Construction Of An Efficient Expression Vector For Mycobacterium Neoaurum And Its Application On The Synthesis Of Androst-1,4-diene-3,17-dione

4-androstene-3,17-dione(AD) and androst-1,4-diene-3,17-dione(ADD) are important intermediates for synthesis of steroid drugs. The synthesis of AD and ADD is mainly by biotransformation of sterols. The cholesterol oxidase(Cho M) and 3-ketosteroid-△1-dehydrogenase(KSDD) are the key emzymes of microbial sterol metabolism in Mycobacterium neoaurum. Previously, strain Mycobacterium neoaurum JC-12, capable of transforming phytosterols to AD and ADD, was isolated from soil and preserved in our lab. The Cho M and KSDD of M. neoaurum JC-12 have already been studied. Therefore, based on the previous work, Cho M and KSDD were co-expressed to improve the ADD production in this work.(1) Plasmid p MF41 was used to overexpress Cho M(Cho M1 and Cho M2) and KSDD in M. neoaurum JC-12. Cho M2 is the main cholesterol oxidase in Mycobacterium neoaurum JC-12. In shake flask fermentation, the AD and ADD production of recombinant M. neoaurum JC-12/p MF41-cho M2 were 1.17 g/L and 4.98 g/L, increased 27.2% and 2.5% compared to the original strain, respectively. The AD and ADD production of recombinant M. neoaurum JC-12/p MF41-ksdd were 0.59 g/L(decreased 35.9%) 5.27 g/L(increased 8.3%).(2) To construct p MTac, p ACE promoter on p MF41 was replaced by tac promoter, and then four recombinants were constructed for verifying the function of p MTac, which were M. neoaurum JC-12/p MF41-gfp, M. neoaurum JC-12/p MTac-gfp, M. neoaurum JC-12/p MF41-ksdd and M. neoaurum JC-12/p MTac-ksdd. Fluorescence detection results showed that much more green fluorescent protein(GFP) was expressed in M. neoaurum JC-12/p MTac-gfp than M. neoaurum JC-12/p MF41-gfp. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/p MTac-ksdd, which was 6.53-fold compared to M. neoaurum JC-12. Optimization of the promotor of p MTac by substitution of its-10 region sequence of tac promoter was carried out, and then p MTac-2 was selected for an favorable candidate. The KSDD activity of M. neoaurum JC-12/p MTac-2-ksdd was 5.16 U/mg, which was 15.12-fold compared to the original strain. The whole-cell of M. neoaurum JC-12/p MTac-2-ksdd could transformed 1 g/L AD to ADD in a percent conversion of 82% in 14 h, which was 10.7-fold compared to M. neoaurum JC-12. Whereas the whole-cell of M. neoaurum JC-12/p MTac-2-cho M2 transformed 1 g/L cholesterol in a percent conversion of 56% in 24 h, which was 2.1-fold compared to M. neoaurum JC-12. In shake flasks, the AD and ADD production of recombinant M. neoaurum JC-12/ p MTac-2-cho M2 were 1.43 g/L and 5.18 g/L, increased 55.4% and 6.7% compared to the original strain, respectively; The AD production of recombinant M. neoaurum JC-12/ p MTac-2-ksdd was 0.12 g/L, decreased 86.9% compared to the original strain, the ADD production was 6.14 g/L, increased 26.3%.(3) For further improve ADD production, the Cho M2 and KSDD were coexpressed in M. neoaurum JC-12 by p MTac-2. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/p MTac-2-ksdd-cho M2 was improved to 10.68 g/L(increased 27.4%), AD production was 0.62 g/L(decreased 50%).