Engineering mycobacteria artificial promoters and ribosomal binding sites for enhanced sterol production

Abstract

Mycobacteria is important microbial cell factory in the steroid industry, and there is an urgent need to develop synthetic biology toolkit to regulate the expression level of genes in mycobacterial strains. In this study, we first evaluated the adaptation of seven promoters in Mycobacterium neoaurum ATCC 25795, including trp, tac, trc, and tic from Escherichia coli, pm2 from Bacillus subtilis, and hsp60, hsp70 from Mycobacterium. In order to obtain stronger promoters, 10 new artificial promoters were obtained by retaining the core regions with appropriate truncation, and by concatenating these promoters to increase the sigma factor recognition region. Secondly, we constructed a ribosomal binding sites (RBS) library (with 49 RBSs) based on the RBS Calculator-predicted and iGEM design, and verified their regulation on genes expression in Mycobacterium. Since AU-rich sequences increase mRNA stability and/or translation efficiency, we obtained 38 strong RBS and 11 weak RBS compared to the control (the RBS of the plasmid pMV261). Eventually, we chose a high-strength promoter (CP6) and a medium-strength RBS (R1) to drive the overexpression of the genes encoding 3-ketosteroid-9α-hydroxylase (kshA and kshB) in M. neoaurum, leading to a production titer of 9α-hydroxy-androst-4-ene-3,17-dione (9-OHAD) of 5.72 ± 0.05 g/L (with 13.0 g/L phytosterols as the substrates), a significant increase in comparison to the original strain (4.58 ± 0.02 g/L).