文献类型：期刊文献

中文题名：龟裂链霉菌zwf2基因阻断提高土霉素生物合成

英文题名：Disruption of zwf2 gene to improve oxytetraclyline biosynthesis in Streptomyces rimosus M4018

作者：刘志勇[1];郭美锦[1];钱江潮[1];庄英萍[1];张嗣良[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2008

卷号：48

期号：1

起止页码：21

中文期刊名：微生物学报

外文期刊名：Acta Microbiologica Sinica

收录：MEDLINE(收录号：18338571);CSTPCD;;Scopus;北大核心:【北大核心2004】；CSCD:【CSCD2011_2012】；PubMed;

基金：国家基础科学研究项目(2007CB714303);校优秀青年教师科研基金;上海市重点学科建设项目(B505)~~

语种：中文

中文关键词：葡萄糖-6-磷酸脱氢酶;zwf2;龟裂链霉菌M4018;土霉素生物合成

外文关键词：Glucose-6-phosphate dehydrogenase; zwf2; S. rimosus M4018; Oxytetracycline biosynthesis

摘要：葡萄糖-6-磷酸脱氢酶(G6PDH)是链霉菌磷酸戊糖途径中第一个酶("看家"酶),也是形成NADPH的关键酶,由zwf1和zwf2基因编码。以温敏型质粒pKC1139为基础构建了用于阻断龟裂链霉菌zwf2的重组质粒pKC1139-zwf2′,通过大肠杆菌GM2929去甲基化pKC1139-zwf2′后电转至原始龟裂链霉菌M4018感受态细胞,筛选得到转化子。转化子进一步通过PCR鉴定和点杂交印迹分析鉴定,证明是zwf2基因阻断的阳性突变子命名为M4018-Δzwf2。以原始菌株为对照,突变子摇瓶发酵结果表明:突变子的葡萄糖-6-磷酸脱氢酶酶活是原始菌的50%左右,但土霉素生物合成水平则提高了27%;在细胞生长方面,二者均在第4d进入生长稳定期而开始大量合成土霉素,发酵结束时细胞菌体浓度基本相同,但突变子的单位菌丝体土霉素生物合成能力则提高了31%。因此,zwf2的阻断有利于土霉素的生物合成,而对细胞生长没有明显影响。
Genes of zwfl and zwf2 encode two isozymes of glucose-6-phosphate dehydrogenase （G6PDH） of Streptomyces, respectively. G6PDH is the first enzyme in the oxidative pentose phosphate pathway （PPP） and the key enzyme for NADPH generation.Based on thermal sensitive plasmid pKCl139, a recombinant plasmid pKC1139-zwf2＇ was constructed and verified with restriction enzyme digestion. The plasmid pKC1139-zwf2＇ was electropolated into competent Streptmyces rimosus M4018 cells after it was demethylated by E.coli GM2929. Transformants grown on Tryptone Soya Agar （TSA） plate containing 500ug/mL apramycin were selected, and identified using dot hybridization analysis and PCR amplification with apramycin resistant gene as primers. A positive clone was then selected and designated M4018-Δ zwf2. With parent strain S. rimosus M4018 as control, mutant M4018-Δzwf2 was cultured in shaking flask. Specific acitivity of G6PDH of M4018-Δ zwf2 was only half of that of parent strain whereas yield of oxytetracycline （OTC） of mutant was 27% higher, to the mutant had a similar biomass profileto that of the control biosynthesis started when the growth entered stationary phase on the 4th day. However, specific oxytetracycline production of mutant was 31% higher thanthat of the parent strain, indicating that zwf2 disruption could enhance oxytetracycline biosynthesis in S. rimosus M4018-Δ zwf2.