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RISS 인기검색어
- 검색키워드
- 발행기관 : Sun Moon Univ. (검색결과 13 건)
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Shrestha, Pramod Sun Moon Univ. 2008 국내박사
Nineteen cytochrome P450s (CYP) along with two ferredoxins (FDX) and four ferredoxin reductases (FDR) have been reported in Streptomyces peucetius ATCC 27952. However, genome annotations need to be regularly updated if the information they contain is to remain accurate and relevant. Following the homology searches based on conserved signature sequences necessary for CYP, revised analyses of S. peucetius ATCC 27952 genome database available in our laboratory revealed four more CYPs and correction in sequences of few previously known CYPs making twenty three CYPs in total. We also found complete sequences of three pseudo CYP whose heme-binding motifs were either missing or unidentified. Moreover, one FDR and two previously reported FDXs were found to be mistaken. Fdx808 reported as ferredoxin has been found as NADH oxidoreductase, experimentally. We also identified four more FDRs and six new FDXs. So, seven FDRs and six FDXs were found to be present totally in S. peucetius ATCC 27952 to support twenty three cytochrome P450s. Even though numbers of FDXs and FDRs have been reported, sufficient details about the relevant homologous electron transfer system in Streptomyces P450s is unclear. Since CYPP166B1 was clustered with putative ferredoxin FDX6, we expressed and purified six ferredoxin reductase along with them and envisaged the primary pathway NADH→FDR4→FDX6→CYP166B1 for dealkylation of 7- ethoxycoumarin, however, we are unsuccessful to express FDR1. Consequently, further mechanistic studies with the identification of endogenous substrate for CYP166B1 would give more clear explanation. As we know that cytochrome P450s are responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. We expressed several CYPs from S. peucetius and CYP105F2 overexpress in E. coli BL21 (DE3) pLysS, indicated by alkali treated assay, could have activity with oleandomycin. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA further signified its broad substrate specificity towards structurally diverse compounds. Invitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation. Further, a biotransformation system was designed to co-express CYP107P3 (CSP4), cytochrome P450, from S. peucetius, along with CamA (putidaredoxin reductase) and CamB (putidaredoxin) from Pseudomonas putida, the necessary reducing equivalents; in a class I type electron-transfer system in E. coli BL21 (DE3). This was carried out using two plasmids with different selection markers and compatible origins of replication. The study results showed that this biotransformation system was able to mediate the O-dealkylation of 7- ethoxycumarin. Key words: Streptomyces peucetius ATCC 27952, Cytochrome P450, Sequence analysis, Ferredoxin/Ferredoxin reductase, electron transport system, whole-cell biotransformation
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Biosynthesis of spectinomycin from Streptomyces spectabilis and heterologous expression
Laxmi Prasad Thapa Sun Moon Univ. 2008 국내박사
Spectinomycin is an aminocyclitol antibiotic produced by Streptomyces spectabilis and may be regarded as part of a subclass of aminoglycoside antibiotics. Spectinomycin is composed of spectinamine which is derived from myo-inositol and actinospectose derived from TDP-glucose. It is bacteriostatic and exerts its antibacterial effect by binding to the 30S ribosome which disrupts bacterial protein synthesis. Spectinomycin is primarily effective against Gram-negative bacteria such as Pasteurella haemolytica, Pasteurella multocida and Haemophilus somnus. It has limited activity against a number of Gram-positive bacteria. Generally, it is not active against anaerobic bacteria. The heterologous expression of the SPC8 cosmid, containing the spectinomycin biosynthetic gene cluster was carried out in S. venezuelae YJ003 and the production of spectinomycin and its derivatives were confirmed by ESI-mass spectrometry, LC-mass spectrometry, high performance liquid chromatography of isolated secondary metabolites from S. venezuelae YJ003/ SPC8, respectively. Similarly, the pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the resistance genes and regulatory genes. It was heterologously expressed in S. venezuelae YJ003 and the isolated compound was analyzed by ESI-mass spectrometry, LC-mass spectrometry, high performance liquid chromatography, mass-mass spectrometry, and shows a molecular weight of 485 amu as kanamycin A. The analysis of 17 kb nucleotide sequence of spectinomycin biosynthesis gene cluster of S. spectabilis revealed 16 open reading frames (ORFs) and which include biosynthetic genes, resistance genes and regulatory genes. Among them, three genes myo-inositol-1-monophosphatase (spcA), myo-inositol dehydrogenase (spcB) and aminotransferase (spcS2) were cloned into pSET152 vector from the spectinamine biosynthesis pathway for the construction of recombinant plasmid pSLT and expressed into S. venezuelae YJ003. The isolated secondary metabolite from S. venezuelae YJ003/pSLT was confirmed by ESI-mass spectrometry, LC-mass spectrometry and high performance liquid chromatography, and shows a molecular weight of 179 amu as streptamine. The production of the streptamine from strain S. venezuelae YJ003/pSLT proved that these three genes spcA, spcB and spcS2 are sufficient for the biosynthesis of streptamine from glucose-1-phosphate pathway and it is also concluded that myo-inositol dehydrogenase and aminotransferase has dual character. Similarly, the enzyme spcM, which encodes as an N-methyltransferase in the biosynthetic gene cluster of S. spectabilis ATCC27741 catalyzes the transfer of a methyl group from S-Adenosyl-L-methionine to the amino groups of streptamine in biosynthetic pathway of spectinomycin. The plasmid PSEM was constructed by cloning spcM gene in pSET152 vector and integrated on the ribosome of S. kanamyceticus. The engineered aminoglycosides: N-methylated kanamycin was analyzed by ESI-mass, high performance liquid chromatography and tandem mass-mass spectrometry, has molecular weight of 527 amu, respectively. The minimal inhibitory concentration of the trimethylated kanamycin also compared with standard kanamycin A and found slightly lower activity than standard kanamycin A. The production of the tri-methylated kanamycin A proved that spcM acts as a flexible N-methyltransferase. The production of the streptamine by co-expressing the genes spcA, spcB and spcS2 may help to generate the hybrid aminoglycoside antibiotics. Similarly, the production of the tri-methylated kanamycin by integrating the spcM (N-methyltransferase) gene in the ribosome of S. kanamyceticus suggested that the methylation may be takes place after the glycosylation step in the biosynthesis pathway of spectinomycin. The production of spectinomycin and kanamycin by heterologous expression of their respective gene cluster SPC8 and pSKC2 concluded that these gene clusters contain all necessary genes for the biosynthesis of spectinomycin and kanamycin A, respectively.
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