• Journal of Microbiology and Biotechnology
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• 제7권6호
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• pp.373-377
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• 1997

The gene for isopenicillin N synthase (cyclase; IPNS) was cloned from Lysobacter lactamgenus using DNA probe amplified with primers based on the consensus sequences of isopenicillin N synthase genes of other ${\beta}$-lactam-producing microorganisms. The genomic library of L. lactamgenus using pUC18 plasmid cloned at the SacI site were screened with the PCR-generated DNA probe and three positive clones were isolated. Enzyme activities in E. coli clones were confirmed by bioassay and HPLC assay. Throughout the functional mapping, it was observed that the gene for isopenicillin N synthase is located at the 1.3-kb XhoI-BamHI fragment of insert of positive clones. Nucleotide sequencing at both ends of the XhoI-BamHI fragment revealed that IPNS of L. lactamgenus has the common amino acid sequences at amino- and carboxy-termini.

• Archives of Pharmacal Research
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• 제15권3호
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• pp.234-238
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• 1992

Using cell-free extract of Lysobacter lactamgenus, enzymatic conversion of $\delta$-L-($\alpha$-aminoadiphyl)-L-cysteinyl-D-valine (ACV) the first substrate of $\beta$-lactam biosynthesis, into antibiotic compounds was attempted. In high performance liquid chromatographic (HPLC) analysis, the biosynthetic intermediates for cephalosporin antibiotics including isopenicillin N, deacetoxycephalosporin C, deacetylcephalosporin C and unknown cephem compound were detected in reaction mixtures. It implies that cephabacin compounds from L lactamgenus could be produced by biosynthetic routes through penicillin ring formation and its expansion to cephalosporin ring, likely as cephalosporin C from Cephalosporium or cephamycin C from Streptomyces. Among biosynthetic enzyme in cell-free extract, the ring formation activity (isopenicillin N synthetase activity) was separated in 50-60% of ammonium sulfate fraction, and ring expansion activity (deacetoxycephalosporin C synthetase activity) was found to be in 40-50% fraction. The partially purified isopenicillin N synthetase could convert as much as 90% ACV to isopenicillin N during 6-hour reaction.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.635-638
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• 2004

An ATP-binding-cassette (ABC) transporter gene in the cephabacin biosynthetic gene cluster of Lysobacter lactamgenus was characterized. The amplified orf10 (cpbJ) gene was subcloned into pET-28a(+) vector and expressed in E. coli BL21(DE3) strain by 0.5 mM IPTG at $30^{\circ}C$. The membrane fraction of recombinant E. coli cells was separated by ultracentrifugation, and solubilized using 2.5% octyl-$\beta$-D-glucoside. Using the solubilized membrane fraction, the artificial proteoliposomes were reconstituted and analyzed for the biological activity of CpbJ protein. Upon measuring ATPase activity, the proteoliposome made from recombinant E. coli membrane proteins showed slightly higher activity than that from host E. coli membrane proteins. In the measurement of membrane transport activity, the reconstituted proteoliposome of recombinant E. coli membrane proteins exhibited higher activity when both substrates of cephalosporin C and L-Ala-L-Ser were applied, compared to the case of cephalosporin C or L-Ala-L-Ser only. It implies that the CpbJ protein is an ABC transporter secreting cephabacin antibiotics synthesized in cytoplasm.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.160.1-160.1
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• 2003

In order to confirm the biological function of ORF10 in cephabacin biosynthesis gene cluster of Lysobacter lactamgenus as an ATP-binding-cassette (ABC) transporter, the gene for ORF10 was amplified and subcloned into pET-28a(+) expression vector. After gene induction with 0.5 mM IPTG at 30~! and further cultivation at $30^~$ !. for 8 hr, a lot of the recombinant ORF10 protein was produced as soluble form in cytoplasmic fraction as well as a membrane protein in the membrane fraction as likely as other ABC transporters. (omitted)

• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.153-159
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• 2001

In order to clone the peptide synthetase gene form Lysobacter lactamgenus IFO 14,288, the gene fragments were amplified using primers for the adenylation domain and the thionylation domain of the peptide synthetase genes in other organisms by polymerase chain reaction (PCR). The resulting 0.5-kb fragment was cloned in a pGEM-T vector, and the nucleotide sequences were determined. Six different PCR products were obtained; three were identified to be a part of L-$\alpha$-aminoadipyl-L-cysteinyl-D-valine (ACV) synthetase and three to be other peptide synthetases. Using each of the two different classes of PCR products as mixed probes, a cosmid library of L. lactamgenus chromosomal DNA constructed in a pHC79 vector was screened by an in situ hybridization procedure, and one positive clone was selected which was bound by peptide synthetase gene fragments as well as ACV synthetase gene fragments. The partial sequence analysis formt he obtained pPTS-5 cosmid showed th presence of more than two open reading frames. These were for two putative membrane transporters, which were homologous with several integral membrane proteins including the ABC transporter ATP-binding protein of E. coli (YbjZ) and the metal ion uptake protein of Bacillus subtilis (YvrN). A 45% homology was also found between the two transporter proteins at the carboxy terminus. Through a hydropathy analysis and transmembrane analysis. 4-5 transmembrane domains were found in these two proteins. When the genes were expressed in Escherichia coli, the gene products inhibited the hose cell growth, probably due to the disturbance of the membrane transport system.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.427-433
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• 2008

The cephabacins produced by Lysobacter lactamgenus are ${\beta}$-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, ${\beta}$-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of $His_6$-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-L-Ala-L-$^3H$-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with $^{14}C$-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.