Studies On The 3-Ketosteriod-1-Dehydrogenation Of 9-Fluoro Steroid Hormone By Different Microorganisms

Steroid drugs have been widely used in clinic application.9-fluoro steroid hormones such as dexamethasone,betamethasone,triamcinolone and so on are carbohydrate corticosteroids that regulate the metabolism of normal water and salt in the body,promote protein breakdown and hepatic glycogen transformation,body fluid volume and osmotic balance,anti-shock and anti-allergic aspects of an important role.9?,11?-Epoxypregn-1,4-diene-17?,21-diol-3,20-dione?IV?is a key precursor for the production of 9-fluoro steroids hormones.In this study,Nocardioides sp.NS413,Mycobacterium sp.MS136 and E.coli BL21?DE3?were used as the starting strain,and the steroid compound 9?,11?-Epoxypregn-4-ene-17?,21-diol-3,20-dione 21-acetate?I?as the substrate for the production of 9-fluoro steroids by microbial whole cell transformation and cell lysate conversion.And the reaction mechanism and reaction conditions of different transformation systems were optimized.5 g/L substrate?was transformed by Nocardioides sp.NS413 with common fermengtation,when the MCD?Methyl-?-cyclodextrin?was not found in the fermentation broth,the reaction sequence was mainly?,??9?,11?-Epoxypregn-1,4-diene-17?,21-diol-3,20-dione 21-acetate?,?.After the addition of MCD in the fermentation broth,the reaction sequence was mainly?,??9?,11?-Epoxypregn-4-ene-17?,21-diol-3,20-dione?,?.The conversion rate of the product??64.8%?was59.2%higher than that of the MCD-free aqueous system?40.7%?in the two-phase?oil phase-water phase?transformation medium N3 with MCD at 40h.While substrate?was transformed by Nocardioides sp.NS413 resting cell,the production was only?.The conversion of III was the highest at 43.6%when bacteria cultured in the first seed culture and the absence of carbon and nitrogen source of the fermentation medium.Whole cells of Mycobacterium sp.MS136 could only convert?to?,and?can be effectively converted to?by cellular lysates.The reaction order is that?is spontaneously hydrolyzed to?and?undergoes C_1,2-dehydrogenation reaction to?.In order to improve the productivity of?,the key genes kstD,kstD3 and kstD_M encoding C_1,2-dehydrogenase?KSTD?were overexpressed in Mycobacterium sp.MS136 to enhance the C_1,2-dehydrogenation reaction rate,and the results showed that1 g/L substrate?can be converted by recombinant strain MS136-kstD_M cellular lysates at pH 7.0,the productivity of?reached 78.4%after 45 h,which is 38.9%higher than original strain.E.coli recombinant strain BL21?DE3?-kstD,BL21?DE3?-kstD3,BL21?DE3?-kstD_M cell lysate could convert substrate?to II and?.The conversion rate?79.5%?of?by BL21?DE3?-kstD_M was lower than that of mycobacterium recombinant strain MS136-kstD_M?92.8%?.