Soil bacteria from the actinomycetes called anthracyclinones (ACs), some of which have powerful therapeutic activity. ACs have complicated structures that are produced by enzymes found in the bacteria. This project will “mix-and-match” these enzymes to make novel ACs expected to have antibiotic and anticancer activity. The overall goal fo the project is to develop the BIOPOLYMER® BIOBricks POLYketide Metabolic EngineeRing platform In this work, we engineered early stage enzymes composing the “minimal polyketide synthase” (minPKS) from three different pathways: (1) the C-21 doxorubicin pathway (dpsABGCD genes) and the C-21 aclacinomycin pathway (aknBCDE2F genes) (2) the C-20 nogalamycin pathway (snoa123), and (3) the C-19N oxytetracycline pathway (oxyABCD). We expressed the minPKS enzymes from these three pathways in actinomycete “superhosts” that are genetically engineered for heterologous expression of biosynthetic pathways: S. lividans K4-114, Streptomyces coelicolor M1146, Streptomyces coelicolor M1152, and Streptomyces coelicolor M1154. In this work, we expressed both wildtype and codon-optimized versions of the constructs under the control of different promoters to compare the impact of codon adaptation on aromatic polyketide production titer. We discovered that S. coelicolor M1154 was the best chassis for production of aromatic polyketides and that expression of minPKS operons from the kasOp* promoter lead to significant polyketide accumulation in shake flasks. We also discovered that utilization of the native translational coupling between the dimeric KS subunits, and utilization of the wildtype codon preference, were significant for reconstitution of the minPKS enzyme complexes in vivo. These efforts resulted in S. coelicolor M1154 strains that produced greater than 120 mg/L SEK15 in shake flask cultures.

Biopolymer: Biobricks polyketide metabolic enginering of minimal polyketide synthases in streptomyces coelicolor

ACS Fall 2021

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