Study On Function Of 3-ketosteroid-9?-hydroxylases In Arthrobacter Simplex

Arthrobacter simplex commonly used for steroid 1-dehydrogenation in industry.In the process of steroids dehydrogenation,the degradation of dehydrogenation products affects the efficient accumulation of steroid 1-dehydrogenation products in industrial production.3-ketosteroid-9?-hydroxylase is the key enzyme in the degradation of steroid 1-dehydrogenation products.In this article,to clarify the effect of different KSHA isoenzymes in the process of dehydrogenation product degradation,the bioinformatics analysis,RT-PCR technique,and the gene overexpression strategies were used to analyze the catalytic function of different KSHA isozyme in the dehydrogenation.product degradation process by the A.simplex,which could guide us to build a strain which can stably accumulate the dehydrogenation product by the molecular biology methods.It could provide certain theoretical basis and guidance for constructing a strain which can't degrade the dehydrogenation product.In this article,the sequence alignment and relationship between KSHA with other steroid transformation strains was analysed by the bioinformatics analysis.The KSHAs from A.simplex showed a certain sequence conservation,while the amino acid sequence between isozymes showed a great difference,it means different KSHA may show significant difference on the function.The transcriptional level of kshA gene was analyzed by RT-PCR.The transcription level of kshA3 was significantly up-regulated in presences of substrates AD and ADD,which was 6.7 times and 5.2 times compared with the control group.kshAl and kshA2 were not induced by AD,ADD and CA.So KSHA3 may play a key role in product degradation.In order to further clarify the effect of different KSHA isozymes in dehydrogenation product degradation,three A.simplex overexpressed kshA were constructed using E.coli-Arthrobacter sp.shuttle plasmid pART2.The strain which overexpress the kshA3 gene catalytic the degradation of ADD was improved obviously,the time of completely degradation was shorten 50 percent than the control strain,It further proves that the kshA3 maybe is the key enzyme in the process of degrading dehydrogenation product.In order to further clarify the KSHAs catalytic ability,the strategy of genetic heterologous expression was used.The kshAl-3 and kshB1-2 from A.simplex was expressed in E.coli,respectively.The strain which express kshA and kshB gene were mixed.Toward the ultrasonic broken,the crushing fluid was added the substrate AD,The 9?-OH-AD was not generated towards the detecting of HPLC.This could be due to the lack of coenzyme.Or the E.coli's environment is diffrent from the A.simplex.The plasmid pK18mobsacB was used to constructed the suicide plasmids respectively which harbor the kshAl,kshA2 and kshA3's upstream and downstream.The plasmid was integrated into the genome of A.simplex through the homologous recombinant screening.However,the results showed that the plasmid did not integrate to the correct site by PCR verification,and the reverse screening marker SacB did not play a role in this strain.The reasons need to be further studied.