Ke Liu,Yang Gao,Zhen-Hai Li,Min Liu,Feng-Qing Wang,Dong-Zhi Wei
Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum , including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.
Liu Kun,Wang Feng-Qing,Liu Ke,Zhao Yunqiu,Gao Bei,Tao Xinyi,Wei Dongzhi
Progesterone is one of the classical hormone drugs used in medicine for maintaining pregnancy. However, its manufacturing process, coupled with organic reagents and poisonous catalysts, causes irreversible environmental pollution. Recent advances in synthetic biology have demonstrated that the microbial biosynthesis of natural products, especially difficult-to-synthesize compounds, from building blocks is a promising strategy. Herein, overcoming the heterologous cytochrome P450 enzyme interdependency in Mycolicibacterium neoaurum successfully constructed the CYP11A1 running module to realize metabolic conversion from waste phytosterols to progesterone. Subsequently, progesterone yield was improved through strategies involving electron transfer and NADPH regeneration. Mutant CYP11A1 (mCYP11A1) and adrenodoxin reductase (ADR) were connected by a flexible linker (L) to form the chimera mCYP11A1-L-ADR to enhance electron transfer. The chimera mCYP11A1-L-ADR, adrenodoxin (ADX), and ADR-related homolog ARH1 were expressed in M. neoaurum, showed positive activity and produced 45 mg/L progesterone. This electron transfer strategy increased progesterone production by 3.95-fold compared with M. neoaurum expressing mCYP11A1, ADR, and ADX. Significantly, a novel inorganic–biological hybrid system was assembled by combining engineered M. neoaurum and InP nanoparticles to regenerate NADPH, which was increased 84-fold from the initial progesterone titer to 235 ± 50 mg/L. In summary, this work highlights the green and sustainable potential of obtaining synthetic progesterone from sterols in M. neoaurum.
危险品化学品经营许可证（带存储）