請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/21213
標題: Pseudonocardia autotrophica BCRC12444的cytochrome P450 monooxygenase基因以及脂肪酸代謝基因串
Cytochrome P450 monooxygenase gene and a gene cluster for the fatty acid catabolism from Pseudonocardia autotrophica BCRC12444
作者: 陳昭賢
Chen, Chao-Hsien
關鍵字: Pseudonocardia autotrophica
Pseudonocardia autotrophica
cytochrome P450 monooxygenase
gene expression
bioconversion
fatty acid catabolism
gene regulation
cytochrome P450 monooxygenase
基因表現
生物轉換
脂肪酸代謝
基因調控
出版社: 分子生物學研究所
摘要: (一) Pravastatin是一種高脂血症治療劑,可降低血中膽固醇,商業化製造包括兩個步驟,首先利用微生物生產compactin,接著將compactin 氫氧化而產生pravastatin。 Compactin生成可由Penicillium citrinum大量生產而得,然而,compactin的氫氧化必需靠cytochrome P450的催化才會轉變成pravastatin。如果利用化學合成法將compactin轉變成pravastatin,其效果不佳,易將部份產物轉成其他異構物,而且所需的成本非常的高。若用微生物來轉換compactin,可有效的在6 b位置氫氧化而形成pravastatin。在本研究希望尋找具有高轉換能力的菌種,選殖其cytochrome P450 monooxygenas的基因,利用轉形菌株大量轉換compactin形成pravastatin,來替代化學合成法的傳統方式。利用HPLC方法分析,雖然許多細菌都具有轉換compactin的能力,但只有P. autotrophica BCRC 12444及Steptomyces griseolus BCRC 13677真正能將compactin轉成pravastatin,雖然S. griseolus BCRC 13677的轉換能力較P. autotrophica BCRC 12444強,但較不穩定,pravastatin易再轉成其他產物,故選擇P. autotrophica做為標的菌株。針對此菌設計cytochrome P450 monooxygenase 基因的退化性引子,利用PCR方法找到一屬於此基因的DNA片段,利用此片段做為探針,以plaque hybridization方法,由 P. autotrophica的基因庫篩選二個含cytochrome P450 monooxygenase 基因的轉殖株,經定序及上網分析比對,整合出一含有六個基因的DNA片段,六個基因分別為acyl-CoA ligase (fadA)、enoyl-CoA hydratase (fadB)、transcriptional regulator (fadR)、cytochrome P450 monooxygenase (fadC)及ferredoxin (fadD)及hypothetical protein基因。純化的cytochrome P450 monooxygenase及ferredoxin無法將將compactin轉換成pravastatin,以RNA即時定量 PCR方法測試fadC基因的表現,發現此基因的表現不會因為compactin的誘導而提升。 (二) 從Pseudonocardia autotrophyca BBRC12444篩選到一段參與脂肪酸代謝的基因串,以各種DNA序列分析方法及引子延伸反應分析,此基因串除了六個ORF外,還包含一個雙向的啟動子。此fad基因串上被確定的五個基因分別命名為fadA、fadB、fadR、fadC及 fadD,基於其胺基酸的相似度,各基因的產物分別為acyl-CoA ligase (FadA)、enoyl-CoA hydratase (FadB)、transcriptional regulator (FadR)、cytochrome P450 monooxygenase (FadC)及ferredoxin (FadD)。以膠體泳動遲滯技術法及DNase I footprinting分析法,證實調節蛋白FadR可結合在位於fadR及fadC之間的雙向的啟動子區域的操縱子序列上,顯示此蛋白可調控脂肪酸的代謝作用。以即時定量PCR的方法證實了長鏈脂肪酸可誘導fadA、fadB、fadR及fadC基因的表現,而葡萄糖則會抑制這四個基因的表現,所有的數據均顯示此fad基因串與脂肪酸的代謝有關。
(1) Cholesterol is synthesized from acetyl-coenzyme A (Co A) in a pathway that includes more than 20 enzymatic steps. The rate-limiting step of this pathway is catalyzed by 3-hydroxy-3-methygultaryl (HMG)-CoA reductase. Pravastatin is an effective and tissue-selective hypolipidemic drug that inhibits the enzyme in cholesterol synthesis and is commonly used as cholesterol lowering therapeutic medicine. Pravastatin is produced by a two-step fermentation process. The first step is production of compactin by microorganisms and the second is a hydroxylation step that converts compactin to provastatin. Penicillium citrinum can be used as a compactin-producing strain. The cytochome P450-dependent monooxygenase systems are found to participate in the hydroxylation mechanism. Although many microorganisms were found with potently convertible properties, P. autotrophica could potently convert compactin to pravastatin and was used to clone the p450 monooxygenase gene from it. Three degenerate primers were designed from the conserved region of p450 monooxygenase gene. A DNA fragment was amplified from P. autotrophica using nested polymerase chain reaction. DNA sequence analysis revealed that this DNA fragment is part of p450 monooxygenase gene and can be used as a probe to screen for the clones that harbored the cytochrome P450 monooxygenase gene from P. autotrophica genomic library. Two positive clones that contained the plasmid were obtained. Analysis of nucleotide sequence of the inserts revealed that contained six ORFs predicted as acyl-CoA ligase (fadA), enoyl-CoA hydratase (fadB), transcriptional regulator (fadR), cytochrome P450 monooxygenase (fadC), ferredoxin (fadD) and hypothetical protein genes. The cytochrome p450 monooxygenase and ferredoxin genes were cloned into pET32a expression vector and expressed in E. coli Nova Blue. After purification by affinity chromatography, we found that these two enzymes could not convert compactin to pravastatin. Real-time quantitative PCR assays also found that the expression of genes in the cluster could not be induced by compactin. (2) Genes involved in fatty acid degradation (fad) were isolated from Pseudonocardia autotrophyca BBRC12444. Six open reading frames (ORFs) and a bi-directional promoter region were identified by DNA sequence analyses and primer extension. The fad gene cluster included five ORFs, designated fadA, fadB, fadR, fadC and fadD. Base on their amino acid sequence identity, the gene products were identified as acyl-CoA ligase (FadA), enoyl-CoA hydratase (FadB), transcriptional regulator (FadR), cytochrome P450 monooxygenase (FadC) and ferredoxin (FadD). Regulatory protein, FadR, could bind to an operator sequence located in the divergent promoter region between fadR and fadC genes, implicating the control of fatty acids degradation. The real-time quantitative PCR assays revealed that the expression of the fadA, fadB, fadR, and fadC genes was induced by long chain fatty acids and repressed by glucose. All results demonstrated that the fad gene cluster participated in the pathway of the fatty acid catabolism.
URI: http://hdl.handle.net/11455/21213
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