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

Abstract

Branched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis in In this study, we found that the operon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of the operon was upregulated 5-fold in a gene deletion strain (Δ strain) and decreased 3-fold in a overexpression strain (WT/pIB-), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion of significantly improved the Δ strain's growth rate, whereas overexpression repressed WT/pIB- growth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion of and the BcdhE1 gene (genes in the operon) resulted in lower growth rates of Δ and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that the operon is involved in BCAA degradation. Both overexpression (WT/pIB-) and inactivation (Δ strain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB- was decreased by 48%. Lastly, we explored the applications of engineering and in an industrial high-erythromycin-producing strain. deletion in industrial strain E3 (E3) improved erythromycin production by 20%, and the overexpression of in E3Δ (E3Δ/pIB-) increased erythromycin production by 39% compared with 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 E3. We describe a operon involved in BCAA degradation in 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.

A common approach for absolute quantification of short chain CoA thioesters in prokaryotic and eukaryotic microbes

“…From our data, we conclude that methylmalonyl-CoA is formed from succinyl-CoA by methylmalonyl-CoA mutase, eventually as response to TCA cycle activity. Propionyl-CoA, the potentially alternative source for methylmalonyl-CoA via propionyl-CoA carboxylase was proven absent so that this route can be excluded, matching with the fact that propionyl-CoA typically occurs as catabolic intermediate during the degradation of odd-chain fatty acids [38,39] and branched-chain amino acids [40], not present here.…”

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

“…A similar phenomenon was also found in Sac. erythraea , in which four of the hitherto reported TFRs played the negative role in regulation of erythromycin biosynthesis ( Wu et al, 2014b , 2016 , 2019 ; Xu et al, 2018 ). Only the TFR, SACE_7301, was shown to trigger the transcription of eryAI and ermE by interacting with their promoter regions for positively regulating the erythromycin production ( Wu et al, 2014a ).…”

“…Next, several types of TFs were subsequently reported to be involved in erythromycin production in Sac. erythraea , mainly including the families of TetR and Lrp as well as nutrient-sensing regulators ( Wu et al, 2014a , b , 2016 , 2019 ; Liu et al, 2017 , 2019 ; Xu et al, 2018 , 2019b ). This uncovered the complicated mechanism for controlling erythromycin biosynthesis with reciprocal (interactive regulation) or cascaded (hierarchical regulation) modes, which enable us to understand and manipulate the regulatory network governing erythromycin biosynthesis for titer improvement.…”

“…A total of 97 TFRs were encoded by the Sac. erythraea genome ( Wu et al, 2016 ), and only five of them (SACE_3986, SACE_7301, SACE_3446, PccD, and SACE_5754) have been successively reported to control the biosynthesis of erythromycin so far ( Wu et al, 2014a , b , 2016 , 2019 ; Xu et al, 2018 ). These investigations show varied molecular mechanisms for regulating erythromycin production (negatively or positively) or the ery cluster (directly or indirectly).…”

The Expression of NOX From Synthetic Promoters Reveals an Important Role of the Redox Status in Regulating Secondary Metabolism of Saccharopolyspora erythraea

“…Via carboxylation or the TCA cycle, acetyl-CoA is converted to malonyl-CoA or (2S)-methylmalonyl-CoA, which is the necessary precursor for the biosynthesis of the reddish pigment and erythromycin, respectively (Li et al, 2020). Furthermore, the TCA cycle is the main source of (2S)-methylmalonyl-CoA in S. erythraea (Hong et al, 2016;Xu et al, 2018). The biosynthesis of erythromycin generates twice as much NADH compared to pigment production (Li et al, 2020).…”