【摘要】：Nonomuraea sp.ATCC 39727可以通過次級代謝生產(chǎn)A40926 B組分,該物質(zhì)可以作為生產(chǎn)新型糖肽類抗生素達巴萬星的前體,這種抗生素與替考拉寧結(jié)構(gòu)類似,在臨床試驗中,其活性更強,半衰期更長。本研究在實驗室前期經(jīng)過誘變改造、原生質(zhì)體誘變得到的突變菌株A416-B4的基礎(chǔ)上,對其進行基因工程改造,以期簡化生產(chǎn)工藝并提高A40926 B產(chǎn)量,為工業(yè)化生產(chǎn)奠定基礎(chǔ)。首先,本研究針對突變株A416-B4進行了接合轉(zhuǎn)移(基因敲除)遺傳操作系統(tǒng)的優(yōu)化。在優(yōu)化后遺傳操作系統(tǒng)的基礎(chǔ)上,構(gòu)建敲除乙酰轉(zhuǎn)移酶基因dbv23敲除質(zhì)粒pKdbv23,利用同源重組雙交換敲除dbv23基因,得到工程菌A416-△dbv23。對該工程菌進行發(fā)酵驗證,發(fā)現(xiàn)其發(fā)酵主產(chǎn)物為A40926 B組分,而不再產(chǎn)生A40926 PB組分,且A40926 B組分搖瓶發(fā)酵單位達到1577 mg/L,較出發(fā)菌株A416-B4提高了94%。構(gòu)建含兩個調(diào)節(jié)基因dbv3、dbv4的整合型表達質(zhì)粒,將兩個質(zhì)粒整合到A416-△dbv23中,得到二株工程菌株:A416-△dbv23-dbv3、A416-△dbv23-dbv4。對工程菌的發(fā)酵情況進行考察,確定導(dǎo)入dbv4基因效果略優(yōu)于dbv3,A416-△dbv23-dbv4的發(fā)酵單位最高,搖瓶單位達到1707 mg/L,較A416-△dbv23提高了8.2%,較出發(fā)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.
【學(xué)位授予單位】：上海醫(yī)藥工業(yè)研究院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78;TQ927

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