Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea

Erythromycins produced by Saccharopolyspora erythraea have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.

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

ABSTRACTBranched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis inSaccharopolyspora erythraea. In this study, we found that thebkdoperon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of thebkdoperon was upregulated 5-fold in apccDgene deletion strain (ΔpccDstrain) and decreased 3-fold in apccDoverexpression strain (WT/pIB-pccD), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion ofpccDsignificantly improved the ΔpccDstrain's growth rate, whereaspccDoverexpression repressed WT/pIB-pccDgrowth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion ofgdhA1and the BcdhE1 gene (genes in thebkdoperon) resulted in lower growth rates of ΔgdhA1and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that thebkdoperon is involved in BCAA degradation. Bothbkdoverexpression (WT/pIB-bkd) andpccDinactivation (ΔpccDstrain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB-pccDwas decreased by 48%. Lastly, we explored the applications of engineeringpccDandbkdin an industrial high-erythromycin-producing strain.pccDdeletion in industrial strainS. erythraeaE3 (E3pccD) improved erythromycin production by 20%, and the overexpression ofbkdin E3ΔpccD(E3ΔpccD/pIB-bkd) increased erythromycin production by 39% compared withS. erythraeaE3 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 strainS. erythraeaE3.IMPORTANCEWe describe abkdoperon involved in BCAA degradation inS. 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.