Precursor-Directed Biosynthesis of Novel Triketide Lactones

Precursor‐directed biosynthesis was used to produce different triketide lactones (R‐TKLs) in a fermentation process. Plasmids expressing engineered versions of the first subunit of 6‐deoxyerythronolide B synthase (DEBS1) fused to the terminal DEBS thioesterase (TE) were introduced into three different Streptomyces strains. The DEBS1 protein fused to TE had either an inactivated ketosynthase domain (KS1°) or a partial DEBS1 lacking module 1 but containing module 2 (M2+TE). Different synthetic precursors were examined for their effect on R‐TKL production. An overproducing strain of S. coelicolor expressing the M2+TE protein was found to be best for production of R‐TKLs. Racemic precursors were as effective as enantiomerically pure precursors in the fermentation process. The R group on the precursor significantly affected titer (propyl ≫ chloromethyl > vinyl). The R‐TKLs were unstable in fermentation broth at pH 6–8. A two‐phase fermentation with a pH shift was implemented to stabilize the products. The fermentation pH initially was controlled at optimal values for cell growth (pH 6.5) and then shifted to 5.5 during production. This doubled peak titers and stabilized the product. Finally, the concentration of synthetic precursor in the fermentation was optimized to improve production. A maximum titer of 500 mg/L 5‐chloromethyl‐TKL was obtained using 3.5 g/L precursor.