Precursor-directed biosynthesis of novel triketide lactones

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Fermentation Technology

Background:

• Triketide lactones (R-TKLs) are valuable natural products with diverse biological activities.
• Current production methods often face challenges with low yields and product instability.
• Precursor-directed biosynthesis offers a promising strategy for tailoring natural product synthesis.

Purpose of the Study:

• To engineer Streptomyces strains for enhanced production of R-TKLs using precursor-directed biosynthesis.
• To investigate the impact of different engineered DEBS1 variants and synthetic precursors on R-TKL yield.
• To optimize fermentation conditions, including pH and precursor concentration, for maximum R-TKL production and stability.

Main Methods:

• Genetic engineering of Streptomyces strains to express modified 6-deoxyerythronolide B synthase (DEBS1) fused to thioesterase (TE).
• Fermentation of engineered strains with various synthetic precursors to assess R-TKL production.
• Implementation of a two-phase fermentation strategy with a pH shift to improve product stability.
• Optimization of precursor concentration and fermentation parameters.

Main Results:

• An S. coelicolor strain expressing a partial DEBS1 (M2+TE) showed superior R-TKL production.
• The R group on the precursor significantly influenced R-TKL titer, with propyl being most effective.
• A two-phase fermentation (pH 6.5 then 5.5) doubled peak titers and stabilized the R-TKLs.
• Optimized precursor concentration yielded a maximum titer of 500 mg/L for 5-chloromethyl-TKL.

Conclusions:

• Precursor-directed biosynthesis is a viable strategy for producing diverse R-TKLs.
• Strain engineering and fermentation optimization are critical for achieving high yields and stability.
• The developed method provides a robust platform for scalable R-TKL production.