Metabolic Engineering Of Microorganisms Producing Steroid Drug Intermediates ADD And TS

Due to its significant physiological activity, steroid drugs have been widely used in clinic application and become the second categories of drugs after antibiotic drugs. Androst-4-ene-3,17-dione（AD） and androst-1,4-diene-3,17-dione（ADD）, as the important steroid durg intermediates, could be synthesized to almost all the steroid drugs by chemical modification. Thus, they become more important in the pharmaceutical industry. However, there are many problems existed in the microbial steroid transformation, such as the low substrate concentration, the long bioconversion duration, the investigation on the microbial sterol metabolic mechanism is lack, etc. All these problems inhibited the development of microbial sterol transformation in China. Therefore, in this paper, the key enzymes in the sterol conversion process was targeted. We identified the key enzymes involved in the phytosterol transformation process to ADD in Mycobacterium neoaurum, including 3-ketosteroid-Δ1-dehydrogenase（KSDD）, cholesterol oxidase（ChoM）, steroid C27 monooxygenase（SMO） and 3-ketosteroid 9α-hydroxylase（KSH）, and the ADD production was increased dramatically by using metabolic engineering strategy. Besides, we expanded the kind of sterol metabolic intermediates. By using the synthetic biology, we realized the microbial synthesis of the important steroid drug testosterone. The main contents are as follows:1. M. neoaurum ST-095 and its mutant M. neoaurum JC-12, capable of transforming phytosterol to AD and ADD, with different molar ratios of ADD/AD, this difference was related to the enzyme activity of KSDD, which catalyzes the C1,2 dehydrogenation of AD to ADD specifically. Thus, we analyzed the primary structure of KSDDI（from M. neoaurum ST-095） and KSDDII（from M. neoaurum JC-12）. We found the difference was the mutation of V366 S, which increased the enzyme affinity and catalytic efficiency by their heterologous expression in Bacillus subtilis. The functional difference between KSDDI and KSDDII in phytosterol biotransformation was revealed by gene disruption and complementation. These results proved that the different ADD/AD molar ratios of these two M. neoaurum strains were due to the differences in KSDD. KSDD structure analysis revealed that the V366 S mutation could possibly play an important role in stabilizing the active center and enhancing the interaction of AD and KSDD.2. Expression of ChoM1 and ChoM2 genes from M. neoaurum JC-12 in B. subtilis was confirmed by SDS-PAGE and enzyme activity analysis. Enzyme properties showed that the optimum pH and temperature for both ChoM1 and ChoM2 was 7.5 and 37°C, and both enzymes were stimulated by Mg2+ and Mn2+. Compared with ChoM1, ChoM2 showed higher enzyme affinity and catalytic efficiency. Whole-cells of the recombinant strains were firstly used as catalysts, the recombinant strains B. subtilis 168/pMA5-choM1 and B. subtilis 168/pMA5-choM2 catalyzed the bioconversion of cholesterol to 4-cholesten-3-one with a percentage conversion of 67% and 83% after 24 h. Further, the ChoM2 was over-expressed in M. neoaurum JC-12 to strengthen the uptake of sterol and its transformation efficiency. Finally, the ADD production reached 4.73 g·L^-1, increased by 37.1%. This result incitated that it was an effective way by enhancing the ChoM enzyme acitivity to facilitate the efficient sterol bioconversion.3. Three isoenzymes of SMO were firstly characterized from M. neoaurum as the key enzyme in the sterols C27-hydroxylation. By enzyme kinetic analysis and gene disruption and complementation, we found that among these three isoenzymes, SMO2 exhibits the strongest function in sterols catabolism. Further, the SMOs were over-expressed in M. neoaurum JC-12 to strengthen sterol transformation efficiency. The results showed that the expression of SMO1, SMO2 and SMO3 could increased ADD yield from 3.48 g·L^-1 to 4.49 g·L^-1, 4.96 g·L^-1 and 4.13 g·L^-1, increased by 29.0%,42.5% and 18.7%, respectively. And over-expression of SMO2 was more effective on enhancing ADD production. Thus, it was an efficient way by improving SMO enzyme acitivity to enhance ADD yield.4. Metabolic engineering strategy was applied to improve the sterol transformation efficiency to ADD in M. neoaurum JC-12. KSH which catalyzes the ADD catabolism, was firstly disrupted to block the ADD degradation pathway, and the ADD yield was increased from 3.45 g·L^-1 to 4.57 g·L^-1 and no longer decreased. Then ChoM2, SMO2 and KSDD were coexpressed to strengthen the sterol metabolic flux to ADD. Finally, the ADD production of recombinant strain JC-12S2-choM2-ksdd improved from 6.55 g·L^-1 to 12.40 g·L^-1, increased by 89.3%. To shorten the fermentation duration and enhance ADD productivity, in a 5-L fermentor fermentation process of the recombinant strain was optimized. Fructose used as the initial carbon source could eliminate the lag phase and the fermentation duration was decreased. Further, two stage fermentation was carried out and the final ADD production reached 20.1 g/L, which is the highest reported ADD production, with productivity increased from 0.074 g·（L·h）^-1 to 0.112 g·（L·h）^-1. Thus, this strategy provides a possible way in enhancing the ADD production in pharmaceutical industry.5. The microbial synthesis of steroid intermediates testosterone（TS） was further investigated. We optimized the gene codons of human 17β-hydroxysteroid dehydrogenase 3（17β-HSD3） and realized its functional expression in Pichia pastoris GS115. The engineered P. pastoris/17β-HSD3 P exhibited good selectivity for efficient conversion of AD to TS. Moreover, Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase（G6PDH） was coexpressed with 17β-HSD3 P to strengthen the NADPH regeneration system into the pathway from AD to TS. By optimization of the transformation conditions from AD to TS and applying the fed-batch strategy, the coexpressed system P. pastoris/17β-HSD3P-G6 PDH produced TS of 11.6 g·L^-1, which is the highest reported yield using a bioconversion method. The P. pastoris system coexpression target enzyme and the cofactor regeneration enzyme may be helpful for enhancing the production of other steroids. In order to further realize the transformation of phytosterol to TS, we optimized the gene codons of human 17β-HSD3 and realized its expression in M. neoaurum ZADF-4, which produced AD as the main product. However, although the 17β-HSD3 was well expressed, it didn’t show any enzyme activity, and the recombinant strain also can’t transform phytosterol to TS.