Regulation Of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene Expression In Comamonas Testosteroni

Chemicals such as steroids (e.g estradiol (E2) and oral contraceptive etc.) , polycyclic aromatic hydrocarbons (PAHs) and insecticide have been discharged into environment, which have already caused ecological destruction and various diseases such as infertility, cancer increased. Among these environmental hormones, steroid is quite stable and its hormonal activity much more harmful than other compounds in the environment. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic. C. testosteroni strains are able to use steroids (testosterone, progesterone, bile acid) as sole carbon and energy sources. Complete assimilation of steroids is achieved through a complex pathway involving many enzymatic steps of oxidation responsible for the breakdown of the steroid nucleus, C. testosteroni therefore play a significant role in the bioremediation of these stablely hormonally active compounds in the environment. However the catabolic enzymes for these compounds metabolism in C. testosteroni are not constitutively expressed but are induced by their respective substrates. For empowering C. testosteroni to degrade those compounds more effectively, we cloned 3α-HSD/CR and its activator genes from C. testosteroni. Then, its vectors were constructed and had been expressed in Escherichia coli. We constructed two genetically engineered bacteria of Comamonas testosteroni. The results were as follows:1. 3α-HSD/CR and its activator genes were cloned from C. testosteroni by PCR with Comamonas testosteroni genomic DNA. Then two expression vector pET-15b-3α and pET-15b-act1 were constructed respectively and expressed in E. coli BL21. All the above provided the foundation for protein purifing and antibody preparing.2. The activator gene from Comamonas testosteroni was amplied by PCR with Comamonas testosteroni genomic DNA. The PCR fragments were cloned to plasmid pKtacl (containing tac promoter) and two plasmids: pKtac1-act1 (containing 564bp total activator gene) and pKtac1 -act2 (containing 409bp partial activator gene) were obtained. The recombinant plasmid pKtac1-act2 was found mutation on promoter -10 consensus sequence (TATAAT-+TATGTT) by DNA sequencing. The inserts of pKtacl-act1 and pKtacl -act2 were subcloned into pKtacl and yielded pKtacl -act3 (-10 consensus sequence is TATA AT, containing 409bp activator gene) , pKtacl-act4 (-10 consensus sequence is TATGTT, containing 564bp activator gene) .3. The four recombinants were transformed into Escherichia coli HB101. The results indicated: 1) plasmids with TATA AT -10 consensus sequence had higher promoter activity than that of plasmids with TATGTT -10 consensus sequence in E. coli; 2) overexpression of activator from C. testosteroni was toxic to cells so pKtacl-ac/J which contains 409bp activator gene is unstable in E. coli HB101; 3) -10 consensus sequence down mutation (pKtac 1-ac/2, TATAAT-+TATGTT) and tac promoter fragment disappeared(pKtacl -act3 after 4 inoculations) from the cells revealed the self-defence mechanism off. coli HB101.4. The pKtacl -actl (containing 564bp total activator gene) and pKtacl-ac/2 (containing 409bp partial activator gene) were integrated into C. testosteroni chromosome through homologous recombination by electroporation respectively. Genetically engineered bacteria C. testosteroni+pKtac\-actl and C. /es/as/erom+pKtacl-ac/2 were gained by antibiotic and PCR selection then detected by Southern hybridization. The total cell lysate of C. testosteroni^ pKtacl-ac/7 and C. /estas/erom+pKtacl-ac/2 were extracted to detect the quantity of 3a-HSD/CR using ELISA. The results indicated that two genetically engineered bacteria could constitutively expressed 3a-HSD/CR gene evidently without the presence of substrates such as testosterone.5. Two genetically engineered bacteria were inoculated for 50 days and their total cell lysate were extracted per five days to detect the quantity of 3a-HSD/CR and activator using ELISA. The results indicated: the expression of activator and 3a-HSD/CR are quite high and stable when C. testosteroni^ pKtacl-ac/7 inoculated in LB medium containing antibiotic, while both unstable when inoculated in LB medium without antibiotic; the same as C. /es/os/erowj+pKtacl-ac/2. C. tes/as/erom+pKtacl-ac/./ was more stable than C. /es/as/erom+pKtacl-ac/2 in LB medium without antibiotic. From the results we could preliminary conclude that the expression of 3o-HSD/CR was regulated by the activator gene.6. The RT-PCR results indicted: genetically engineered bacteria C. testosteroni+pKtac\-act] and C. testosteroni+pKtacl-act2 constitutively expressed 3a-HSD/CR which is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds; at the same time 4-hydroxy-2-oxovalerate aldolase and 2-hydroxypenta-2,4-dienoate hydratase also constitutively expressed. These enzyme are both founctioned in the catabolism of AHs and steroids.7. The preliminary results of two-dimensional gel electrophoresis showed: genetically...