[Background]Mycobacterium sp. LY-1 has become a dominant strain in industrial production because of its ability to metabolize natural phytosterols into important steroid drug intermediates. CRISPR/Cas9 as an efficient gene editing technology is the key to improving the yield and traits of industrial strains through metabolic engineering. However, due to the toxicity resulted from the high expression of Cas9 and the few available expression elements that have been reported in Mycobacterium, the moderate expression of Cas9 protein in Mycobacterium is greatly limited. [Objective] The endogenous expression elements were selected to activate the expression and reduce the toxicity of Cas9. [Methods] We used the online Berkeley Drosophila Genome Project (BDGP) to predict the endogenous expression elements from the transcriptome data of Mycobacterium genes in literature and available studies. The intensity of each expression element was assessed with enhanced green fluorescent protein as the reporter, and the expression of Cas9 protein was initiated with the expression elements of different intensities. [Results] Twenty-three expression elements with different expression intensities were obtained. The medium and weak expression elements reduced the toxicity of Cas9 to Mycobacterium sp. LY-1 and realized the moderate expression of Cas9 in the strain. [Conclusion] The endogenous expression element library of Mycobacterium sp. LY-1 was established, which laid a good foundation for the subsequent construction of CRISPR/Cas9 tools and the expression regulation of key enzymes in Mycobacterium.
