Functional Identification Of 17?-hydroxysteroid Dehydrogenase And Its Application In Boldenone Synthesis

Boldenone is an important protein assimilating androgen steroid,which can steadily and continuously increase muscle and strength.Now it is mainly synthesized by1,4-androstenedione(ADD)through chemical method.Compared with the boldenone chemical synthesis,biotransformation has the advantages of mild transformation conditions,less environmental pollution and less by-products.However,17?-hydroxysteroid dehydrogenase(17?-HSD)for the synthesis of boldenone,is rarely identified or the identified17?-HSDs do not have the activity of catalyzing 17?-reduction reaction,resulting in the low utilization rate of the enzyme,so it is necessary to screen 17?-hydroxysteroid dehydrogenase with higher activity.In this paper,17?-HSD from many different strains were analysed and screened,and the recombinant enzyme HSDBo with high 17?-reduction activity was obtained.It was expressed in the host with efficient accumulation of steroid intermediates(ADD),and a coenzyme self circulation system was constructed,which realized the one-step transformation from the simple raw material phytosterol to the steroid drug boldenone.(1)Firstly,17?-hydroxysteroid dehydrogenase genes were screened,and its amino acid sequence were used for homology analysis and sequence alignment.Seven kinds of17?-HSDs from fungi and bacteria were selected and expressed in Escherichia coli.SDS-PAGE showed that the recombinant protein could be expressed successfully.Furthermore,the conversion of the substrate ADD for each recombinant 17?-HSD were investigated.It was found that the specific activity of HSDBo was the highest,2.27 times that of HSDCl,indicates that HSDBo had higher catalytic potential.(2)The HSDBo enzymatic properties were studied and found that the optimal temperature of HSDBo was 37?,the optimal pH was 7.5,and it was a non-metallic ion dependent enzyme.The coenzyme dependent type of HSDBo was identified.It was found that NADPH was the optimal cofactor of HSDBo during the reduction of ADD to boldenone.(3)The glucose-6-phosphate dehydrogenase(G6PDHmn)from M.neoaurum was connected with HSDBo in series to construct co-expressed recombinant M.neoaurum JC-12/pMV261-HSDBo-G6PDHmn,and the level of intracellular cofactor was determined during the transformation process.It was found that after co-expression of coenzyme,intracellular NADP~+and NADPH were basically at the same level,realizing the recycling of intracellular cofactor,compared with M.neoaurum JC-12/pMV261-HSDBo,the conversion increased by 40.74%.(4)The conditions of transforming phytosterol to boldenone by recombinant M.neoaurum JC-12/pMV261-HSDBo-G6PDHmn were optimized,including cosolvent,substrate concentration,glucose supplement,and fermentation in 5 L fermentor.The optimal conditions for the transformation were as follows:using methylated-?-cyclodextrin and Tween 80 as cosolvent and substrate concentration of 10 g·L~-11 and addition of 15 g·L~-11 glucose on the third day of the fermentation,3.34 g·L~-11 boldenone was synthesized,which realized one-step transformation from phytosterol to boldenone,and provided an economic and simple method for the industrial production of boldenone.