Studies On The Microbial Multi-Step Functional Transformation Of Steroidal Compounds

Steroids are widespread in nature and thousands of steroidal compounds have been identified. Over 250 sterols and related compounds have been reported to occur in plants, insects, vertebrates, low eukaryotes such as yeasts and fungi. Most steroids are used as hormone drugs which are widely used in anti-tumor, anti-inflammatory, anti-microbial, anti-convulsant and anti-allergy. There are about 300 kinds of approved steroidal drugs to date, and this number tends to grow. Steroid drugs are one of the most marketed medicines and the output is second only to antibiotics.The physiological activity of steroids depends on their structure and the synthesis mainly uses dioscin as raw material. At present the main method is chemical synthesis method, but some sites can hardly to achieve through chemical method. Biotransformation can occur at almost every position of steroids. The reactions of steroids include redox, hydrolysis, acylation, esterification, isomerization, halogenation, the side chain degradation and so on, so microbial steroid transformation is a powerful tool for generating novel steroidal drugs and key intermediates. The esterification is one of the important microbial steroid transformations and testolactone can be obtained by microbial transformation through Baeyer-Villiger monooxygenation in ring D of androstenedione.Testolactone is reported to be a steroid aromatase inhibitor which blocks the transformation of testosterone to estradiol and reduces synthesis of estrone. Testolactone and its derivatives clinically used as anticancer drugs and treatment of male infertility and other diseases.In this study, a rapid specific strains screening method was established. Androstenedione (4AD) and dehydroepiandrosterone (DHEA) were used as initial screening substrates. About 80 strains, which could convert the substrates, were isolated by using enrichment culture method and 3 stains could convert the substrates to sole product. The SCI 301 strain, which transformed a series of steroids to testolactone with high isolated yield, was identified as Fusarium oxysporum by the morphology and molecular biological identification. The reaction conditions were optimized by using androstenedione (4-AD) as substrate. The optimal temperature, pH and co-solvent were 30℃, pH 8.0 and 2%DMSO, respectively. After the strain was incubated for 24-30 h,4-AD was added as a DMSO solution. The reaction was carried out for 15h, and testolactone was isolated with 83% yield. Progesterone, androsta-1,4-diene-3,17-dione (ADD), dehydroepiandrosterone (DHEA), pregnenolone and testosterone were also transformed to testolactone, with isolated yield of 93%,82%,78%,76% and 98%, respectively. The intermediates of ADD and boldenone were detected during these transformations, and a possible transformation sequence was proposed.