The Analysis Of The Genetic Differences Of KsdD In Mycobacterium Neoaurum

Androst-4-ene-3,17-dione(AD)and androst-1,4-diene-3,17-dione(ADD)is an important steroid drug intermediates.In the preliminary study we found that there aresignificant differences on the ratio of ADD to AD between Mycobacterium neoaurum TCCC 11028(MNR)and M.neoaurum TCCC 11028 M3(MNR M3),there is only one base difference(C413T)on 3-ketosteroid-?1-dehydrogenase(KsdD)gene,resulting in 138 amino acid changes(S138L)of KsdD.The influences of changes in KsdD structure on KsdD activity,which was caused by one base differences in ksdD gene,through ksdD-MNR and ksdD-MNR M3 gene exogenous expression in Escherichia coli BL21(DE3),gene deletion and replacement in MNR M3,as well as the transcription level of KsdD in MNR and MNR M3 was investigated.This study may serve as a basis for future studies on the structural analysis and catalytic mechanism of dehydrogenase.AD transformation experiments were carried out in genetically engineered bacteria,the results of TLC showed that AD was more easily transformed into ADD with genetically engineered bacteria E.coli BL21(DE3)/pET-22b(+)-ksdD-MNR than with E.coli BL21(DE3)/pET-22b(+)-ksdD-MNR.Therefore,the activity of KsdD-MNR M3 was low,and the KsdD activity of the engineered strains was similar to that of the original MNR and MNR M3 strains,initially confirmed that the differences between the KsdD structure did effect on the KsdD activity,which was caused by 413 base difference of ksdD genes.The gene knockout and replacement of ksdD were designed according to the two-step method of unmarked knockout principle.The results of steroid transformation experiments in the presence of HP-?-CD showed that MNR M3?ksdD was only produced AD,but no ADD was detected.This result indicated that the strain completely lost KsdD activity.The ADD/AD ratios with MNR M3 ?ksdD::ksdD-MNR and wild-type MNR M3 were 31.54 and 0.15,respectively.Confirmed that when ksdD-MNR M3 was replaced by ksdD-MNR,the ADD/AD ratio of MNR M3?ksdD::ksdD-MNR returned to the similar level of the MNR strain(28.08).The effect of S138L on the structure of the KsdD enzyme was considered the primary cause of the fluctuations in the ADD/AD ratio.Therefore,the activity differences between ksdD-MNR and ksdD-MNR M3 were indeed caused by the structure of the KsdD enzyme,and the nucleotide differences in the 413 base considerably affected KsdD activity.The mechanism by which residue mutations alter enzyme activity may be connected with the crystal structure of KsdD from Rhodococcus erythropolis SQ1.The results showed that as a key amino acid residue in the active center position,Ser-138 played an important role in maintaining the active center in the hydrophobic environment of KsdD.The expression levels of KsdD enzyme in MNR and MNR M3 strains by qPCR experiments was analyzed,the results showed that ksdD gene expression levels of MNR was 71%of MNR M3 strain,indicating that the KsdD enzyme of MNR and MNR M3 at the same transcription level,not a main factor of the differences on the ADD/AD ratio.