| 3β,7α,15α-Trihydroxy-5-androsten-17-one(7α,15α-di OH-DHEA) is a key intermediate in the synthesis of drospirenone, which can be obtained by introducing two hydroxyls in C7 and C15 of DHEA through microbial transformation. Drospirenone is the main component of ―Yasmin‖, the fourth generation of oral contraceptives. Because Yasmin exhibits superior advantages of good contraceptive effect, weight control and skin care, it has become the best-selling oral contraceptive since it appeared on the market in 2000. However, the large-scale production of 7α,15α-di OH-DHEA is hindered by poor catalytic activity, low substrate concentration and conversion efficiency in the biotransformation of DHEA into 7α,15α-di OH-DHEA by Colletotrichum lini ST-1. Therefore, in order to improve the conversion rate of 7α,15α-di OH-DHEA, a series of works including analysis of hydroxylation specificity of C. lini ST-1, establishment of CDs biotransformation system, research of coenzyme regeneration, exploration of P450 pre-induction and relevant mechanism are studied. The main contents are as follows:1. Analysis of the substrate spectrum and hydroxylation specificity of C. lini ST-1. A range of structurally diverse compounds including estranes, androstanes, and pregnanes were selected as substrates. The results demonstrated that C. lini ST-1 had a broad substrate spectrum which could undergo hydroxylation on C7, C11 and C15, the type of substituent group of C3 and C17 had remarkable influence on hydroxylation sites. After extraction and purification, ten kinds of transformation products were obtained. Among them, 11α,15α-dihydroxyandrost-4-en-3,17-dione, 11α,15α-dihydroxy-canrenone and 11α,15α-dihydroxy-16α,17α-epoxyprogesterone were firstly reported, and dihydroxylation at C11 and C15 was a new type of steroids hydroxylation. Moreover, a dihydoxylation mechanism of C. lini ST-1 firstly hydroxylation at a site with better affinity and then hydroxylation at another site was proposed. Compared with other strains that had ability of hydroxylation, C. lini ST-1 showed the higher conversion rates on a variety of substrates. The molar conversion rate of 7α,15α-di OH-DHEA could reached to 83.6% when the concentration of DHEA was 4 g·L-1.2. Establishment of CDs complexation transformation system. With the addition of M-β-CD or HP-β-CD at the molar ratio of 1:1, the concentration of DHEA was increased to 10 g·L-1 from 4 g·L-1, and the conversion rates of total products were enhanced to 74.9%(8.06 g·L-1) and 78.4%(8.44 g·L-1) from 63.6%(6.91 g·L-1), respectively. Therefore, the effects of CDs on DHEA biotransformation were analyzed systematically. XRD, DSC, phase solubility and thermodynamic parameters(?G, ?H, ?S and Ks) analysis showed that an inclusion complex was formed between CDs and DHEA at a molar ratio of 1:1, which remarkably increased the solubility of DHEA. SEM, TEM and the analysis of composition and content of cell fatty acid demonstrated that the cell permeability could be improved by the addition of CDs. Therefore, the mechanisms of CDs complexation technique for enhancing the conversion rate included not only solubilizing of steroids, but also increasing cell permeability for both substrate and product.3. Effects of pre-induction of P450(key enzyme of steroids hydroxylation) on conversion rate. The results showed the molar conversion rate of 7α,15α-di OH-DHEA could be enhanced to 58.4% and 56.0% from 36.2% respectively by the induction with 0.6% ethanol or 0.5% DHEA for 12 h. Moreover, it was found the induction of ethanol had some relationship with its hydroxyl group, and a negative correlation was observed between inductive effect and the length of carbon chain.4. Analysis of the inductive mechanism through comparative proteomic. The results revealed 50 and 59 differential proteins were obtained with the induction of ethanol or DHEA respectively. Most of differential proteins were related on steroids metabolism, redox reaction, energy metabolism and transport proteins by functional clustering. Based on the results of i TRAQ analysis, the significantly up-regulated P450 was obtained, which was laid a foundation for the subsequent amplification P450 gene, enhancement of P450 expression and construction of recombinant engineering strains. Meanwhile, proteomic analysis showed the expression of NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase and other redox related proteins were significantly up-regulated, which suggested NADPH was an important factor in the process of DHEA biotransformation. Moreover, q RT-PCR was used to evaluate the transcriptional level of the key differential proteins. The results of q RT-PCR were consistent with the analysis of i TRAQ, which demonstrated the induction of ethanol or DHEA was achieved through the increase of proteins expression in transcriptional level.5. Establishment of a dual-cosubstrate coupled system for NADPH regeneration. Based on the results of i TRAQ analysis and the experiment of coenzyme precursor, low intracellular NADPH/NADP ratio was demonstrated to be a limiting factor during the process of DHEA biotransformation. Then a dual cosubstrate-coupled system consisting of nicotinic acid(20 mmol·L-1) and glucose(15 g·L-1) was investigated in C. lini ST-1, and the molar conversion rate of 7α,15α-di OH-DHEA was dramatically increased to 65.2% after transformation with 42 h. Further, optimization of the conversion process including CDs complexation, pre-induction and coenzyme regeneration were performed through statistical method. The optimal conversion conditions were as follows: HP-β-CD/DHEA molar ratio 0.94, ethanol concentration 0.525%(v/v), glucose 16.05 g·L-1. Under the optimal conditions, the molar conversion rate of 7α,15α-di OH-DHEA could reach to 70.2% at the substrate concentration of 10 g·L-1. |
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