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.

Every year, seafood waste produced globally contains about 10 million tons of wasted crab, shrimp and lobster shells, which are rich in chitin resources. The exploitation and utilization of chitin resources are of great significance to environmental protection, economic development and sustainable development. Lytic polysaccharide monooxygenases (LPMOs) can catalyze polysaccharides by oxidative breakage of glycosidic bonds and have catalytic activity for chitin and cellulose, so they play an important role in the transformation of refractory polysaccharides into biomass. Although there have been many studies related to LPMOs, the research related to lytic chitin monooxygenases (LCMs) is still very limited. The specific catalytic mechanism of LCMs has not been fully elucidated, which poses a challenge to their application in industrial biomass conversion. This review introduces the present situation of resource development and utilization in chitin, the origin and classification of different LCMs families, the structural characteristics of LCMs and the relationship between structure and function. The research results related to activity detection, screening, preparation and transformation of LCMs were summarized and discussed. Finally, the synergistic effect of LCMs and chitin enzyme on biomass degradation was reviewed, and the existing problems and future research directions were pointed out. This is the first review focusing on Chitin-Active LPMOs in recent years, intending to provide a reference for applying chitin degradation enzymes system in the industry.