Widening the bottleneck: Heterologous expression, purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli

Bioorg Med Chem. 2020 Oct 15;28(20):115686. doi: 10.1016/j.bmc.2020.115686. Epub 2020 Aug 15.

Abstract

Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable clinical success, the biosynthetic prowess of these enzymes has been stymied by 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. To begin filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KS-CLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer (Kr). Using E. coli as a heterologous host, we obtained soluble proteins in titers signifying improvements over previous KS-CLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKS-CLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. These results lay important groundwork for optimizing the interaction between KrKS-CLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.

Keywords: Acyl carrier protein; Ketosynthase chain length factor; Polyketide biosynthesis; Type II polyketide.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Chloroflexi / enzymology*
  • Escherichia coli / metabolism*
  • Polyketide Synthases / biosynthesis*
  • Polyketide Synthases / isolation & purification
  • Polyketide Synthases / metabolism

Substances

  • Polyketide Synthases

Supplementary concepts

  • Ktedonobacter racemifer