龟裂链霉菌zwf2基因阻断提高土霉素生物合成

Improvement of oxytetraclyline biosynthesis in S.rimosus M4018 with zwf2 disruption

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。

Abstract:

Genes of zwf1 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 pKC1139, 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 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-△zwf2. With parent strain S. rimosus M4018 as control, mutant M4018-△zwf2 was cultured in shaking flask. Specific acitivity of G6PDH of M4018-△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 Oxytetracycline 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-△zwf2.