Study On The Physiological Function And Steroid Transformation Mechanism Of CYP105 Family Genes In Streptomyces Virginiae IBL14

Streptomyces.virginiae IBL14 is a strain of actinomycetes isolated from activated sludge from a steroidal drug factory.The strain can grow on media having sterol compounds(including diosgenin,estradiol,deoxycorticosterone,cholesterol,ergosterol,etc.)as the sole carbon source and produce a variety of new sterol compounds through secondary metabolism.To systematically illuminate the physiological function and enzymatic mechanism of CYP superfamily genes in S.virgziniae IBL14,the whole genome of S.virginiae IBL14 was sequenced by using Illumina HiSeq 2000 sequencing platform and the data obtained by sequencing were filtered,assembled and evaluated.Further,all genes of the S.virginiae IBL14 genome were predicted using Glimmer and the function was annotated by blasting the protein sequences of the genes with the following databases:KEGG,SwissProt,NR and GO,To study the physiological functions and enzymatic mechanisms of the CYP 105 family genes in this study,the five CYP 105 family genes in the S.virginiae IBL14 genome were firstly predicted and analysized by bioinformatics methods,then knocked out by the type I-B-svi CRISPR-Cas system in S.virginiae IBL14 itself and cloned into E.coli,respectively and finally the biotransformation of the five CYP enzymes were carried out and identified.Based on the results of bioinformatics analysis,we found that there are 31 cytochrome P450 genes in the S.virginiae IBL1 genome,among which svh001,svu001,svu003,svu004 and svu005 belong to CYP105 family genes.Genes svh001,svu003 and svu004,identified as CYP105C1 subfamily members,have very high homology with extremely conservative and similar secondary structure,suggesting that they probably have similar physiological functions and enzymatic mechanisms.Gene svu005 is identified as a CYP105D1 subfamily member.Unlike svh001,svu003,svu004 and svu005,gene svu001 does not belong to any known subfamily of CYP105 despite that it contains all thirteen a superhelix structure but is longer than others(499 aa in svu001,about 400 aa in others).All of the CYP105 family genes contain K-helix and Heme binding motifs.The Heme binding motifs of svh0019 svu003,svu004,svu005 are extremely conserved,among which the Heme binding motifs of svu003 and svu004 are identical.The K-helix of the five genes is basically the same too.It is worth noting that 1-helix is considered to be a conserved motif in all CYP family membes,but not found in svu001.In conclusion,we identified genes svh001,svu003 and svu004 as CYP105C1,svu005 as CYP105D1 and svu001 as a member of a new CYP 105 subfamily.After all genes of the S.virginiae IBL14 genome were predicted and annotated,we found that the genome contains a cas gene locus harboring 6 cas genes,i.e.,cas7,cas5,cas3,cas4,casl and cas2.At the.same time based.on the analytical results of CRISPRfinder program online by entering the whole genome sequence of S.virginiae IBL14,we found that the genome of S.virginiae IBL14 contains 18 CRISPR candidates,among which CRISPR 1,2,and 3 are confirmed CRISPRs.Further,based on the analytical results of the gene composition and architecture of the cas gene locus,the CRISPR-Cas system in the S.virginiae IBL14 genome was named type?-B-svi CRISPR-Cas system.Lukily,we demonstrated experimentally that the type?-B-svi CRISPR-Cas system could be programmed for self-targeting genome editing of S,virginiae IBL14 and the genome editing tool based on the type ?-B-svi CRISPR-Cas system could be used for non-self-targeting genome editing in prokaryotic species too,suggesting that it is possible for this type ? Cas3 system to replace the current commercial type ? Cas9 system in many cases.Experimentally,by designing a gene editing plasmid(harboring g-DNA and t-DNA)and transforming it into S.virginiae IBL14 protoplasts,we obtained five mutants with the single-deletion of the five CYP105 family genes,espectively.Further the physiological functions of the five mutants were studied by growth curve measurement,physiological morphological analysis and bioinformatics analysis.The results are as follows:i)the type ?-B-svi CRISPR-Cas system can be used for self-targeting genome editing of S.virginiae IBL14 with advantages of easy operation,taking short time and high efficiency;ii)the removals of the five genes are not lethal to the strain IBL14 but significantly affect the growth and physiological changes in the cell,especially the large amount accumulation of pigments in both liquid and solid cultures(e.g.,mutant IBL14?svu004);iii)complex secondary metabolic products of S.virginiae IBL14 can inhibit effectively the growth of microorganisms,especially fungi;iv)the knockouts of the five CYP105 family genes significantly fluence the production of antibiotics and antifungal agents of the strain IBL14 by affecting the metabolic pathway of polyketone.Further,we successfully cloned the five genes into Escherichia coli JM109(DE3),optimized fermentation conditions to achieve soluble expression of the five recombinant proteins and investigated the biotransformation functions of the five proteins by using a large number of sterol compounds as a substrate.The results show as follows:i)all of the products of the CYP105 family genes are hydroxylases and can hydroxylate estradiol to estriol;ii)Svu004 can hydroxylate estradiol to 2-hydroxy-estradiol and estriol at the same time;iii)Svh01,Svu001,Svu003 and Svu004 can hydroxylate Vitamin D3 to 25-hydroxy-vitamin D3;iv)Svh01 and Svu004 can biotransform 16,17-epoxy progesterone to two kinds of new compounds,differencing from the known 11-hydroxylated products;v)Svu005 with the abiliity of hydroxylation at C9 of androstenedione can catalyze benzophenone to 4-hydroxybenzophenone;vi)Svu001 can hydroxylate arginine to N-?-hydroxy-L-arginine,which involves in arginine metabolic pathway;vii)the three C subfamily proteins exhibit a certain similarity in substrate specificity,differing from Svu001 and Svu005.This study revealed the subfamily localization and evolutionary relationship of the CYP105 gene of S.virginiae IBL14,explored the substrate region of the CYP105 family gene and deepened the understanding of the molecular mechanism of the hydroxylation reaction.The results of this study can help to transform and synthesize new hydroxylase to improve the enzyme reactivity and environmental adaptability.In the specific sites of sterol substances,the specific stereocomplexes introduce hydroxyl groups and produce new steroids and drugs Body;promote the transformation and degradation of steroidal contaminants.