Engineering of a TetR family transcriptional regulator BkdR enhances heterologous spinosad production in B4 chassis.

UNLABELLED

Precursor supply plays a significant role in the production of secondary metabolites. In bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl- and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl- and propionyl-CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in B4, which binds directly to the promoter region of the neighboring operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between and . When was deleted, we found an enhanced supply of malonyl- and methylmalonyl-CoA precursors in B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into B4. When supplemented with propionate to provide propionyl-CoA, the novel -deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium.

IMPORTANCE

In this study, we describe a operon involved in short-chain acyl-CoA carboxylation in B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for chassis engineering to enhance heterologous polyketide yield.