BACKGROUND
cytochrome p <dig>  genes refer to such genes that encode a superfamily of iron-containing hemoproteins with a maximum absorption spectrum near 450 nm, often characterized by conserved cys residue in hydrophobic pocket  <cit> .
most of the orfs of cyp have three distinct characteristics used often for their identification and analysis, i.e., the i-helix of putative cyps , the conserved exxr motif located in the k-helix and the cytochrome p <dig> cysteine heme-iron ligand signature motif   <cit> .
according to a widely-accepted taxonomy, cyps within a family share more than 40% amino acid identity and members of subfamilies share more than 55% amino acid identity  <cit> .
occasionally, the decision to accept a sequence in a known family depends greatly on how it clusters on a tree, not so much on the absolute amino acid identity  <cit> .
cyps have been confirmed existing in all eukaryotic  and prokaryotic organisms   <cit> .
they often are monooxygenases involved in oxidation of a range of endogenous compounds, such as cholesterol, lipids and steroidal hormones, as well as xenobiotics such as drugs and toxic chemicals in environment  <cit> .
cyps catalyse diverse reactions, including c-h hydroxylation, epoxidation, hetero-atom oxidation, aromatic ring oxidation and dealkylation  <cit> .
in the catalytic reaction process of p <dig> monooxygenase, one atom of o <dig> is inserted into substrate while the other is reduced to h2o.
cyp genes responsible for secondary metabolism are often laid in antibiotic biosynthetic gene clusters to catalyze stereo- and region- specific reaction of substrates to related derivatives.
the biotransforming capabilities of bacterial cyps have been widely elucidated.
p450soy  from streptomyces griseus was involved in the degradation of a diverse array of complex agrochemicals and environmental pollutants  <cit> .
cyp105c <dig> from actinomycete spp.
had the ability to transform benanomicin a into two derivatives, 10-hydroxybenanomicin a and 11-o-demethylbenanomicin  <cit> .
the functions of related cyp <dig> family members have been reported.
cyp107e from micromonospora griseorubida was found to govern the hydroxylation and epoxidization in mycinamicin biosynthesis  <cit> , p <dig> terf  from streptomyces platensis to catalyze hydroxylation of terfenadine  <cit>  and hydroxylase pikc  of streptomyces venezuelae to convert narbomycin to picromycin  <cit> .
cyp <dig> of mycobacterium tuberculosis demonstrated omega-hydroxylase activity of relevant methyl-branched lipids  <cit> .
ybdt  of bacillus subtilis was involved in fatty acid beta-hydroxylation  <cit> .
cyp <dig> of nocardia farcinica ifm <dig> had the functions of the o-dealkylation and ortho-hydroxylation of formononetin  <cit>  and 154h <dig> from clostridium acetobutylicum performed biocatalytic reactions with different aliphatic and aromatic substrates  <cit> .
genome sequencing is an effective way to predict and annotate all the possible cyps genes in an organism.
streptomyces coelicolor a <dig> , a typical strain which is often used for the study of physiological function and antibiotic production, is the first streptomyces species sequenced in  <dig>  its linear chromosome is  <dig>  mb  <cit>  which contains  <dig> open reading frames  with  <dig> putative cyps  <cit> .
s. avermitilis, known for producing the antiparasitic agent avermectin, contains  <dig> orfs with  <dig> putative cyps in the 9 mb chromosomes  <cit> .
the genome of streptomyces peucetius atcc <dig> with the size of  <dig>  mb contains  <dig> putative cyps  <cit> .
s. virginiae ibl <dig>  isolated from activated sludge for treatment of waste from a steroidal drug factory, is an effective degradative strain of various steroidal compounds, including progesterone, isotestosterone, dihydrotestosterone, hydrocortisone, cholesterol and ostrone  <cit> .
to comprehensively understand the function of cyps of s. virginiae ibl <dig> in degradation and biotransformation of diosgenin, the whole genome sequencing of s. virginiae ibl <dig> isolated by our lab was carried out for the first time.
using in silico technology, we predict and annotate all of the putative cyps of s. virginiae ibl <dig> and analyze these cyps evolutionarily and functionally via comparison with those of other streptomyces species.
furthermore, functions and characteristics of cyp genes svh <dig> and cyp svu <dig> in this strain are experimentally identified and analyzed.
RESULTS
genome sequencing and cyps in s. virginiae ibl14
by in silico analysis of newly-sequenced s. virginiae ibl <dig>  <dig>  mb genome,  <dig> orfs are identified and the total gc content exceeds 70%.
the annotated results via rpsblast display that there are a total of  <dig> putative cyps in the genome of this strain ibl <dig>  contributing to approximately  <dig> % of all the coding sequences.
the number of cyps is identical to that in s. avermitili and almost two times as that in s. coelicolor a <dig> and s. peucetius atcc <dig> .
such high level of cyp diversity suggests the high diversity of the secondary metabolism pathways in s. virginiae ibl <dig>
the  <dig> out of  <dig> putative cyps of s. virginiae ibl <dig> belong to  <dig> previously-reported cyp families, i.e.,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> and another to an unknown family, as shown in table  <dig>  among the all, the cyp121a , cyp <dig> , cyp <dig> , cyp <dig> , cyp154h , cyp <dig> , cyp <dig> , cyp <dig> , cyp <dig>  and cyp <dig>  are firstly reported in s. virginiae, and especially, cyp107m, cyp185a and cyp247a have been found rarely in streptomycete spp.
the svu <dig>  svu <dig> and svu <dig> have lower identity with other family members  while others show more than 63% identity to cyps of other organisms.
it’s worth noting that the svu <dig> presumably belongs to a new cyp family since no close homologue is found in genbank except that in streptomyces sp.
mg <dig> with 66% identity.
a the name of the putative cyps in s. virginiae ibl <dig>
b amino acid number of putative cyps.
c closest homologs in genbank and the family classification of cyps searched in cyped.
d number of amino acid overlap, which exceeds the protein size, is due to the introduction of gaps during blast comparison.
e the highest percent identity for a set of aligned segments to the same subject sequence.
features of cyps from s. virginiae ibl14
table  <dig> displays the three characteristic motifs of cyps of s. virginiae ibl <dig>  the critical residues are highlighted with bold fonts, which are threonine  in gxxtt motif of i-helix, glutamic acid  and arginine  in exxr motif of k-helix and cysteine  in the gxxxcxg heme-binding domain signature, respectively.
from the table  <dig>  we can find the i-helix is absent in svu <dig>  and the i-helix and k-helix missing in svu <dig>   <cit> , which reflects i-helix is not absolutely essential for the cyp structure.
the  <dig> members of cyp <dig> family svu <dig> /157a and svu <dig> /157c do not have arginine residue in k-helix like the cyp157c <dig> from s. coelicolor a <dig> having a motif e  <dig> qslw  <cit>  and the cyp157a <dig> and cyp157c <dig> from s. avermitilis exhibiting a 257evlw motif and a 257eqslw motif  <cit> .
the cyp <dig> family proteins that lack consensus exxr motifs but genetically are linked to their upstream conservons imply that they have functions linked to the upstream pathway  <cit> .
besides, svu <dig>  svu <dig>  svu <dig>  svu <dig> and svu <dig> do not strictly follow the gxxxcxg motif of heme-binding.
multiple alignments and phylogenetic analysis
the phylogenetic tree of the combined cyps of s. virginiae ibl <dig>  s. avermitilis ma- <dig>  s. venezuelae atcc  <dig> and streptomyces sp.
mg <dig> is presented in figure  <dig>  from figure  <dig>  we can find almost of all the cyps in s. virginiae ibl <dig> are closely related to their homologues.
more than  <dig> of cyps from s. virginiae ibl <dig> are close to those from streptomyces sp.
mg <dig> and the member  of new cyp family found in s. virginiae ibl <dig> is only close to streptomyces sp.
mg <dig>  these results indicate that the cyps from s. virginiae ibl <dig> are closer to those from streptomyces sp.
mg <dig> than those from other streptomyces spp, including s. avermitilis ma- <dig> and s. venezuelae atcc  <dig>  for the four species of s. virginiae ibl <dig>  sp.
mg <dig>  avermitilis ma- <dig> and s. venezuelae atcc  <dig>  the families cyp  <dig> and cyp <dig>  have more closely evolutionary relationship.
further, the paralogous relationship of the  <dig> cyps in s. virginiae ibl <dig> was generated with the neighbor-joining methods .
from figure  <dig>  we can find that svh <dig> and svu <dig> and svu <dig> as well as svu <dig> and svu <dig> in s. virginiae ibl <dig> have the closest homologous evolutionary relationship, respectively.
it’s worth noting that most members belonging to the same cyp family are clustered together as expected, e.g., the  <dig> members of cyp <dig> family.
the prediction of functions of cyps in s. virginiae ibl14
a high identity over 70% among different protein sequences reasonably suggests that they may hold similar function  <cit> .
as shown in the table  <dig>  we can find a sum of  <dig> cyp sequences of s. virginiae ibl <dig> have best matches to those of other streptomyces, which are helpful in function prediction.
cyp <dig> and cyp <dig> are the most studied bacterial cytochromes which are associated with the degradation and biotransformation of a diverse array of xenobiotics and antibiotic biosynthesis.
analysis of cyps sequence of s. virginiae ibl <dig> shows that there are  <dig> cyps belonging to cyp <dig>  five to cyp <dig>  four to cyp <dig>  three to cyp <dig>  two to cyp <dig> and one to each other family, which indicates the diversity and importance of the two groups cyp <dig> and cyp <dig>  the predicted functions of several putative cyps in s. virginiae ibl <dig>  combined with reported experimental evidences, were listed in table  <dig>
cyps in s. virginiae ibl <dig> and their ferredoxin reductase and ferredoxin
the catalytic activity of cyps depends greatly on individual ferredoxin or/and ferredoxin reductase associated with.
it was reported that there are three, six and four ferredoxin reductase genes and six, nine and two ferredoxin genes in s. coelicolor a <dig> , s. avermitilis and s. peucetius, respectively.
in s. coelicolor a <dig>  only cyp105d <dig> is arranged in an operon with a ferredoxin gene  <cit> .
in s. peucetius cyp147f is clustered with ferredoxin reductase  <cit> .
in s. avermitilis both cyp105p <dig> and cyp105d <dig> are clustered with ferredoxin, cyp147b <dig> is arranged in an operon with a ferredoxin and ferredoxin reductase, cyp105q <dig> is associated in an operon containing both a ferredoxin and ferredoxin reductase, and cyp <dig> is fused to a p <dig> reductase  <cit> .
three ferredoxin reductase genes and seven ferredoxin genes are found in s. virginiae ibl <dig> after annotation of s. virginiae ibl <dig> genome.
that is, the activities of many of the cyps in s. virginiae ibl <dig> are supported by different combinations with the three ferredoxin reductases and seven ferredoxins.
also in s. virginiae ibl <dig>  svu <dig> , svh <dig>  and svu <dig>  is found to cluster with ferredoxin svf <dig>  svf <dig> and svf <dig>  respectively and svu <dig>  clustered with ferredoxin reductase svfr <dig> and ferredoxins svf <dig>  the facts suggest that the functional realization of cyps svu <dig>  svh <dig>  svu <dig> and svu <dig> needs the participation of electron transfer.
the result of homology analysis by blast-searching the genbank are listed in the table  <dig>
a the name of gene in s. virginiae ibl <dig>
b homologues searched in genbank.
regulatory elements and functional genes clustered with cyps
the cyps in s. peucetius atcc <dig> clustered with regulatory elements were reported  <cit> .
in the annotations of gene arrangement around the putative cyps on the s. virginiae ibl <dig> chromosome, svu <dig>  svu <dig> and svu <dig> were found to cluster with the genes of atp/gtp binding proteins   <cit> , svu <dig>  svu <dig> to cluster with lysr-family transcriptional regulator   <cit> , svu <dig> to cluster with two component transcriptional regulators and luxr family   <cit> , svu <dig> to cluster with a transcriptional regulator, arac family   <cit>  and two component transcriptional regulators, luxr family and svu <dig> to cluster with the atp-binding protein fbpc and tetr-family transcriptional regulators   <cit> .
as described above, the cyps in s. virginiae ibl <dig> chromosome are responsible for the transcriptional regulation of many functional genes related with primary, secondary metabolism, as well as the responses to environmental factors as expected.
besides, cyps are clustered with other functional genes.
svh <dig> is adjacent to the genes of mdlb, abc-type multidrug transport system, atpase and permease components, which may be involved in the transportation of substrates  <cit> .
svu <dig> lies next to alcohol dehydrogenase, suggesting that svu <dig> may take part in alcohol bioconversion and biodegradation.
svu <dig> is next to  <dig>  5-dopa dioxygenase which is a member of the class iii extradiol dioxygenase family  to cleave aromatic rings between a hydroxylated carbon and an adjacent non-hydroxylated carbon), suggesting that the combination of svu <dig> and  <dig>  5-dopa dioxygenase may be responsible in biodegradation of substrates with aromatic rings.
svu <dig> is adjacent to mbth-like protein which is found in known antibiotic synthesis gene clusters  <cit> .
the cholesterol oxidase chol from s. virginiae ibl <dig> in the bioconversion and biodegradation of diosgenin responsible for the conversion of diosgenin to diosgenone  via a couple of c3-dehydrogenation and c4-5-isomerization was reported  <cit> .
in s. virginiae ibl <dig> the gene encoding svu <dig>  clusters with the genes of putative ferredoxin svfr <dig> and cholesterol oxidase , suggesting that the cytochrome p <dig> joins with the cholesterol oxidase chol to catalyze the oxidation of cholesterol and its structural analogs.
in conclusion, cyps from s. virginiae ibl <dig> may have multiple functions in secondary metabolism, including hydroxylation, dehydrogenation, ring-cleavage, transportation, etc.
functional identification and characteristics of svh <dig> and svu022
to elucidate all putative cyps’ functions in s. virginiae ibl <dig>  four cyp genes of the strain ibl <dig> were firstly selected.
among them, the functional identities of cyp genes svh <dig> and svu <dig>  has been finished.
the cytochrome p <dig> svh <dig>   <cit>  belongs to the class i  p <dig> system based on a taxonomic split, in which electrons are transferred from nadph or nadh to ferredoxin reductase and ferredoxin.
sequence analysis revealed the complete sequence of svh <dig> with atg as the start codon has 70% g + c content.
the sequence of possible ribosome-binding site is located on the upstream of svf <dig> .
both svh <dig> and svf <dig> contain 1200 bp and 243 bp, respectively, based on sequence analysis.
to obtain the expressed products of them, both svh <dig> and svf <dig> sequences were first ligated into a pet22b vector in a cluster to generate the expression plasmid pet22b-svh01-svf <dig> that was then cloned into e. coli jm <dig>  to form a recombinant strain e. coli ibl <dig> .
the pcr results of svh <dig> and svf <dig> from the recombinant strain e. coli ibl <dig> were analyzed by gel electrophoresis  and also confirmed by gene sequencing.
the svu <dig> with a g + c content of 73%  consists of  <dig> nucleotides.
similarly, the complete sequence of svu <dig> was first inserted to the shuttle plasmid phcmc <dig> to form the recombinant plasmid phcmc05-svu <dig>  and then cloned in b. subtilis wb800n  to produce the recombinant strain b. subtilis ibl  <dig> .
the pcr result of svu <dig> from the recombinant strain b. subtilis ibl  <dig> is shown in figure 3c.
svh <dig>  is a peptide of  <dig> amino acids, with a molecular weight of  <dig>  kda and a pi value of  <dig>  estimated by the expasy .
to obtain its expressed product and study product characteristics, the recombinant strain e. coli ibl <dig> was incubated and induced.
the expression of svh <dig> was shown in figure 4a.
from the sds-page, we can find that the two distinctly additional protein bands should be svh <dig> with an about mw of 44 kda and svf <dig> with an about mw of  <dig>  kda, respectively.
the further functional identification of the svh01/fcpc of s. virginiae ibl <dig>  hydroxylating the c25-tertiary carbon of diosgenin to form isonuatigenone, was experimentally confirmed  <cit> .
svu <dig>  is a deduced protein of  <dig> amino acids which shares 91% identity with that in streptomyces sp.
mg <dig>  the estimation of mw and pi of svu <dig> are  <dig>  kda and  <dig> , respectively.
similarly, the recombinant strain b. subtilis ibl  <dig> was incubated and induced to study the product expression and its characteristics.
the expressed result of svu <dig> from the recombinant strain b. subtilis ibl  <dig> was shown in figure 4b.
the sds-page displays a distinct protein band with about mw of  <dig>  kda as expected.
the further experimental results from tlc, hplc and lc/ms indicated that the cyp svu <dig> enables to biotransform β-estradiol into estriol.
figure  <dig> shows the profiles of the biotransformation of β-estradiol by strains b. subtilis wb800n and b. subtilis ibl  <dig> in hplc.
the functional identification of the svu <dig>  and svu <dig>  is in progress.
CONCLUSIONS
s. virginiae ibl <dig> contains  <dig> putative cyps,  <dig> ferredoxins and  <dig> ferredoxin reductases in its  <dig>  mb linear chromosome.
most of the cyps in s. virginiae ibl <dig> belong to the cyp <dig>  family and cyp <dig>  family.
compared phylogenetically with cyps from  <dig> typical streptomycete spp., s. virginiae ibl <dig> appears to be closest to those of streptomyces sp.
mg <dig>
further analysis showed that among all of the  <dig> cyps in s. virginiae ibl <dig>  three cyps are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three cyps with atp/gtp binding proteins, four cyps arranged with transcriptional regulatory genes and one cyp locates on the upper of atp-binding protein and transcriptional regulators as well as four cyps associated with other functional genes involved in secondary metabolism and degradation.
the new characteristics found in cyps from s. virginiae ibl <dig> suggest that the exxr motif in the k-helix is not absolutely conserved in cyp <dig> family as reported  <cit>  and i-helix not absolutely essential for the cyp structure.
particularly, one new family was found based on the cyp svu <dig> in s. virginiae ibl <dig> which shares 66% identity only to that from streptomyces sp.
mg <dig>
two recombinant strains e. coli ibl <dig>  and b. subtilis ibl  <dig>  were constructed and subsequently their functions were identified, respectively.
experimental results showed that both cyp svh <dig> and cyp svu <dig> are two hydroxylases, capable of bioconverting diosgenone into isonuatigenone and β-estradiol into estriol, respectively.

 cytochrome p genes refer to such genes that encode a superfamily of iron-containing hemoproteins with a maximum absorption spectrum near 450 nm, often characterized by conserved cys residue in hydrophobic pocket. most of the orfs of cyp have three distinct characteristics used often for their identification and analysis, i.e., the i-helix of putative cyps, the conserved exxr motif located in the k-helix and the cytochrome p cysteine heme-iron ligand signature motif. according to a widely-accepted taxonomy, cyps within a family share more than 40% amino acid identity and members of subfamilies share more than 55% amino acid identity. occasionally, the decision to accept a sequence in a known family depends greatly on how it clusters on a tree, not so much on the absolute amino acid identity. cyps have been confirmed existing in all eukaryotic and prokaryotic organisms. they often are monooxygenases involved in oxidation of a range of endogenous compounds, such as cholesterol, lipids and steroidal hormones, as well as xenobiotics such as drugs and toxic chemicals in environment. cyps catalyse diverse reactions, including c-h hydroxylation, epoxidation, hetero-atom oxidation, aromatic ring oxidation and dealkylation. in the catalytic reaction process of p monooxygenase, one atom of o is inserted into substrate while the other is reduced to h2o. cyp genes responsible for secondary metabolism are often laid in antibiotic biosynthetic gene clusters to catalyze stereo- and region- specific reaction of substrates to related derivatives. the biotransforming capabilities of bacterial cyps have been widely elucidated. p450soy from streptomyces griseus was involved in the degradation of a diverse array of complex agrochemicals and environmental pollutants. cyp105c from actinomycete spp. had the ability to transform benanomicin a into two derivatives, 10-hydroxybenanomicin a and 11-o-demethylbenanomicin. the functions of related cyp family members have been reported. cyp107e from micromonospora griseorubida was found to govern the hydroxylation and epoxidization in mycinamicin biosynthesis, p terf from streptomyces platensis to catalyze hydroxylation of terfenadine and hydroxylase pikc of streptomyces venezuelae to convert narbomycin to picromycin. cyp of mycobacterium tuberculosis demonstrated omega-hydroxylase activity of relevant methyl-branched lipids. ybdt of bacillus subtilis was involved in fatty acid beta-hydroxylation. cyp of nocardia farcinica ifm had the functions of the o-dealkylation and ortho-hydroxylation of formononetin and 154h from clostridium acetobutylicum performed biocatalytic reactions with different aliphatic and aromatic substrates. genome sequencing is an effective way to predict and annotate all the possible cyps genes in an organism. streptomyces coelicolor a, a typical strain which is often used for the study of physiological function and antibiotic production, is the first streptomyces species sequenced in its linear chromosome is  mb which contains open reading frames with putative cyps. s. avermitilis, known for producing the antiparasitic agent avermectin, contains orfs with putative cyps in the 9 mb chromosomes. the genome of streptomyces peucetius atcc with the size of  mb contains putative cyps. s. virginiae ibl isolated from activated sludge for treatment of waste from a steroidal drug factory, is an effective degradative strain of various steroidal compounds, including progesterone, isotestosterone, dihydrotestosterone, hydrocortisone, cholesterol and ostrone. to comprehensively understand the function of cyps of s. virginiae ibl in degradation and biotransformation of diosgenin, the whole genome sequencing of s. virginiae ibl isolated by our lab was carried out for the first time. using in silico technology, we predict and annotate all of the putative cyps of s. virginiae ibl and analyze these cyps evolutionarily and functionally via comparison with those of other streptomyces species. furthermore, functions and characteristics of cyp genes svh and cyp svu in this strain are experimentally identified and analyzed.  genome sequencing and cyps in s. virginiae ibl14 by in silico analysis of newly-sequenced s. virginiae ibl  mb genome, orfs are identified and the total gc content exceeds 70%. the annotated results via rpsblast display that there are a total of putative cyps in the genome of this strain ibl contributing to approximately % of all the coding sequences. the number of cyps is identical to that in s. avermitili and almost two times as that in s. coelicolor a and s. peucetius atcc. such high level of cyp diversity suggests the high diversity of the secondary metabolism pathways in s. virginiae ibl the out of putative cyps of s. virginiae ibl belong to previously-reported cyp families, i.e.,,,,,,,,,,,, and another to an unknown family, as shown in table  among the all, the cyp121a, cyp, cyp, cyp, cyp154h, cyp, cyp, cyp, cyp and cyp are firstly reported in s. virginiae, and especially, cyp107m, cyp185a and cyp247a have been found rarely in streptomycete spp. the svu svu and svu have lower identity with other family members while others show more than 63% identity to cyps of other organisms. it’s worth noting that the svu presumably belongs to a new cyp family since no close homologue is found in genbank except that in streptomyces sp. mg with 66% identity. a the name of the putative cyps in s. virginiae ibl b amino acid number of putative cyps. c closest homologs in genbank and the family classification of cyps searched in cyped. d number of amino acid overlap, which exceeds the protein size, is due to the introduction of gaps during blast comparison. e the highest percent identity for a set of aligned segments to the same subject sequence. features of cyps from s. virginiae ibl14 table  displays the three characteristic motifs of cyps of s. virginiae ibl the critical residues are highlighted with bold fonts, which are threonine in gxxtt motif of i-helix, glutamic acid and arginine in exxr motif of k-helix and cysteine in the gxxxcxg heme-binding domain signature, respectively. from the table  we can find the i-helix is absent in svu and the i-helix and k-helix missing in svu, which reflects i-helix is not absolutely essential for the cyp structure. the members of cyp family svu /157a and svu /157c do not have arginine residue in k-helix like the cyp157c from s. coelicolor a having a motif e qslw and the cyp157a and cyp157c from s. avermitilis exhibiting a 257evlw motif and a 257eqslw motif. the cyp family proteins that lack consensus exxr motifs but genetically are linked to their upstream conservons imply that they have functions linked to the upstream pathway. besides, svu svu svu svu and svu do not strictly follow the gxxxcxg motif of heme-binding. multiple alignments and phylogenetic analysis the phylogenetic tree of the combined cyps of s. virginiae ibl s. avermitilis ma- s. venezuelae atcc and streptomyces sp. mg is presented in figure  from figure  we can find almost of all the cyps in s. virginiae ibl are closely related to their homologues. more than of cyps from s. virginiae ibl are close to those from streptomyces sp. mg and the member of new cyp family found in s. virginiae ibl is only close to streptomyces sp. mg these results indicate that the cyps from s. virginiae ibl are closer to those from streptomyces sp. mg than those from other streptomyces spp, including s. avermitilis ma- and s. venezuelae atcc for the four species of s. virginiae ibl sp. mg avermitilis ma- and s. venezuelae atcc the families cyp and cyp have more closely evolutionary relationship. further, the paralogous relationship of the cyps in s. virginiae ibl was generated with the neighbor-joining methods. from figure  we can find that svh and svu and svu as well as svu and svu in s. virginiae ibl have the closest homologous evolutionary relationship, respectively. it’s worth noting that most members belonging to the same cyp family are clustered together as expected, e.g., the members of cyp family. the prediction of functions of cyps in s. virginiae ibl14 a high identity over 70% among different protein sequences reasonably suggests that they may hold similar function. as shown in the table  we can find a sum of cyp sequences of s. virginiae ibl have best matches to those of other streptomyces, which are helpful in function prediction. cyp and cyp are the most studied bacterial cytochromes which are associated with the degradation and biotransformation of a diverse array of xenobiotics and antibiotic biosynthesis. analysis of cyps sequence of s. virginiae ibl shows that there are cyps belonging to cyp five to cyp four to cyp three to cyp two to cyp and one to each other family, which indicates the diversity and importance of the two groups cyp and cyp the predicted functions of several putative cyps in s. virginiae ibl combined with reported experimental evidences, were listed in table  cyps in s. virginiae ibl and their ferredoxin reductase and ferredoxin the catalytic activity of cyps depends greatly on individual ferredoxin or/and ferredoxin reductase associated with. it was reported that there are three, six and four ferredoxin reductase genes and six, nine and two ferredoxin genes in s. coelicolor a, s. avermitilis and s. peucetius, respectively. in s. coelicolor a only cyp105d is arranged in an operon with a ferredoxin gene. in s. peucetius cyp147f is clustered with ferredoxin reductase. in s. avermitilis both cyp105p and cyp105d are clustered with ferredoxin, cyp147b is arranged in an operon with a ferredoxin and ferredoxin reductase, cyp105q is associated in an operon containing both a ferredoxin and ferredoxin reductase, and cyp is fused to a p reductase. three ferredoxin reductase genes and seven ferredoxin genes are found in s. virginiae ibl after annotation of s. virginiae ibl genome. that is, the activities of many of the cyps in s. virginiae ibl are supported by different combinations with the three ferredoxin reductases and seven ferredoxins. also in s. virginiae ibl svu, svh and svu is found to cluster with ferredoxin svf svf and svf respectively and svu clustered with ferredoxin reductase svfr and ferredoxins svf the facts suggest that the functional realization of cyps svu svh svu and svu needs the participation of electron transfer. the result of homology analysis by blast-searching the genbank are listed in the table  a the name of gene in s. virginiae ibl b homologues searched in genbank. regulatory elements and functional genes clustered with cyps the cyps in s. peucetius atcc clustered with regulatory elements were reported. in the annotations of gene arrangement around the putative cyps on the s. virginiae ibl chromosome, svu svu and svu were found to cluster with the genes of atp/gtp binding proteins, svu svu to cluster with lysr-family transcriptional regulator, svu to cluster with two component transcriptional regulators and luxr family, svu to cluster with a transcriptional regulator, arac family and two component transcriptional regulators, luxr family and svu to cluster with the atp-binding protein fbpc and tetr-family transcriptional regulators. as described above, the cyps in s. virginiae ibl chromosome are responsible for the transcriptional regulation of many functional genes related with primary, secondary metabolism, as well as the responses to environmental factors as expected. besides, cyps are clustered with other functional genes. svh is adjacent to the genes of mdlb, abc-type multidrug transport system, atpase and permease components, which may be involved in the transportation of substrates. svu lies next to alcohol dehydrogenase, suggesting that svu may take part in alcohol bioconversion and biodegradation. svu is next to 5-dopa dioxygenase which is a member of the class iii extradiol dioxygenase family to cleave aromatic rings between a hydroxylated carbon and an adjacent non-hydroxylated carbon), suggesting that the combination of svu and 5-dopa dioxygenase may be responsible in biodegradation of substrates with aromatic rings. svu is adjacent to mbth-like protein which is found in known antibiotic synthesis gene clusters. the cholesterol oxidase chol from s. virginiae ibl in the bioconversion and biodegradation of diosgenin responsible for the conversion of diosgenin to diosgenone via a couple of c3-dehydrogenation and c4-5-isomerization was reported. in s. virginiae ibl the gene encoding svu clusters with the genes of putative ferredoxin svfr and cholesterol oxidase, suggesting that the cytochrome p joins with the cholesterol oxidase chol to catalyze the oxidation of cholesterol and its structural analogs. in conclusion, cyps from s. virginiae ibl may have multiple functions in secondary metabolism, including hydroxylation, dehydrogenation, ring-cleavage, transportation, etc. functional identification and characteristics of svh and svu022 to elucidate all putative cyps’ functions in s. virginiae ibl four cyp genes of the strain ibl were firstly selected. among them, the functional identities of cyp genes svh and svu has been finished. the cytochrome p svh belongs to the class i p system based on a taxonomic split, in which electrons are transferred from nadph or nadh to ferredoxin reductase and ferredoxin. sequence analysis revealed the complete sequence of svh with atg as the start codon has 70% g + c content. the sequence of possible ribosome-binding site is located on the upstream of svf. both svh and svf contain 1200 bp and 243 bp, respectively, based on sequence analysis. to obtain the expressed products of them, both svh and svf sequences were first ligated into a pet22b vector in a cluster to generate the expression plasmid pet22b-svh01-svf that was then cloned into e. coli jm to form a recombinant strain e. coli ibl. the pcr results of svh and svf from the recombinant strain e. coli ibl were analyzed by gel electrophoresis and also confirmed by gene sequencing. the svu with a g + c content of 73% consists of nucleotides. similarly, the complete sequence of svu was first inserted to the shuttle plasmid phcmc to form the recombinant plasmid phcmc05-svu and then cloned in b. subtilis wb800n to produce the recombinant strain b. subtilis ibl. the pcr result of svu from the recombinant strain b. subtilis ibl is shown in figure 3c. svh is a peptide of amino acids, with a molecular weight of  kda and a pi value of estimated by the expasy. to obtain its expressed product and study product characteristics, the recombinant strain e. coli ibl was incubated and induced. the expression of svh was shown in figure 4a. from the sds-page, we can find that the two distinctly additional protein bands should be svh with an about mw of 44 kda and svf with an about mw of  kda, respectively. the further functional identification of the svh01/fcpc of s. virginiae ibl hydroxylating the c25-tertiary carbon of diosgenin to form isonuatigenone, was experimentally confirmed. svu is a deduced protein of amino acids which shares 91% identity with that in streptomyces sp. mg the estimation of mw and pi of svu are  kda and, respectively. similarly, the recombinant strain b. subtilis ibl was incubated and induced to study the product expression and its characteristics. the expressed result of svu from the recombinant strain b. subtilis ibl was shown in figure 4b. the sds-page displays a distinct protein band with about mw of  kda as expected. the further experimental results from tlc, hplc and lc/ms indicated that the cyp svu enables to biotransform β-estradiol into estriol. figure  shows the profiles of the biotransformation of β-estradiol by strains b. subtilis wb800n and b. subtilis ibl in hplc. the functional identification of the svu and svu is in progress.  s. virginiae ibl contains putative cyps, ferredoxins and ferredoxin reductases in its  mb linear chromosome. most of the cyps in s. virginiae ibl belong to the cyp family and cyp family. compared phylogenetically with cyps from typical streptomycete spp., s. virginiae ibl appears to be closest to those of streptomyces sp. mg further analysis showed that among all of the cyps in s. virginiae ibl three cyps are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three cyps with atp/gtp binding proteins, four cyps arranged with transcriptional regulatory genes and one cyp locates on the upper of atp-binding protein and transcriptional regulators as well as four cyps associated with other functional genes involved in secondary metabolism and degradation. the new characteristics found in cyps from s. virginiae ibl suggest that the exxr motif in the k-helix is not absolutely conserved in cyp family as reported and i-helix not absolutely essential for the cyp structure. particularly, one new family was found based on the cyp svu in s. virginiae ibl which shares 66% identity only to that from streptomyces sp. mg two recombinant strains e. coli ibl and b. subtilis ibl were constructed and subsequently their functions were identified, respectively. experimental results showed that both cyp svh and cyp svu are two hydroxylases, capable of bioconverting diosgenone into isonuatigenone and β-estradiol into estriol, respectively. 
streptomyces virginiae ibl is an effective degradative strain for many steroidal compounds and can also catalyze the c hydroxylation of diosgenin, the first-ever biotransformation found on the f-ring of diosgenin. c. one new subfamily was found as per the fact that the cyp svu in s. virginiae ibl shares 66% identity only to that from streptomyces sp. the analytical results of blastp showed that the strain ibl contains cyps, ferredoxins and ferredoxin reductases in its  mb linear chromosome. experimental results showed that both cyp svh and cyp svu are two hydroxylases, capable of bioconverting diosgenone into isonuatigenone and β-estradiol into estriol, respectively. mg further analysis showed that among all of the cyps in s. virginiae ibl three cyps are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three cyps with atp/gtp binding proteins, four cyps arranged with transcriptional regulatory genes and one cyp located on the upstream of an atp-binding protein and transcriptional regulators as well as four cyps associated with other functional genes involved in secondary metabolism and degradation. cyps from s. virginiae ibl are phylogenetically closed to those of streptomyces sp. these characteristics found in cyps from s. virginiae ibl show that the exxr motif in the k-helix is not absolutely conserved in cyp family and i-helix not absolutely essential for the cyp structure, too. to completely elucidate the hydroxylation function of cytochrome p genes found during biotransformation of steroids by s. virginiae ibl the whole genome sequencing of this strain was carried out via sequencing systems.
 as well known, both natural and synthetic steroidal compounds are powerful endocrine disrupting compounds which can cause reproductive toxicity and affect cellular development in mammals and thus are generally regarded as serious contributors to water pollution. streptomyces virginiae ibl is an effective degradative strain for many steroidal compounds and can also catalyze the c hydroxylation of diosgenin, the first-ever biotransformation found on the f-ring of diosgenin.  to completely elucidate the hydroxylation function of cytochrome p genes found during biotransformation of steroids by s. virginiae ibl the whole genome sequencing of this strain was carried out via sequencing systems. the analytical results of blastp showed that the strain ibl contains cyps, ferredoxins and ferredoxin reductases in its  mb linear chromosome. cyps from s. virginiae ibl are phylogenetically closed to those of streptomyces sp. mg and streptomyces sp. c. one new subfamily was found as per the fact that the cyp svu in s. virginiae ibl shares 66% identity only to that from streptomyces sp. mg further analysis showed that among all of the cyps in s. virginiae ibl three cyps are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three cyps with atp/gtp binding proteins, four cyps arranged with transcriptional regulatory genes and one cyp located on the upstream of an atp-binding protein and transcriptional regulators as well as four cyps associated with other functional genes involved in secondary metabolism and degradation.  these characteristics found in cyps from s. virginiae ibl show that the exxr motif in the k-helix is not absolutely conserved in cyp family and i-helix not absolutely essential for the cyp structure, too. experimental results showed that both cyp svh and cyp svu are two hydroxylases, capable of bioconverting diosgenone into isonuatigenone and β-estradiol into estriol, respectively. biotransformationcytochrome p450ferredoxinferredoxin reductasegene sequencingsecondary metabolism 