达巴万星前体A40926 B生产菌种的基因工程改造

发布时间：2018-10-11 11:06

【摘要】：Nonomuraea sp.ATCC 39727可以通过次级代谢生产A40926 B组分,该物质可以作为生产新型糖肽类抗生素达巴万星的前体,这种抗生素与替考拉宁结构类似,在临床试验中,其活性更强,半衰期更长。本研究在实验室前期经过诱变改造、原生质体诱变得到的突变菌株A416-B4的基础上,对其进行基因工程改造,以期简化生产工艺并提高A40926 B产量,为工业化生产奠定基础。首先,本研究针对突变株A416-B4进行了接合转移(基因敲除)遗传操作系统的优化。在优化后遗传操作系统的基础上,构建敲除乙酰转移酶基因dbv23敲除质粒pKdbv23,利用同源重组双交换敲除dbv23基因,得到工程菌A416-△dbv23。对该工程菌进行发酵验证,发现其发酵主产物为A40926 B组分,而不再产生A40926 PB组分,且A40926 B组分摇瓶发酵单位达到1577 mg/L,较出发菌株A416-B4提高了94%。构建含两个调节基因dbv3、dbv4的整合型表达质粒,将两个质粒整合到A416-△dbv23中,得到二株工程菌株:A416-△dbv23-dbv3、A416-△dbv23-dbv4。对工程菌的发酵情况进行考察,确定导入dbv4基因效果略优于dbv3,A416-△dbv23-dbv4的发酵单位最高,摇瓶单位达到1707 mg/L,较A416-△dbv23提高了8.2%,较出发菌株A416-B4提高了110%。
[Abstract]:Nonomuraea sp.ATCC 39727 can produce component A40926B by secondary metabolism, which can be used as precursor of dabavanxin, a new glycopeptide antibiotic, which is similar to teicoplanin in structure and has stronger activity and longer half-life in clinical trials. On the basis of mutagenesis and protoplast mutagenesis of mutant strain A416-B4 in the early stage of laboratory, genetic engineering was carried out to simplify the production process and increase the yield of A40926B, thus laying a foundation for industrial production. First of all, the genetic operating system of A416-B4 was optimized for conjugation transfer (knockout). On the basis of the optimized genetic operating system, the dbv23 knockout plasmid pKdbv23, of knockout acetyltransferase gene was constructed, and the engineering strain A416- dbv23. was obtained by using homologous recombination double exchange knockout dbv23 gene. The main product of the fermentation was A40926B, but no A40926 PB was produced, and the fermentation unit of A40926B in shaking flask reached 1577 mg/L, which was 94 494% higher than that of the original strain A416-B4. An integrated expression plasmid containing two regulatory genes, dbv3,dbv4, was constructed, two plasmids were integrated into A416- dbv23, and two engineering strains, A416- dbv23-dbv3,A416- dbv23-dbv4., were obtained. The fermentation of engineering bacteria was investigated. The results showed that the effect of introducing dbv4 gene was slightly better than that of dbv3,A416- dbv23-dbv4. The shaking flask unit reached 1707 mg/L, and increased 8.2% compared with A416- dbv23, and 110% higher than the original strain A416-B4.
【学位授予单位】：上海医药工业研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;TQ927

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