Elucidating the mechanism of fluorinated extender unit loading for improved production of fluorine-containing polyketides

Omer Ad, Benjamin W. Thuronyi, Michelle C.Y. Chang

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

Polyketides are a large family of bioactive natural products synthesized by polyketide synthase (PKS) enzyme complexes predominantly from acetate and propionate. Given the structural diversity of compounds produced using these two simple building blocks, there has been longstanding interest in engineering the incorporation of alternative extender units. We have been investigating the mechanism of fluorinated monomer insertion by three of the six different modules of the PKS involved in erythromycin biosynthesis (6-deoxyerythronolide B synthase, DEBS) to begin understanding the contribution of different steps, such as enzyme acylation, transacylation, C-C bond formation, and chain transfer, to the overall selectivity and efficiency of this process. In these studies, we observe that inactivation of a cis-acyltransferase (AT) domain to circumvent its native extender unit preference leads concurrently to a change of mechanism in which chain extension with fluorine-substituted extender units switches largely to an acyl carrier protein (ACP)-independent mode. This result suggests that the covalent linkage between the growing polyketide chain and the enzyme is lost in these cases, which would limit efficient chain elongation after insertion of a fluorinated monomer. However, use of a standalone trans-acting AT to complement modules with catalytically deficient AT domains leads to enzyme acylation with the fluoromalonyl-CoA extender unit. Formation of the canonical ACP-linked intermediate with fluoromalonyl-CoA allows insertion of fluorinated extender units at 43% of the yield of the wild-type system while also amplifying product yield in single chain-extension experiments and enabling multiple chain extensions to form multiply fluorinated products.

Original languageEnglish (US)
Pages (from-to)E660-E668
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number5
DOIs
StatePublished - Jan 31 2017
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Fluorine
  • Natural products
  • Polyketide synthase
  • Synthetic biology

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