Functional Identification Of Curvularia Lunata CICC 40301 Steroidal Hydrolyase Gene

Selective microbial hydroxylation of steroids streamlines the synthesis of steroid drugs and key intermediates and obviate multiple protection groups manipulations.Curvularia lunata is industrially used to catalyze the 11β-hydroxylation of 11-deoxycortisol(RS)to produce anti-inflammatory hydrocortisone.Curvularia lunata CICC 40301 was not able to convert RS to produce anti-inflammatory hydrocortisone,but showed higher activity on progesterone.Due to the poor understanding of the genetic background for Curvularia lunata used in the steroid industry,it is formidable to rationally engineer efficient and robust production strains for large-scale steroid hydroxylation.In order to explore the progesterone metabolites and the genetic basis of steroid hydroxylation specificities in Curvularia lunata 40301,this thesis firstly clarified that the products of progesterone bioconversion from Curvularia lunata were 14α-hydroxylprogesterone(53.6%)and 7α-,14α-dihydroxylprogesterone(33.7%)which are both important drug steroid compound intermediates.The expression of the hydroxylase was shown to be strongly induced by steroid substrates and a strategy combining RNA-seq,qRT-PCR and yeast functional expression was used to search for responsible steroid hydroxylase gene(s).Two highly inducible candidate P450 genes(DN3109 and DN4923)were isolated and recombinant yeast cells expressing DN4923 was encoded the target steroid hydroxylase CYP5103B5.Bioinformatics analysis showed that Curvularia lunata 40301 is prone to genetic variation,caused the difference in DNA level among different individuals in the same species.Further simulation of CYP5103B5 tertiary structure and substrate binding pocket,the use of site-directed mutation technology,directional transformation of CYP5103B5.The mutant G362P/V364G was able to specifically convert progesterone to 14α-hydroxylprogesterone(100%,as opposed to 7α-,14α-dihydroxylprogesterone).The identification of hydroxylase CYP5103B5 will help elucidate the structural basis underlying regio-and stereoselectivity of steroid hydroxylation reaction,which is key to engineering more efficient industrial biocatalysts for greener process of steroid drug production.