Effect of TetR Family Transcriptional Regulator PccD on Phytosterol Metabolism of Mycolicibacterium

Androstenedione (AD) is an important intermediate for the production of steroidal drugs. The process of transforming phytosterols into AD by <i>Mycolicibacterium</i> is mainly the degradation process of the phytosterol side chain, and the excessive accumulation of propionyl-CoA produced by <i>Mycobacterium</i> will produce toxic effects, which seriously restricts the transformation performance of strains. In this study, <i>Mycolicibacterium</i> sp. LZ2 (Msp) was used as the research object to study the transcription factor PccD of the TetR family, which has the role of propionyl-CoA metabolism regulation. By constructing overexpression and deletion strains of <i>pccD</i>, it was confirmed that <i>pccD</i> had an inhibitory effect on the transcription of propionyl-CoA carboxylase genes (<i>pccA</i> and <i>pccB</i>). Electrophoretic Mobility Shift Assay (EMSA) and DNase I footprint analysis demonstrated that PccD is directly involved in the transcriptional regulation of <i>pccA</i> and <i>pccB</i> and is a negative transcriptional regulator of the <i>pcc</i> operon. In the study of phytosterol transformation, the growth rate and bacterial viability of Msp-Δ<i>pccD</i> were higher than Msp, but the growth of Msp-<i>pccD</i> was inhibited. As a result of testing of intracellular propionyl-CoA levels and AD production yields, it was found that lower propionyl-CoA levels and higher AD production yields were observed in Msp-Δ<i>pccD</i>. The results expand the cognition of propionyl-CoA metabolism regulation and provide a theoretical basis and reference for the rational transformation of phytosterol transformation strains and secondary metabolite synthesis strains with propionyl-CoA as a substrate, which has important research significance.