Studies On Screening Of Gibberella Intermedia And Its Bioconversion Conditions For The Preparation Of3β,7α,15α-trihydroxy-5-androsten-l7-one

Drospirenone, an analogue of17α-spironolactone, is a unique progestogen. It was developedby the German Schering company as successful fourth-generation oral contraceptives. Yasmin, acombined oral contraceptive, containing drospirenone and ethinylestradiol, has reliablecontraceptive effect as well as it has seldom side effect. It has become the best-selling female oralcontraceptives since its first launch in Europe2000.3β,7α,15α-trihydroxy-5-androsten-17-one(7α,15α-diOH-DHEA) is an important drug intermediate for the synthesis of drospirenone, and itcould be obtained by dehydroepiandrosterone (DHEA) hydroxylation. The research of this workinclude the strain screening, the purification and identification of the transformation products, andthe optimization of fermentation conditions for the formation of7α,15α-diOH-DHEA. The mainresults were as follows:1. A strain Gibberella intermedia CA3-1which could hydroxylate DHEA to7α,15α-diOH-DHEA was screened out by bioconversion tests in shake bottle from the strainspreserved in laboratory. The main two transformation products were purified using HPLCpreparation column and silica gel column chromatography, and their structures were elucidated as7α,15α-diOH-DHEAand7α-OH-DHEAby HPLC, MS, IR, NMR and melting point analysis.2. The effects of medium composition and culture conditions on the dihydroxylation of DHEAby G. intermedia CA3-1were studied. The optimized medium composition was as follows (g·L-1):glucose15, yeast extract16, corn syrup15and K2HPO40.15. The optimized bioconversionconditions for the formation of7α,15α-diOH-DHEA were as follows: initial pH of6.5, inoculumvolume of10%, transformation temperature of30℃and liquid volume of30mL/250mL. In thisoptimum conditions, the7α,15α-diOH-DHEA yield was45.3%at the substrate DHEA additionalconcentration of8.0g·L-1, which was25.5%higher than that of the original conditions.3. The key factors that affect the bioconversion process were investigated. Firstly, to promotethe dissolution of substrate, the effects of organic solvents, surfactants and plant oils on productyield were studied. The results indicated that the2%rapeseed oil could enhance7α,15α-diOH-DHEA yield to63.1%, which was45.2%higher than that of control. The mechanismof the enhancement was also studied. The rapeseed oil could stabilize the pH, change thepermeability of cells and contribute to cell growth. Secondly, the induction of transformation wasstudied. The0.8%benzene could increase the product yield as inducer. The adding time of inducerwas12th hour, and the length of induce time was12hours. Then the optimal7α,15α-diOH-DHEAyield was57.5%, increased by25.5%. Finally, an efficient NADPH regeneration method wasestablished using12g·L-1xylose as the co-substrate. The results showed that the optimal7α,15α-diOH-DHEAyield was61.0%, which was33.3%higher than that of control.