Enhancement of acyl-CoA precursor supply for increased avermectin B1a production by engineering meilingmycin polyketide synthase and key primary metabolic pathway genes.

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 μg/mL - 4.36-fold higher than WT value (48.9 μg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 μg/mL. Overexpression of fatty acid β-oxidation pathway genes in the strain further increased B1a titer to 452.8 μg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 μg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.