Enzymatic Engineering Modification Of P450scc And Research On C14-OH Active Steroids Assisted By Biocatalysis

Steroid drugs,also known as steroids,occupy an important position in the clinic.Pregnenolone is an important precursor of steroid hormones,and can synthesize hydrocortisone,progesterone and other drugs or drug intermediates that have important industrial value.The current industrial production of pregnenolone is generally obtained through the use of naturally derived steroid compounds such as cholesterol,diosgenin or stigmasterol through various organic synthesis reactions.These methods are cumbersome and have low production efficiency.The P450 side chain cleavage enzyme(P450scc)can cleave the bond between the 20 and 22 positions of the cholesterol side chain into pregnenolone.Most of the P450 sccs found so far exist in mitochondria of eukaryotes and are insoluble.It is difficult to study their reactivity.In Japanese Patent Report,two P450 sccs derived from bacteria are expressed and purified in E.coli.It can selectively cooperate with the electron transport matching constructed in vitro,and at the same time study its active site,make amino acid mutations,can study its activity more efficiently,and bring new breakthroughs in the industrialization of steroid hormone biosynthesis.By synthesizing the P450A/P450 B gene,we constructed it into the E.coli system,successfully expressed the protein and completed the in vitro P450 scc activity study.A variety of electron transport chain systems have been constructed,and P450A/P450 B activity has been successfully measured,but the enzyme activity is low in efficiency and cannot applied in industrial production.We have engineered proteins from the perspective of enzyme engineering.Three sites with important regulatory effects on selectivity,namely F84,F88,and L253,were mutated.After mutation,position 84 was still active,so saturation mutation was performed on position 84.The effect of F84 G and F84 L is more obvious.The conversion rate of the substrate4-cholesten-3-one increased from 11.55% to 35.34% and 32.87%,respectively.Hydroxylation is one of the most important reactions for the functionalization of steroids.C14 hydroxylated steroids are a unique type of natural products with important physiological activity and pharmaceutical value.The chemical methods to achieve hydroxylation steps are complex and lengthy,and the yield is low,so biocatalysis and biotransformation have become very important synthetic tools.In this work,we directly construct the steroid C14-OH through the fungal Thamnostylum piriforme NBRC6117 biocatalysis,expand the substrate range of biofermentation,build a C14-OH steroid library with diverse structures,and provide a rich source for the synthesis of natural products in the later period.At the same time,the 14α-OH gene was excavated from the perspective of genetic engineering.By analyzing the transcriptome,20 P450 genes were screened and constructed into Aspergillus oryzae NSAR1 for heterologous expression and biotransformation.It is more effective to improve the hydroxylation efficiency of the strain.In order to enhance the potential of the method to be applied in industry,starting from the problems of substrate solubility and strain conversion substrate loading limit,the feed concentration was increased from 0.25 g / L to 1.0 g / L,while ensuring product yield The rate is above 40%.