Genetic engineering for Cholesterol Transformation System in Streptomyces

链霉菌胆固醇转化系统的基因工程

• 批准号: 01480036
• 负责人: MUROOKA Yosikatu
• 金额: $ 3.39万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480036/
• 关键词: Streptomyces; Cholesterol oxidase; Transformation of cholesterol; Genetic engineering; Cytochrome P-450; コレステロ-ル酸化酵素; コレステロ-ル転換系; Pー450

Streptomyces species produce a large variety of medically important metabolites and extracellular enzymes, in particular cholesterol oxidase. Cholesterol oxidase catalyzes the oxidation of cholesterol to 4-cholesten-3-one, with the reduction of oxygen to hydrogen peroxide. The enzyme is commonly used for the enzymatic transformation of cholesterol and is alo used for quantitation of levels of cholesterol in clinical specimens.Recently, we cloned the gene involved in the production of cholesterol oxidase (choA) from Streptomyces sp. strain SA-COO. Cells of Streptomyces lividans carrying a plasmid that includes the choA gene overproduce extracellular Cholesterol oxidase when cultured in appropriate medium.We sequenced the complate cholesterol oxidase gene. The sequence of the choA gene revealed that the gene of cholesterol oxidase starts from a GTG initiation codon and terminates at a TAA termination codon. In the 3'-flanking region of choA, a long palin dromic sequence, which may act as … More a transcription terminator, was found. The gene encodes a mature cholesterol oxidase of 504 amino acids with a caluculated Mr of 54,913.The nucleotide sequence of the promoter region of the gene for choA was determined. We found an open reading frame (choP) that has a high degree of similarity to the sequence of the gene for cytochrome P-450s from heman and Pseudomonas species. We determined the product of the choP gene was cytochrome P-450-like protein.We have cloned the chromosomal DNA, which is adjoined upstream and down stream of the choP-choA operon, by guided genomic walking method developed by us. These clones may be involved in cholesterol metabolism.During the study on the decomposition of cholesterol and secretion of heterologous gene products in Streptomyces, we found secretory overproduction of cholesterol oxidase in Streptomyces lividans by a recombinant multi-copy shuttle vector, the results of which are potential useful for production of heterologous gene product extracellularly bu using Streptomyces host-vector system. Less

链霉菌产生多种医学上重要的代谢物和胞外酶，特别是胆固醇氧化酶。胆固醇氧化酶催化胆固醇氧化成4-胆甾烯-3-酮，同时氧被还原为过氧化氢。该酶通常用于胆固醇的酶促转化，也可用于临床标本中胆固醇水平的测定。最近，我们从链霉菌中克隆了与胆固醇氧化酶(ChoA)产生有关的基因。菌株SA-COO。在适当的培养条件下，携带含有ChoA基因的变铅青链霉菌细胞会产生过量的胞外胆固醇氧化酶，我们对其进行了测序。该基因的序列分析表明，胆固醇氧化酶基因起始于GTG起始密码子，终止于TAA终止密码子。在Choa的3‘侧翼区，一个长的Palin序列，可能起到…的作用发现了更多的转录终止子。该基因编码504个氨基酸的成熟胆固醇氧化酶，计算的MR为54,913。测定了该基因启动子区的核苷酸序列。我们发现了一个开放阅读框(CHOP)，它与海曼和假单胞菌的细胞色素P-450基因序列高度相似。利用自行开发的基因组步行法，克隆了CHOP-Choa操纵子上游和下游邻接的染色体DNA。在对链霉菌中胆固醇分解和外源基因产物分泌的研究中，我们发现了重组多拷贝穿梭载体在变青链霉菌中分泌过量的胆固醇氧化酶，这一结果为利用链霉菌宿主载体系统生产外源基因产物奠定了基础。较少

项目成果

Istvan Molanr: "Secretory overproduction of the Streptomyces cholesterol oxidase by Streptomyces lividans with a recombinant multiーcopy vector"

Istvan Molanr：“利用重组多拷贝载体，浅青紫链霉菌分泌过量的链霉菌胆固醇氧化酶”

Yoshikatsu Murooka: "Biotechnology Advances (分担執筆)" Pergamon Press Plc.,Oxford,UK, 29 (1990)

Yoshikatsu Murooka：“生物技术进展（合著者）”Pergamon Press Plc.，牛津，英国，29（1990）

Istvan Molnar et al.: ""Guided genomic walking", a systematic approach to cloning intermediate-sized restriction fragments extending outside the boundaryies of known sequences"

Istvan Molnar 等人：““引导基因组行走”，一种克隆延伸到已知序列边界之外的中等大小限制性片段的系统方法”

Yoshikatsu, Murooka: "Biotechnology Advances" Pergamon Press Plc., Oxford, UK. Vol8. 29-57 (1990)

Yoshikatsu, Murooka：“生物技术进步”Pergamon Press Plc.，牛津，英国。

Istv^^'an Moln^^'ar: "“Guided genomic walking",a systematic approach to cloning intermediateーsized restriction fragments extending outside the boundaries of known sequences"

Istv^^an Moln^^ar：““引导基因组行走”，一种克隆延伸到已知序列边界之外的中等大小限制性片段的系统方法”