枯草桿菌F29-3豐原素合成酶基因的研究

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Title:	枯草桿菌F29-3豐原素合成酶基因的研究 Study of Fengycin Synthetase Genes from Bacillus subtilis F29-3
Authors:	傅惠絹 Hui-Chuan Fu
Contributors:	林翠品劉世東嘉南藥理科技大學：生物科技研究所
Keywords:	豐原素枯草桿菌 F29-3Fengycin Bacillus subtilis F29-3
Date:	2004
Issue Date:	2008-10-15 17:20:55 (UTC+8)
Abstract:	豐原素（Fengycin）是由炫饃黖筦29-3所產生的一種脂胜肽類抗真菌的抗生素，它的結構含有十個胺基酸和一長鏈的脂肪酸。胜肽的部分是由 FenC、FenD、FenE、FenA、FenB等五個豐原素合成酶，以非核醣體的方式產生。在過去的研究顯示 FenD含有FenD1和FenD2兩個胺基酸的間隔區，每個間隔區包含 condensation、adenylation及 thiolation domain;此外 FenD2的C端還具有 epimerization domain。此研究以鎳螯合的親合性管柱純化大腸桿菌表現含有 condensation、adenylation及 thiolation domain 的FenD1 (CAT-FenD1)和含有 adenylation domain 的FenD2 (AD-FenD1)，生化反應的結果證明FenD1和FenD2分別可以活化豐原素第三及第四個胺基酸－L-Tyr和 L-Thr。在研究Iturin的起始胜肽合成例子中一般認為可能需要acyl-CoA synthetase和 acyl-carrier protein幫助脂肪酸部分的合成，而且由Tn917轉位實驗及序列分析在 fen operon 下游有fenL基因，對於豐原素的合成是需要的，而且它的弁鈱cyl-CoA synthetase。因此本研究將破壞fenL來證明fenL基因的重要性。同時以大腸桿菌大量表現FenL蛋白質並且利用親和性管柱進行純化70 kDa的FenL。 Fengycin is a lipopeptidic antifungal antibiotic produced by Bacillus subtilis F29-3. This antibiotic contains ten amino acids and a long-chain fatty acid residue. This peptide is synthesized nonribosomally by five fengycin synthetases, including FenC, FenD, FenE, FenA and FenC. Earlier studies show that FenD contains two amino acid modules, FenD1 and FenD2, each of which contains a condensation, adenylation and thiolation domain, that are typically present in peptide synthetases. In addition, FenD2 also contains an epimerization domain in C-terminal region. A protein containing the condensation, adenylation and thiolation domains of FenD1 (CAT-FenD1) and adenylation domain of FenD2 (AD-FenD2) was expressed in Escherchia coli and purified by Ni-chelate affinity chromatography. Biochemical analysis revealed that FenD1 and FenD2 activate the third and the fourth amino acid in fengycin, L-Tyr and L-Thr, respectively. In the case of iturin synthesis, initiation of peptide synthesis involves fatty acid synthesis and the functiosn of acyl-CoA synthetase and acyl carrier protein. Our earlier Tn917 mutagenesis studies had identified a gene, fenL , which encodes a protein with a sequence similar to acyl-CoA synthetase. Therefore, this investigation mutagenizes fenL and demonstrates the importance of the gene. Meanwhile, the fenL was expressed in Escherchia coli and purified by affinity chromatography with a His resin. The overexpressed recombinant protein has an estimated molecular mass of 70 kDa.
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