| Steroid drugs are the second largest class of drugs after antibiotics.They are widely used clinically in anti-inflammatory,anti-tumor,and treatment of cardiovascular diseases.It was found that the derivatives of steroid hydroxylation have higher pharmacological activity and have better application prospects.Traditional chemical methods are difficult to introduce hydroxyl groups at specific sites of steroid parent nuclei,while microbial methods can effectively solve this challenge.In order to explore more steroid hydroxylating strains,this study was conducted to investigate the biotransformation of 13 different structures of steroid drug intermediates,including 17α-hydroxy progesterone,using 11 strains with steroid hydroxylation ability.The transformation products were isolated,purified and structurally identified to generate four steroidal hydroxylated derivatives.Through the screening of hydroxylated strains,it was found for the first time that Colletotrichum lini SF-307 could convert 17α-hydroxy progesterone to produce 11α,17α-dihydroxy progesterone with the best conversion effect.Optimization of the transformation process of the strain to improve its ability to produce 11α,17α-dihydroxy progesterone.The main findings of the study are as follows:（1）Screening of strains for the conversion of steroid drug intermediates by biological hydroxylation.In this study,11 strains of steroid hydroxylating bacteria were selected from laboratory collections and the biotransformation of 13 steroid compounds were studied.The conversion products were verified by high performance liquid chromatography and mass spectrometry analysis,and it was preliminarily confirmed that Rhizopus nigricans,Gibberella intermedia and Absidia coerulea could convert androstenedione to produce 11α-hydroxy androstenedione,and R.nigricans has the highest hydroxylation capacity.R.nigricans,Rhizopus stolonifer and Bacillus subtilis could convert progesterone to produce 11α-hydroxy progesterone,and R.nigricans has the highest hydroxylation capacity.Colletotrichum lini,R.nigricans,R.stolonifera,G.intermedia and A.coerulea could convert 17α-hydroxy progesterone to produce 11α,17α-dihydroxy progesterone,and C.lini has the highest hydroxylation capacity.Streptomyces roseochromogenes could convert prednisolone to produce16α-hydroxy prednisolone.The above four transformation products were separated and purified by preparative high performance liquid chromatography and their structures were characterized by Nuclear Magnetic Resonance.They were identified as 11α-hydroxy androstenedione,11α-hydroxy progesterone,11α,17α-dihydroxy progesterone and 16α-hydroxy prednisolone.It was found for the first time that C.lini SF-307 have a significant 11α-hydroxylation capacity for17α-hydroxy progesterone.The conversion product of C.lini SF-307,11α,17α-dihydroxy progesterone,has excellent pharmacological activity and application potential as an important intermediate in the biosynthesis of prednisone acetate.At present,the current biosynthesis process of 11α,17α-dihydroxy progesterone suffers from long conversion cycle and many by-products.Therefore,C.lini SF-307 was subsequently selected as the study object to optimize the 11αhydroxylation process for the conversion of 17α-hydroxy progesterone.（2）Optimization of media components for the transformation of 17α-hydroxy progesterone by C.lini SF-307.The optimal composition of the transformation medium was determined by single-factor and orthogonal tests:15 g·L-1 soluble starch,1.8 g·L-1 ammonium chloride,0.6 g·L-1 magnesium chloride,and 3 g·L-1 corn pulp.The conversion of 17α-hydroxy progesterone was 93.5%when the substrate was fed at a concentration of 0.5 g·L-1 and 1%（v/v）of ethanol was added for substrate solubilization.The highest product concentration was 220.4mg·L-1,which was 87.7%higher than before optimization.（3）Process optimization of C.lini SF-307 conversion of 17α-hydroxy progesterone.The optimal transformation conditions for the strains were determined by a single-factor study:temperature 35°C,starting p H 6.5,inoculum level 5%,loading volume 50 m L/250 m L,and transformation time 56 h.Through studying the effect of adding different kinds of organic solvents on the conversion efficiency of the substrate,the results showed that ethanol was the best co-solvent.When 2%（v/v）ethanol was added for substrate solubilization,the substrate conversion was 94.9%and the maximum concentration of 11α,17α-dihydroxy progesterone could reach 301.1 mg·L-1,which was increased by 120.1%compared with that before optimization.In response to the insufficient supply of coenzyme NADPH in the substrate hydroxylation reaction,a coenzyme regeneration method was constructed using 15 g·L-1 of glucose as the sugar co-substrate.Under this condition,the substrate conversion was 94.3%,and the concentration of 11α,17α-dihydroxy progesterone could be elevated to 323.2 mg·L-1,which was 16.7%higher than before optimization.The results of the substrate feeding process showed that when the substrate was added to the reaction system in batches,the substrate conversion reached 98.5%and the product concentration could be further increased to 339.8mg·L-1,which was 9.8%higher than the control.It was also found that the substrate tolerance of C.lini SF-307 to 17α-hydroxy progesterone was maximized at a substrate feeding concentration of 7.5 g·L-1.The highest concentration of 11α,17α-dihydroxy progesterone was1099.7 mg·L-1 with 67.8%substrate conversion under the conditions of batch addition of substrate. |
