Abstract:Mycobacterium neoaurum JC-12 is an important strain to transform phytosterolsto steroid hormones. Traditional gene editing methods are inefficient, long cycles, complicated to manipulate and resistance labels required. The successful application of CRISPR(clustered regular interspaced short palindromic repeats)/Cas9 system in many non-model strains provides a referencefor this study. A CRISPR-Cas system for genome editing of Mycobacterium neoaurum JC-12 was constructed by combining non-homologous end joining(NHEJ). Targeted sgRNAs were respectively designed for the knockout and transcriptional activation of anthrone-dehydrogenase(KSDD) synthesizing androst-1,4-diene-3,17-dione(ADD) by catalyzing 4-androstene-3,17-dione(AD)on the Mycobacterium neoaurum JC-12 genome. The results showed that the ksdd gene was successfully knocked out and the enzyme activity of KSDD decreased by 80%. The AD yield of the mutant strain after 96 h was 30% higher than that of the wild-type strains, and no ADD was produced. Furthermore, the transcriptional regulation results indicated that the transcriptional level of ksdd at -29 target site decreased by 38% compared to that of the control, while the transcriptional level of target-83 and-141 increased 2.24 and 3.23 times, respectively. However, the corresponding AD and ADD yields did not change significantly when the -29 site was activated. Notably, there was 26% and 25% increase in the yields of AD and ADD respectively at the -83 site, and 29.5% and 36% increase at the -141 site. This study provides amore extensive and convenient way to further implement genome level metabolic engineering in Mycobacterium neoaurum.
