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Background  Androstenedione (AD) is an important steroid medicine intermediate that is obtained via the degradation of phytosterols by mycobacteria. The production process of AD is mainly the degradation of the phytosterol aliphatic side chain, which is accompanied by the production of propionyl CoA. Excessive accumulation of intracellular propionyl-CoA produces a toxic effect in mycobacteria, which restricts the improvement of production efficiency. The 2-methylcitrate cycle pathway (MCC) plays a significant role in the detoxification of propionyl-CoA in bacterial. The effect of the MCC on phytosterol biotransformation in mycobacteria has not been elucidated in detail. Meanwhile, reducing fermentation cost has always been an important issue to be solved in the optimizing of the bioprocess.    Results  There is a complete MCC in  Mycobacterium neoaurum  (MNR),  prpC ,  prpD  and  prpB  in the  prp  operon encode methylcitrate synthase, methylcitrate dehydratase and methylisocitrate lyase involved in MCC, and PrpR is a specific transcriptional activator of  prp  operon. After the overexpression of  prpDCB  and  prpR  in MNR, the significantly improved transcription levels of  prpC ,  prpD  and  prpB  were observed. The highest conversion ratios of AD obtained by MNR- prpDBC  and MNR- prpR  increased from 72.3 ± 2.5% to 82.2 ± 2.2% and 90.6 ± 2.6%, respectively. Through enhanced the PrpR of MNR, the in intracellular propionyl-CoA levels decreased by 43 ± 3%, and the cell viability improved by 22 ± 1% compared to MNR at 96 h. The nitrogen transcription regulator GlnR repressed  prp  operon transcription in a nitrogen-limited medium. The  glnR  deletion enhanced the transcription level of  prpDBC  and the biotransformation ability of MNR. MNR- prpR /Δ glnR  was constructed by the overexpression of  prpR  in the  glnR -deleted strain showed adaptability to low nitrogen. The highest AD conversion ratio by MNR- prpR /Δ glnR  was 92.8 ± 2.7% at low nitrogen level, which was 1.4 times higher than that of MNR.    Conclusion  Improvement in phytosterol biotransformation after the enhancement of propionyl-CoA metabolism through the combined modifications of the  prp  operon and  glnR  of mycobacteria was investigated for the first time. The overexpress of  prpR  in MNR can increase the transcription of essential genes ( prpC, prpD and prpB ) of MCC, reduce the intracellular propionyl-CoA level and improve bacterial viability. The knockout of  glnR  can enhance the adaptability of MNR to the nitrogen source. In the MNR ΔglnR  strain, overexpress of  prpR  can achieve efficient production of AD at low nitrogen levels, thus reducing the production cost. This strategy provides a reference for the economic and effective production of other valuable steroid metabolites from phytosterol in the pharmaceutical industry.

microbial cell factories

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum