Identification And Gene Modification Of Key Genes About 3-Ketosteroid △~1-Dehydrogenase And 3-Ketosteroid 9α-Hydroxylase Involved In Steroid Nucleus Degradation In Mycobacterium Neoaurum NwIB-01 And Strain Development Via Genetic Manipulation

Due to the significant physiological activity, steroid drugs have been widely used in clinic application as its second place following antibiotic drugs. In contrast to the chemical synthesis, biotransformation provides an alternative method in the production of steroid medicine intermediates and has been used extensively as a common and economical process in the pharmaceutical industry. Phytosterols can be catabolized to a series of steroidal derivatives by Mycobacterium. Among these,4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) and 9a-hydroxy-4-androstene-3,17-dione (9α-OH-AD) are the major products usually applied in the industry due to their essential value in producting various steroid medications. Present domestic research of steroid production strains is just limited to strain isolation and mutation screening, but not involved in the steroid degradation genes at the molecular level, which hinder the development of domestic steroid drug industry. In abroad, rapid progress in this research has been occured. With the development of molecular biology and in-depth studies on steroid degradation system, genetic engineering is surely to play an important role in the production of steroid drugs.We obtained and investigated the key enzyme genes involved in the steroid degradation and modified Mycobacterium neoaurum NwIB-01 at the genetic level in this study. Consequently, strain NwIB-01 exhibited powerful ability of cleaving the side chain specifically from soybean phytosterols to accumulate AD (4-androstene-3,17-dione) and ADD (1,4-androstadiene-3,17-dione). However, the difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. Due to the structural similarity, the AD and ADD mixture complicates their purification and decreases their yield, which impedes the further commercial application of strain NwIB-01. In this paper, we obtained some genes for key enzyme including 3-ketosteroidΔ1-dehydrogenase and 3-ketosteroid 9a-hydroxylase and this is the first report of the genes in Mycobacterium neoaurum. Moreover, We have tackled the problem via the gene disruption and augmentation of the key genes. The KsdDM augmentation mutant showed to be good 1,4-androstadiene-3,17-dione (ADD)-producing strains respectively. The detailed work was introduced as following:1. Strain screening and research on phenotypeStrain NwIB-01 was selected and identified as Mycobacterium neoaurum. It was showed that when cultured in 15 g/1 phytosterols, the yield of ADD reached 4.23 g/1 while accompanied by 1.76 g/1 AD in 96-h-old culture (the molar yield of AD+ADD is 57.8%). 2. Investigation of the key gene 3-ketosteroidΔ1-dehydrogenase (ksdDM)Complete open reading frame of 3-ketosteroidΔ1-dehydrogenase has been obtained from Mycobacterium neoaurum for the first time. The results show that the full length of ksdDM is 1701 bp with GTG as its starting codon, and the upstream sequence of GAAAGG is probably the ribosome binding site. Homology analysis revealed that ksdDM in NwIB-01 is 82% identical to the ksdD nucleotide sequence (Genbank CP000480) in Mycobacterium smegmatis str. MC2155 (NCBI highest homology). In addition, plasmids of pET series were used to the heterologous expression in E. coli BL21(DE3). The KsdDM activity was assayed by the spectrophotometrical method and the maximum activity of the intracellular soluble parts of KsdDM was 0.8 U/mg.3. Study of the key genes coding for 3-ketosteroid 9a-hydroxylase (kshA) and 3-ketosteroid 9α-hydroxylase reducase (kshB)Complete gene sequences of 3-ketosteroid 9a-hydroxylase (kshA) and 3-ketosteroid 9a-hydroxylase reducase (kshB) have been obtained respectively relying on design of degenerate primers and genome walking. The results show that the full length of kshA is 1188 bp with GTG as its start codon, and a 6 bp sequence GGGAGG acts for the corresponding ribosome biding sites. Homology analysis revealed that kshA in NwIB-01 is 85%identical to the 3-ketosteroid 9a-hydroxylase nucleotide sequence (ACV87349) in Mycobacterium smegmatis str. MC2155 (NCBI highest homology). Subsequently, the whole open reading frame of kshA has been cloned and heterologously expressed using vector pET-22b in E.coli and co-expression system of kshA and kshB through vector pET-22b has been finally achieved.4. Gene knock-out of ksdDMThe suicide plasmid of KsdD was constructed with the plasmids p2NIL and pGOAL19. At 96 h after adding phytosterols, the molar ratio of AD:ADD in products of strain NwIB-01 was 1:2.4, while the molar ratio reached 11.5:1 in products of mutant NwIB-02 (ksdDM knock-out mutant).5. Overexpression of ksdDM and construction of industrial strain producing ADD.Mycobacterial replicating vectors pMV261 and pMV306 were used to over-express ksdDM in NwIB-01 and the mutant NwIB-04 by overexpression ksdDM was constructed. For strain NwIB-04, ADD reached the maximum 4.94 g/1 at 96 h of culture, while AD showed no obvious accumulation during the whole time course and only 0.096 g/1 at 96 h. The molar ratio of AD:ADD is 1:51.5 in products of mutant NwIB-04, and the final purity of ADD exceeded 95%.6. Preliminary research on the steroid degradation gene clusterThe genomic fosmid library was built by using plasmid pCC2FOS and the steroid degradation gene cluster was selected using degenerate primers. And about 81 kb fragment of the steroid degradation gene cluster was obtained. Furthermore, some key enzymes involved in steroid degradation have been discerned in this research.In short, this is the first report of the KsdD and Ksh genes in Mycobacterium neoaurum and the first report of gene-augmentation of a sterol catabolic enzyme to construct a sterol pathway intermediate (ADD)-producing strain. This study provides a feasible way to achieve excellent phytosterol-transforming strains with high product purity.