| Steroid hormone drugs are the second largest class of drugs to antibiotics,they have good prospects for industrial development with the demand increasing year by year.9α-hydroxyandrost-4-ene-3,17-dione(9α-OH-AD)is a core steroid drug intermediate which occupies a good market share.As a drug intermediate,it can be further synthetized the anti-inflammatory and anti-allergic glucocorticoid drugs,such as dexamethasone,betamethasone and beclomethasone.Presently,9α-OH-AD biosynthesis methods mainly contain two ways,one method is the androst-4-ene-3,17-dione(AD)produced firstly by one microorganism and then was converted to 9α-OH-AD by other microorganism.The other is that the 9α-OH-AD is directly biotransformated from phytosterols.However,there are still many problems in the process of 9α-OH-AD production by biotransformation methods.The problems,such as low substrate concentrations,more by-products and low yields,are limiting the further industrialization development of 9α-OH-AD.In this study,a strain that could convert phytosterols to 9α-OH-AD was screened.Based on the study of conversion characteristics,the key enzyme gene of 9α-OH-AD was cloned and exogenous expression.To increase the substrate concentration and the yield of the 9α-OH-AD,the key enzyme genes were enhanced expression in the wild strain.The main contents are as follows:(1)Screening and identification of strains which can biotransformate phytosterols into9α-OH-AD.A strain that can transform phytosterols to 9α-OH-AD was screened from soils such as oilseed oil and rape fields.Through 16 S rDNA sequence alignment,morphological,physiological and biochemical properties analysis,the wild strain was identified as Mycobacterium genus and named Mycobacterium sp.LY-1.And then the bioconversion products were isolated and detected by HPLC,LC-MS,1HNMR and13 CNMR.The results showed that there were six kinds of steroids in the conversion products:9α-hydroxy-3-oxo-23,24-bisnorchol-4-en-22-oic acid(9α-OH-BNC),9α,17β-hydroxyandrost-4-ene-3-one(9α-OH-T),9α-OH-AD,9α,22-dihydroxy-23,24-bisnorchol-4-en-3-one(9α-OH-BNA),AD and9α,24-dihydroxychol-4-ene-3-one(9α-OH-DHC).The product 9α-OH-AD accounted for the highest proportion 87.6%.(2)The transformation characteristics of Mycobacterium sp.LY-1.In order to improve the conversion efficiency of phytosterols,the effect of substrate cosolvent soybean oil on the transformation was investigated.The results showed that soybean oil could effectively promote the phytosterols transformation of Mycobacterium sp.LY-1.When the substrate concentration was 15 g·L-1and the adding concentration of soybean oil was 16 mL·g-1,the9α-OH-AD yield increased from 11% of control to 40%.In addition,the study also found thatglucose can promote cell growth to a certain extent,but it will enhance the further conversion of 9α-OH-AD to 9α-OH-T,which is not helpful for the accumulation of9α-OH-AD.Furthermore,the substrate specificity of strain LY-1 was studied.This strain only could convert phytosterols to 9α-OH-AD,whereas it converted AD into ADD.The ability of this strain using AD is far less than using phytosterols.(3)Cloning and exogenous expression of the 9α-OH-AD key enzyme3-ketosteroid-9α-hydroxylase(KSH).The redox bicomponent KshA and KshB of KSH in Mycobacterium sp.LY-1 were successfully tandemconstructed on plasmid pET28a(+)in combination and separation expressed exogenously in Escherichia coli BL21.Further substrate transformation experiments demonstrated that recombinant strain BL21/pET28a(+)-KshAB could synthesize 9α-OH-AD,whereas the BL21/pET28a(+)-KshA and BL21/pET28a(+)-KshB could not.(4)Transformation system optimization and molecular modification of Mycobacterium sp.LY-1.The traditional competent cell culture and preparation method were improved.The transformation of the recombinant plasmid into wild strain Mycobacterium sp.LY-1 was successfully achieved.Based on this work,the redox bicomponent KshA and KshB of KSH in strain Mycobacterium sp.LY-1 were enhanced in separate and combined expression.The results showed that expression of 9α-OH-AD could be improved by the two methods,and the effect of co-expression KshA and KshB on the synthesis of the 9α-OH-AD was the highest.The yield of 9α-OH-AD by the recombinant strain LY-1/pMV261-KshAB was increased to39% with 30 g·L-1 phytosterols,and the substrate concentration was increased nearly 1 times. |
