英語  

CiNii Research

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

Tumescenamide C, a new cyclic lipodepsipeptide, was isolated from a culture broth of an actinomycete Streptomyces sp. KUSC-F05. Tumescenamide C was a congener of tumescenamides A and B, representing a sixteen-membered ring system, consisting of two proteinogenic and three non-proteinogenic amino acids, to which a methyl-branched fatty acid was attached. The planar structure was determined by spectroscopic analysis, while its absolute stereochemistry was determined by chemical degradation and asymmetric synthesis. Tumescenamide C exhibited antimicrobial activity with high selectivity against Streptomyces species. © 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2012.04.075

Web of Science

Scopus

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Antibiotics  

DOI： 10.1038/ja.2012.34

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Scopus

PubMed

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

Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/cbic.201100798

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Scopus

PubMed

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  

DOI： 10.24496/tennenyuki.54.0_79

CiNii Research

記述言語：日本語   出版者・発行元：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

近年，天然物の生合成遺伝子の情報をもとに，有用天然物を獲得する取り組みが行なわれるようになってきた．中でも植物および真菌といった真核生物から単離された生物活性物質の生物合成に関する報告がなされてきた．ここでは，特に真核生物由来の天然物生合成遺伝子を出芽酵母の異種発現系を用いて発現させ，有用物質の生産に挑戦する取り組みについて解説する．

DOI： 10.1271/kagakutoseibutsu.50.163

CiNii Books

CiNii Research

記述言語：日本語   出版者・発行元：日本生物工学会  

コレクション : 国立国会図書館デジタルコレクション > 電子書籍・電子雑誌 > 学術機関 > 学協会

CiNii Books

CiNii Research

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  

Microbes are a rich source of bioactive metabolites; they represent a wide variety of chemical structures and specific biological activities. In our continuing search for bioactive metabolites from microbes, a culture broth of an actinomycete strain of the genus Streptomyces, from which several bioactive secondary metabolites were isolated, was found to contain a novel antimicrobial lipodepsipeptide, designated pentadepsin A (1). Herein, we report the isolation, structure elucidation and biological activity of pentadepsin A. The molecular formula of 1 was established as C37H57N508 on the basis of HR-FABMS and NMR spectral data. The planar structure of 1 was elucidated by 2D NMR analysis; it represented a sixteen-membered ring system, consisting of five amino acids, to which a unique acyl group 2,4-dimethyl heptanoic acid (Dmh) was linked through an amide bond. The absolute stereochemistry of amino acid residues was determined by advanced Marfey's method, while that of Dmh residue was partially elucidated by PGME method. For the full elucidation of the side chain structure, (2S,4R)-Dmh and (2S,4S)-Dmh were synthesized via Evans alkylation. Spectroscopic comparison of Dmh obtained from 1 with the synthesized ones revealed that the side chain of 1 is (2S,4S)-2,4-dimethylheptanoic acid. Pentadepsin A selectively inhibited cell growth of Streptomyces species, while exhibited no growth inhibition against other bacteria tested and no cytotoxic effect mammalian cells. Detailed analysis of the mechanism of action of pentadepsin A is underway.

DOI： 10.24496/tennenyuki.53.0_397

CiNii Books

CiNii Research

記述言語：英語   出版者・発行元：Pediatric Blood and Cancer  

DOI： 10.1002/pbc.22478

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Scopus

PubMed

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  

DOI： 10.24496/tennenyuki.52.0_397

CiNii Research

Web of Science

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

Symbiospirols A, B, and C, long carbon-chain compounds with a molecular weight of 1207 were isolated from the cultured symbiotic dinoflagellate Symbiodinium sp. Their planar structures and partial relative stereochemistries were elucidated based on 1D and 2D NMR spectra and a degradation reaction. Symbiospirols consisted of a C67-linear chain with a β,β′- dihydroxyl ketone moiety, eight hydroxyl groups, one tetrahydropyran ring, and two 1,6-dioxaspiro[4,4]nonane rings. Symbiospirol A had an inhibitory effect against L-phosphatidylserine-induced PKC activation. © 2009 American Chemical Society.

DOI： 10.1021/ol900299x

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Scopus

PubMed

記述言語：日本語   出版者・発行元：天然有機化合物討論会  

Dinoflagellates are widely known as the rich source of biologically active and structural unique secondary metabolites. Among them, large polyols and polyethers composed of a long carbon backbone that is highly functionalized by oxygen, as we say "super-carbon-chain compounds (SCCs)", are some of the most unusual compounds. In our quest for SCCs from marine dinoflagellates, we isolated a novel spiroketal compound, symbiospirol A, from Symbiodinium sp. that was the same strain which produced symbiodinolide. Its planar structure and partial relative stereochemistries were elucidated based on 1D and 2D-NMR and degradation reaction. Symbiospirol A was shown to inhibit PKC activation induced by phosphatidylserine. Symbiodinolide is the typical SCC with a molecular weight of 2,860, which we isolated from the dinoflagellate Symbiodinium sp. The chemical degradation reaction using the 2nd-generation Hoveyda-Grubbs catalyst was employed for the stereochemical assignment at 62-membered macrolactone of symbiodinolide. The absolute configurations at several chiral centers were determined by spectroscopic analysis of MTPA diesters. Further chemical degradation studies using the 2nd-generation Grubbs' catalyst have performed new reaction. In addition, the complete stereochemical assignment of symbiodinolide is in progress. In our continuing search for novel bioactive metabolites from symbiotic marine dinoflagellates, we found that the water layer from the symbiotic dinoflagellate with jellyfish Mastigias papua displayed considerable activity against the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC). Further purification of the water layer guided by the bioassay and mass measurement led to the isolation of an unknown bioactive SCC, which we named symbiopolyol, possessing the molecular weight of 1,220. Studies on the isolation, structure elucidation, and biological activities of symbiopolyol will be described.

DOI： 10.24496/tennenyuki.50.0_35

CiNii Books

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

The structure of zamamistatin, an antibacterial dibromotyrosine derivative, has been revised. Its structure was not exo- or endo-type dimer 1 or 2 which were described previously, but was identical to a dibromophenylpyruvic acid derivative, aeroplysinin-1 (3). © 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetlet.2008.06.125

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Scopus

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  

DOI： 10.24496/tennenyuki.50.0_35

CiNii Research

記述言語：日本語  

J-GLOBAL

記述言語：日本語   出版者・発行元：日本癌学会  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

J-GLOBAL

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：日本語   出版者・発行元：天然有機化合物討論会  

Transforming growth factor-β (TGF-β) facilitates tumor growith and metastasis in advanced cancer. Discovery of novel TGF-β signaling inhibitors may open a new strategy for treatment of cancer patients. However, few clinically-promising drugs that block the TGF-β signaling pathway have been reported. Therefore, we are investigating novel TGF-β signaling inhibitors from natural sources, including microbial metabolites. For the screening assay, we established a luciferase reporter cell line from mink lung epithelial cells, Mv1Lu, to monitor the TGF-β-induced signal. Among over 2000 microbial extracts screened, an extract from an actinomycete Streptomyces sp. was found to inhibit the luciferase activity without cytotoxicity. Three litters of the culture broth were extracted with n-BuOH, filtered and concentrated. An active substance, named tryptopeptin A, was obtained by using silica gel chromatography followed by reverse-phase HPLC, as a colorless solid (3.6mg, 0.023%). Detailed MS and NME spectroscopic analyses revealed that tryptopeptin A was a novel acylated tripeptede, somprising a isovaleric acid, an N-methylvaline, a threonine and a C-terminally modified tryptophan in which carboxylic acid was replaced with an α,β-epoxyketone gropu. The absolute stereochemistries of tryptopeptin A were determined by Marfey's method and a synthetic approach: the physicochemical properties of the synthesized compound were idintical with those of the natural product. Tryptopeptin A potently inhibited TGF-β-dependent luciferase activity without cytotoxicity, suggesting the presence of specific cellular target molecule(s). Structure-activity relationships and modes of action of tryptopeptin A will also be discussed.

DOI： 10.24496/tennenyuki.52.0_397

CiNii Books

J-GLOBAL

記述言語：日本語  

J-GLOBAL