Using chemobiosynthesis and synthetic mini-polyketide synthases to produce pharmaceutical intermediates in Escherichia coli

Abstract

Recombinant microbial whole-cell biocatalysis is a valuable approach for producing enantiomerically pure intermediates for the synthesis of complex molecules. Here, we describe a method to produce polyketide intermediates possessing multiple stereogenic centers by combining chemobiosynthesis and engineered mini-polyketide synthases (PKSs). Chemobiosynthesis allows the introduction of unnatural moieties, while a library of synthetic bimodular PKSs expressed from codon-optimized genes permits the introduction of a variety of ketide units. To validate the approach, intermediates for the synthesis of trans-9,10-dehydroepothilone D were generated. The designer molecules obtained have the potential to greatly reduce the manufacturing cost of epothilone analogues, thus facilitating their commercial development as therapeutic agents.

FIG. 1.

(A) Scheme for the synthesis of trans-9,10-dehydroepothilone D. (B) Production of tetraketide intermediate 3 by the double extension of SNAC 1 with two D-type modules, 4a by extension with a D-G bimodular PKS and 4b by extension with a D-H bimodular PKS. (C) Structures of two-carbon units added by the extension modules used in this work. D-type modules: eryM2, eryM5, eryM6, gldM3, sorM6, lepM10, rifM5, and epoM7; G-type modules: eryM3, rapM3, lepM4, and rapM6; H-type module: pikM6. ery, erythromycin; sor, soraphen; rap, rapamycin; gdm, geldanamycin; rif, rifamycin; lep, leptomycin.

FIG. 2.

Two classes of expression plasmid used to test bimodular interactions in E. coli. pAng vectors contain a CloDF13 replication origin, a streptomycin resistance selection marker, and a P_BAD promoter to drive the expression of LM-Module-LC^eryM2 ORFs. pBru vectors contain a ColE1 replication origin, a carbenicillin resistance selection marker, and a P_BAD promoter to drive the expression of LN^eryM3-Module-TE ORFs. Productive combinations of modules are revealed by the formation of triketide lactones (TKL).

FIG. 3.

Method used to produce polyketide intermediates. PKS modules capable of catalyzing the extension of the unnatural starter unit are identified by feeding the SNAC to bacteria expressing appropriate LN^ery3-Mod-TEs and analyzing for product. Modules active in the first SNAC extension then are reformatted as LN^ery5-Mod-LC^ery2 donor modules in pAngII vectors, and these are coexpressed with appropriate LN^ery3-Mod-TEs from pBru vectors to determine which bimodular combinations can perform two extensions of the SNAC.