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Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis.

ABSTRACT: Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P1 are a class of bacteriostatic agents, which have drawn renewed attention because they have no cross-resistance to commonly used antibiotics. However, their biosynthesis is poorly understood. Here, we perform a stepwise introduction of the nine genes from the proposed gene cluster for helvolic acid into Aspergillus oryzae NSAR1, which enables us to isolate helvolic acid (~20?mg?L-1) and its 21 derivatives. Anti-Staphylococcus aureus assay reveals that the antibacterial activity of three intermediates is even stronger than that of helvolic acid. Notably, we observe an unusual C-4 demethylation process mediated by a promiscuous short-chain dehydrogenase/reductase (HelC) and a cytochrome P450 enzyme (HelB1), which is distinct from the common sterol biosynthesis. These studies have set the stage for using biosynthetic approaches to expand chemical diversity of fusidane-type antibiotics.

SUBMITTER: Lv JM

PROVIDER: S-EPMC5696383 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis.

Lv Jian-Ming JM Hu Dan D Gao Hao H Kushiro Tetsuo T Awakawa Takayoshi T Chen Guo-Dong GD Wang Chuan-Xi CX Abe Ikuro I Yao Xin-Sheng XS

Nature communications 20171121 1

Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P<sub>1</sub> are a class of bacteriostatic agents, which have drawn renewed attention because they have no cross-resistance to commonly used antibiotics. However, their biosynthesis is poorly understood. Here, we perform a stepwise introduction of the nine genes from the proposed gene cluster for helvolic acid into Aspergillus oryzae NSAR1, which enables us to isolate helvolic acid (~20 mg L<sup>-1</sup>) and ...[more]

PMID: 29158519

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Project description:Steryl esters (SEs) serve as a storage pool of sterols that helps to maintain proper levels of free sterols (FSs) in cell membranes throughout plant growth and development, and participates in the recycling of FSs and fatty acids released from cell membranes in aging tissues. SEs are synthesized by sterol acyltransferases, a family of enzymes that catalyze the transfer of fatty acil groups to the hydroxyl group at C-3 position of the sterol backbone. Sterol acyltransferases are categorized into acyl-CoA:sterol acyltransferases (ASAT) and phospholipid:sterol acyltransferases (PSAT) depending on whether the fatty acyl donor substrate is a long-chain acyl-CoA or a phospolipid. Until now, only Arabidopsis ASAT and PSAT enzymes (AtASAT1 and AtPSAT1) have been cloned and characterized in plants. Here we report the identification, cloning, and functional characterization of the tomato (Solanum lycopersicum cv. Micro-Tom) orthologs. SlPSAT1 and SlASAT1 were able to restore SE to wild type levels in the Arabidopsis psat1-2 and asat1-1 knock-out mutants, respectively. Expression of SlPSAT1 in the psat1-2 background also prevented the toxicity caused by an external supply of mevalonate and the early senescence phenotype observed in detached leaves of this mutant, whereas expression of SlASAT1 in the asat1-1 mutant revealed a clear substrate preference of the tomato enzyme for the sterol precursors cycloartenol and 24-methylene cycloartanol. Subcellular localization studies using fluorescently tagged SlPSAT1 and SlASAT1 proteins revealed that SlPSAT1 localize in cytoplasmic lipid droplets (LDs) while, in contrast to the endoplasmic reticulum (ER) localization of AtASAT1, SlASAT1 resides in the plasma membrane (PM). The possibility that PM-localized SlASAT1 may act catalytically in trans on their sterol substrates, which are presumably embedded in the ER membrane, is discussed. The widespread expression of SlPSAT1 and SlASAT1 genes in different tomato organs together with their moderate transcriptional response to several stresses suggests a dual role of SlPSAT1 and SlASAT1 in tomato plant and fruit development and the adaptive responses to stress. Overall, this study contributes to enlarge the current knowledge on plant sterol acyltransferases and set the basis for further studies aimed at understanding the role of SE metabolism in tomato plant growth and development.

Project description:BackgroundSheath rot disease caused by Sarocladium oryzae is an emerging threat for rice cultivation at global level. However, limited information with respect to genomic resources and pathogenesis is a major setback to develop disease management strategies. Considering this fact, we sequenced the whole genome of highly virulent Sarocladium oryzae field isolate, Saro-13 with 82x sequence depth.ResultsThe genome size of S. oryzae was 32.78 Mb with contig N50 18.07 Kb and 10526 protein coding genes. The functional annotation of protein coding genes revealed that S. oryzae genome has evolved with many expanded gene families of major super family, proteinases, zinc finger proteins, sugar transporters, dehydrogenases/reductases, cytochrome P450, WD domain G-beta repeat and FAD-binding proteins. Gene orthology analysis showed that around 79.80 % of S. oryzae genes were orthologous to other Ascomycetes fungi. The polyketide synthase dehydratase, ATP-binding cassette (ABC) transporters, amine oxidases, and aldehyde dehydrogenase family proteins were duplicated in larger proportion specifying the adaptive gene duplications to varying environmental conditions. Thirty-nine secondary metabolite gene clusters encoded for polyketide synthases, nonribosomal peptide synthase, and terpene cyclases. Protein homology based analysis indicated that nine putative candidate genes were found to be involved in helvolic acid biosynthesis pathway. The genes were arranged in cluster and structural organization of gene cluster was similar to helvolic acid biosynthesis cluster in Metarhizium anisophilae. Around 9.37 % of S. oryzae genes were identified as pathogenicity genes, which are experimentally proven in other phytopathogenic fungi and enlisted in pathogen-host interaction database. In addition, we also report 13212 simple sequences repeats (SSRs) which can be deployed in pathogen identification and population dynamic studies in near future.ConclusionsLarge set of pathogenicity determinants and putative genes involved in helvolic acid and cerulenin biosynthesis will have broader implications with respect to Sarocladium disease biology. This is the first genome sequencing report globally and the genomic resources developed from this study will have wider impact worldwide to understand Rice-Sarocladium interaction.

Dataset Information

Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis.

Publications

Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis.

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