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Collaborative Biosynthesis of Maleimide- and Succinimide-Containing Natural Products by Fungal Polyketide Megasynthases.

ABSTRACT: Fungal polyketide synthases (PKSs) can function collaboratively to synthesize natural products of significant structural diversity. Here we present a new mode of collaboration between a highly reducing PKS (HRPKS) and a PKS-nonribosomal peptide synthetase (PKS-NRPS) in the synthesis of oxaleimides from the Penicillium species. The HRPKS is recruited in the synthesis of an olefin-containing free amino acid, which is activated and incorporated by the adenylation domain of the PKS-NRPS. The precisely positioned olefin from the unnatural amino acid is proposed to facilitate a scaffold rearrangement of the PKS-NRPS product to forge the maleimide and succinimide cores of oxaleimides.

SUBMITTER: Sato M

PROVIDER: S-EPMC5673468 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Collaborative Biosynthesis of Maleimide- and Succinimide-Containing Natural Products by Fungal Polyketide Megasynthases.

Sato Michio M Dander Jacob E JE Sato Chizuru C Hung Yiu-Sun YS Gao Shu-Shan SS Tang Man-Cheng MC Hang Leibniz L Winter Jaclyn M JM Garg Neil K NK Watanabe Kenji K Tang Yi Y

Journal of the American Chemical Society 20170405 15

Fungal polyketide synthases (PKSs) can function collaboratively to synthesize natural products of significant structural diversity. Here we present a new mode of collaboration between a highly reducing PKS (HRPKS) and a PKS-nonribosomal peptide synthetase (PKS-NRPS) in the synthesis of oxaleimides from the Penicillium species. The HRPKS is recruited in the synthesis of an olefin-containing free amino acid, which is activated and incorporated by the adenylation domain of the PKS-NRPS. The precise ...[more]

PMID: 28365998

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Project description:BackgroundPolyketides are one of the most important classes of secondary metabolites and usually make good drugs. Currently, heterologous production of fungal polyketides for developing a high potential industrial application system with high production capacity and pharmaceutical feasibility was still at its infancy. Pichia pastoris is a highly successful system for the high production of a variety of heterologous proteins. In this work, we aim to develop a P. pastoris based in vivo fungal polyketide production system for first time and evaluate its feasibility for future industrial application.ResultsA recombinant P. pastoris GS115-NpgA-ATX with Aspergillus nidulans phosphopantetheinyl transferase (PPtase) gene npgA and Aspergillus terrus 6-methylsalicylic acid (6-MSA) synthase (6-MSAS) gene atX was constructed. A specific compound was isolated and identified as 6-MSA by HPLC, LC-MS and NMR. Transcription of both genes were detected. In 5-L bioreactor, the GS115-NpgA-ATX grew well and produced 6-MSA quickly until reached a high value of 2.2 g/L by methanol induction for 20 hours. Thereafter, the cells turned to death ascribing to high concentration of antimicrobial 6-MSA. The distribution of 6-MSA changed that during early and late induction phase it existed more in supernatant while during intermediate stage it mainly located intracellular. Different from 6-MSA production strain, recombinant M. purpureus pksCT expression strains for citrinin intermediate production, no matter PksCT located in cytoplasm or in peroxisomes, did not produce any specific compound. However, both npgA and pksCT transcripted effectively in cells and western blot analysis proved the expression of PPtase. Then the PPTase was expressed and purified, marked by fluorescent probes, and reacted with purified ACP domain and its mutant ACPm of PksCT. Fluoresence was only observed in ACP but not ACPm, indicating that the PPTase worked well with ACP to make it bioactive holo-ACP. Thus, some other factors may affect polyketide synthesis that include activities of the individual catalytic domains and release of the product from the synthase of PksCT.ConclusionsAn efficient P. pastoris expression system of fungal polyketides was successfully constructed. It produced a high production of 6-MSA and holds potential for future industrial application of 6-MSA and other fungal polyketides.

Project description:The pool of fungal secondary metabolites can be extended by activating silent gene clusters of cultured strains or by using sensitive biological assays that detect metabolites missed by analytical methods. Alternatively, or in parallel with the first approach, one can increase the diversity of existing culture collections to improve the access to new natural products. This review focuses on the latter approach of screening previously uncultured fungi for chemodiversity. Both strategies have been practiced since the early days of fungal biodiscovery, yet relatively little has been done to overcome the challenge of cultivability of as-yet-uncultivated fungi. Whereas earlier cultivability studies using media formulations and biological assays to scrutinize fungal growth and associated factors were actively conducted, the application of modern omics methods remains limited to test how to culture the fungal dark matter and recalcitrant groups of described fungi. This review discusses the development of techniques to increase the cultivability of filamentous fungi that include culture media formulations and the utilization of known chemical growth factors, in situ culturing and current synthetic biology approaches that build upon knowledge from sequenced genomes. We list more than 100 growth factors, i.e., molecules, biological or physical factors that have been demonstrated to induce spore germination as well as tens of inducers of mycelial growth. We review culturing conditions that can be successfully manipulated for growth of fungi and visit recent information from omics methods to discuss the metabolic basis of cultivability. Earlier work has demonstrated the power of co-culturing fungi with their host, other microorganisms or their exudates to increase their cultivability. Co-culturing of two or more organisms is also a strategy used today for increasing cultivability. However, fungi possess an increased risk for cross-contaminations between isolates in existing in situ or microfluidics culturing devices. Technological improvements for culturing fungi are discussed in the review. We emphasize that improving the cultivability of fungi remains a relevant strategy in drug discovery and underline the importance of ecological and taxonomic knowledge in culture-dependent drug discovery. Combining traditional and omics techniques such as single cell or metagenome sequencing opens up a new era in the study of growth factors of hundreds of thousands of fungal species with high drug discovery potential.

Dataset Information

Collaborative Biosynthesis of Maleimide- and Succinimide-Containing Natural Products by Fungal Polyketide Megasynthases.

Publications

Collaborative Biosynthesis of Maleimide- and Succinimide-Containing Natural Products by Fungal Polyketide Megasynthases.

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