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Pass-back chain extension expands multimodular assembly line biosynthesis.

ABSTRACT: Modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymatic assembly lines are large and dynamic protein machines that generally effect a linear sequence of catalytic cycles. Here, we report the heterologous reconstitution and comprehensive characterization of two hybrid NRPS-PKS assembly lines that defy many standard rules of assembly line biosynthesis to generate a large combinatorial library of cyclic lipodepsipeptide protease inhibitors called thalassospiramides. We generate a series of precise domain-inactivating mutations in thalassospiramide assembly lines, and present evidence for an unprecedented biosynthetic model that invokes intermodule substrate activation and tailoring, module skipping and pass-back chain extension, whereby the ability to pass the growing chain back to a preceding module is flexible and substrate driven. Expanding bidirectional intermodule domain interactions could represent a viable mechanism for generating chemical diversity without increasing the size of biosynthetic assembly lines and challenges our understanding of the potential elasticity of multimodular megaenzymes.

SUBMITTER: Zhang JJ

PROVIDER: S-EPMC6917876 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Pass-back chain extension expands multimodular assembly line biosynthesis.

Zhang Jia Jia JJ Tang Xiaoyu X Tang Xiaoyu X Huan Tao T Ross Avena C AC Moore Bradley S BS

Nature chemical biology 20191021 1

Modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymatic assembly lines are large and dynamic protein machines that generally effect a linear sequence of catalytic cycles. Here, we report the heterologous reconstitution and comprehensive characterization of two hybrid NRPS-PKS assembly lines that defy many standard rules of assembly line biosynthesis to generate a large combinatorial library of cyclic lipodepsipeptide protease inhibitors called thalassospiramides. W ...[more]

PMID: 31636431

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Project description:BackgroundBetulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory.ResultsIn this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87?±?2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH- or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the ?-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89?±?11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures.ConclusionsHere, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.

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Pass-back chain extension expands multimodular assembly line biosynthesis.

Publications

Pass-back chain extension expands multimodular assembly line biosynthesis.

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