中文摘要：

枯草芽孢杆菌Bacillus subtilis RF1是课题组前期通过基因工程改造得到的一株核黄素高产菌,为了进一步提高核黄素的产量,需要对该菌株进行进一步的基因工程改造.本研究首先将编码的链丝菌素乙酰基转移酶基因sat克隆到p MA5质粒上,构建具有诺瓦丝菌素Norseothricin（NTC）抗性的重组质粒p MA5-sat,实验证实该重组质粒能够用于B.subtilis RF1的抗性筛选.随后将核黄素合成相关的关键酶葡萄糖-6-磷酸脱氢酶编码基因zwf克隆到重组质粒p MA5-sat上,获得重组质粒p MA5-sat-zwf,并成功构建重组菌B.subtilis RF1/p MA5-sat-zwf.结果显示,重组菌胞内葡萄糖-6-磷酸脱氢酶活力比原始菌提高了近50倍,说明葡萄糖-6-磷酸脱氢酶在重组菌中成功过量表达;根据发酵特性分析,重组菌B.subtilis RF1/p MA5-sat-zwf最终核黄素产量达到12.01 g/L,比原始菌B.subtilis RF1提高了30.3%.综上,本研究构建的新型抗性质粒能够成功运用于核黄素生产菌枯草芽孢杆菌的基因工程改造.

英文摘要：

In our previous study, a genetically engineered strain Bacillus subtilis RF1 was constructed for riboflavin production. To enhance riboflavin production, further genetic modification was required to operate on B. subtilis RF1. The sat gene encoded Streptothricin acetyltransferase was inserted into expression vector pMA5, and the recombinant plasmid pMA5- sat was constructed and transformed into B. subtilis RF1. Glucose-6-phosphate dehydrogenase encoded by gene zwfis one of key enzymes involved in pentose phosphate pathway. And gene zwfharbored in the new plasmid pMA5-sat was constructed and was successfully transformed into B. subtilis RF1 to obtain recombinant strain B. subtilis RF1/pMA5-sat-zwf. The intracellular glucose-6-phosphate dehydrogenase activity of recombinant strain B. subtilis RF1/pMA5-sat-zwfwas about 50- fold higher than that of strain B. subtilis RF1. The fermentation results showed that the riboflavin production by recombinant strain B. subtilis RF1/pMA5-sat-zwfreached 12.01 g/L, which was 30.3% higher than that by strain B. subtilis RF1. The results indicate that this new resistance plasmid can be successfully used in riboflavin-producing strain B. subtilis RF 1.