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/ΔglnR 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/ΔglnR 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 MNRΔglnR 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.

中文翻译：

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通过使用新分枝杆菌的转录调节因子PrpR和GlnR增强柠檬酸甲酯循环来提高低氮水平下植物甾醇的生物转化。

背景技术雄烯二酮（AD）是重要的类固醇药物中间体，其通过分枝杆菌降解植物甾醇而获得。AD的生产过程主要是植物甾醇脂族侧链的降解，同时伴随着丙酰基CoA的生产。细胞内丙酰辅酶A的过度积累在分枝杆菌中产生毒性作用，这限制了生产效率的提高。2-甲基柠檬酸循环途径（MCC）在细菌中丙酰辅酶A的解毒中起重要作用。MCC对分枝杆菌中植物甾醇生物转化的影响尚未详细阐明。同时，降低发酵成本一直是生物工艺优化中需要解决的重要问题。结果在新分枝杆菌（MNR）中有完整的MCC，prp操纵子中的prpC，prpD和prpB编码参与MCC的柠檬酸合酶，柠檬酸脱水酶和甲基异柠檬酸裂合酶，而PrpR是prp操纵子的特定转录激活因子。在MNR中prpDCB和prpR过表达后，观察到prpC，prpD和prpB的转录水平显着提高。由MNR-prpDBC和MNR-prpR获得的AD的最高转化率分别从72.3±2.5％增加到82.2±2.2％和90.6±2.6％。通过增强MNR的PrpR，与96小时的MNR相比，细胞内丙酰辅酶A的细胞内水平降低了43±3％，细胞活力提高了22±1％。氮转录调节剂GlnR在氮限制的培养基中抑制prp操纵子转录。glnR缺失增强了prpDBC的转录水平和MNR的生物转化能力。MNR-prpR /ΔglnR是通过在glnR缺失菌株中prpR的过表达而构建的，显示出对低氮的适应性。在低氮水平下，MNR-prpR /ΔglnR的最高AD转化率为92.8±2.7％，是MNR的1.4倍。结论首次研究了通过对分枝杆菌的prp操纵子和glnR进行联合修饰来提高丙酰辅酶A代谢后植物甾醇生物转化的改善。MNR中prpR的过表达可以增加MCC必需基因（prpC，prpD和prpB）的转录，降低细胞内丙酰辅酶A的水平并提高细菌的生存能力。glnR的敲除可以增强MNR对氮源的适应性。在MNRΔglnR菌株中，prpR的过表达可以在低氮水平下实现AD的高效生产，从而降低生产成本。该策略为制药行业从植物甾醇经济有效地生产其他有价值的甾族代谢物提供了参考。