Construction And Application Of Strains For Steroids C1,2-Dehydrogenation With High Efficiency

The ?1-dehydrogenation reactions are typical in the industrial production of steroid drugs.Arthrobacter simplex is usually applied in ?1-dehydrogenation and has been extensively studied for many years.At present,A.simplex is mainly used to produce prednisone acetate(PA)form cortisone acetate(CA).The construction of the A.simplex with high dehydrogenation etticiency is very important for steroids production.In this study,3-ketosteroid-?1-dehydrogenase(KsdD)was overexpressed in A.simplex,and the gene knock out approaches were also perfermed in A.simplex and the results are as follows:The gene of KsdD from A.simple was cloned into pART2,and then transformed into A.simplex for overexpression of KsdD.Seven overexpression engineering strains were successfully constructed using A.simplex as the expression host They were pART2-WT,pART2-W299A,pART2-W299G,pART2-KsdDl,pART2-KsdD2,pART2-KsdD3 and pART2-KsdD4.Whole-cell transformation by these engineering strains were carried out when using five steroid substrates(cortisone acetate,cortisone,androstendione,methyltestosterone,testosterone)as the substrate.The results obtained from high performance liquid chromatography(HPLC)showed that the transformation efficiency of pART2-W299A and pART2-W299G overexpressing strains mutated from tryptophan to alanine and glycine was higher than that of pART2-WT,pART2-KsdD3.The pART2-Ks'dDl,pART2-KsdD2 and pART2-KsdD4 overexpressing strains showed preference for different substrate in transformation.Based on homologous recombination strategy,the plasmid pK18mobsacB was used to constructed the suicide plasmids which harboring upstream and downstream of the KsdD3.The desired gene knockout strains of A.simplex were not achieved after the process of electrotransformation,screening and verification.This perhaps due to that sucrose-fructose gene(SacB)could not play a reverse screening role in A.simplex.The knockout vectors carrying cas9 gene,KsdD3 N20 sgRNA and deleting pART2 plasmid sgRNA were also constructed by CRISPR/Cas9 gene editing technology.The desired gene knockout strains were not achieve affer of A.simplex.Then the knockout vectors was was adjusted and four optimized knockout vectors(pAS481,pAS482,pAS491,pAS492)were obtained.However,the feasibility of the knockout system in A.simplex is still need to be validated.