Identification And Functional Analysis Of Cytochrome P450Complement In Streptomyces Virginiae IBL14

S. virginiae IBL14, isolated from activated sludge for treatment of waste from a steroidal drug factory, is an effective degradation strain of steroidal compounds,including progesterone hydrocortisone and cholesterol. IBL14has the ability of diosgenin bioconversion and biodegradation. In the end, eleven new metabolic products and2new metabolic pathwaysare found:the reactionsincluding C25hydroxylation of diosgenin and double methoxylation at C6. Now, we conjecturethat cytochrome P450s play an important role in these new pathways. To obtain the fragments and identify therelationships of structuresand functions of cytochrome P450s in S. virginiae IBL14, and genome sequencing of IBL14has been finished. The results indicate that the genome sequence is80Mb in linear chromosome containing33cytochrome P450genes. Before genome sequencing, one cytochrome P450momooxygenase gene, fcpc, has been cloned by molecular genetics technology. It can catalyze hydroxylation atC25of diosgenin.Then, the sequence alignment of fcpc and the predicted gene of svh01indicate that they are the same one.The sequence alignment among all CYPs from S. virginiae IBL14, Streptomyces griseus subsp. Griseus NBRC13350, Streptomyces avermitilis and Streptomyces sp. Mgl revealed that the genetic relationships of CYPs from S. virginiae IBL14and that from Streptomyces sp. Mgl are closest. The members of CYP105, CYP107and CYP157in those four strains have highly similarity, respectively.,Svu001may be a new member in CYP family and has60%identity with a CYP sequence from Streptomyces sp. Mgl in Genbank, while the identity is less than25%with the other sequences. To compare with putative CYP sequences and the sequences of the same family in CYPED indicate that almost all the CYPs from S. virginiae IBL14have the conserved domains including the amino acid residue GXXT in I-Helix, ExxR motif in K-Helix and the Cys hydrophobicity pocket.A total of9ferredoxin genes and3ferredoxin reductase genes in S. virginiae IBL14are identified. There are3CYPs in S. virginiae IBL14are found to clusterwith3different ferredoxin genes and svu020is found to cluster with ferredoxin and ferredoxin reductase genes. Additionally, several CYPs cluster with regulator genes and the other functional genes in S. virginiae IBL14.Now, the working of cloning and expression of svh01, which can degrade diosgenin and ethinyloestradiol, in E. coli has been finished. The working of svu022cloning in E. coli and the target protein expressed in B. subtilis has been completed too.