PccD Regulates Branched-Chain Amino Acid Degradation and Exerts a Negative Effect on Erythromycin Production in Saccharopolyspora erythraea

Abstract

ABSTRACT Branched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis in Saccharopolyspora erythraea . In this study, we found that the bkd operon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of the bkd operon was upregulated 5-fold in a pccD gene deletion strain (Δ pccD strain) and decreased 3-fold in a pccD overexpression strain (WT/pIB- pccD ), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion of pccD significantly improved the Δ pccD strain's growth rate, whereas pccD overexpression repressed WT/pIB- pccD growth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion of gdhA1 and the BcdhE1 gene (genes in the bkd operon) resulted in lower growth rates of Δ gdhA1 and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that the bkd operon is involved in BCAA degradation. Both bkd overexpression (WT/pIB- bkd ) and pccD inactivation (Δ pccD strain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB- pccD was decreased by 48%. Lastly, we explored the applications of engineering pccD and bkd in an industrial high-erythromycin-producing strain. pccD deletion in industrial strain S. erythraea E3 (E3 pccD ) improved erythromycin production by 20%, and the overexpression of bkd in E3Δ pccD (E3Δ pccD /pIB- bkd ) increased erythromycin production by 39% compared with S. erythraea E3 in an industrial fermentation medium. Addition of 30 mM valine to industrial fermentation medium further improved the erythromycin production by 23%, a 72% increase from the initial strain S. erythraea E3. IMPORTANCE We describe a bkd operon involved in BCAA degradation in S. erythraea . The genes of the operon are repressed by a TetR regulator, PccD. The results demonstrated that PccD controlled the supply of precursors for biosynthesis of erythromycin via regulating the BCAA degradation and propionyl-CoA assimilation and exerted a negative effect on erythromycin production. The findings reveal a regulatory mechanism in feeder pathways and provide new strategies for designing metabolic engineering to increase erythromycin yield.