Abstract

A82846B, producing by Kibdelosporangium aridum, is an important precursor of the semi-synthetic glycopeptide antibiotic Oritavancin. K. aridum produces three components A82846A, B and C, so it is essential to increase A82846B titer and reduce A82846A and C titers by overexpressing halogenase and glycosyltransferase genes. Firstly, we constructed the genetically engineered strain SIPI-3927-attB harboring artificial attB site via homologous recombination. Secondly, two strains SIPI-3927-C1 and C2 were also constructed by integrating halogenase genes vcm8 and orf10 into artificial attB sites of SIPI-3927-attB, respectively. Meantime, three strains SIPI-3927-C3, C4 and C5 containing overexpressing glycosyltransferase A, B and C genes were obtained, respectively. Through fermentation analyses, the results showed that SIPI-3927-C1 and C2 could increase A82846B ratios, in which SIPI-3927-C1 showed a better performance. Moreover, the titer of SIPI-3927-C3 was highest in those of three strains. Finally, the strain SIPI-3927-C6 was constructed by integrating both orf10-encoded halogenase and orf11-encoded glycosyltransferase A, of which the yield and ratio of A82846B in shake-flask fermentation reached 1200 mg/L and 73.6%, respectively. Besides, the yield and ratio of A82846B in SIPI-3927-C6 grew up to 2520 mg/L and 86.5% in the 5-L fermenter culture, respectively. In conclusion, overexpressing orf10 gene can increase A82846B ratio,while overexpressing orf11 gene can increase A82846B titer as well. The artificial attB site is effective for inserting new genes.

中文翻译：

---

通过人工attB辅助在拟南芥中SIRF-3927的orf10 – orf11基因的过表达增强A82846B的生产

摘要

姬松茸（Kibdelosporangium aridum）生产的A82846B是半合成糖肽抗生素奥利万星的重要前体。K. aridum产生三种成分A82846A，B和C，因此必须通过过量表达卤化酶和糖基转移酶基因来提高A82846B的效价并降低A82846A和C的效价。首先，我们通过同源重组构建了带有人工attB位点的基因工程菌株SIPI-3927- attB。其次，两个菌株SIPI-3927-C1和C2也通过halogenase基因整合构建vcm8和ORF10到人工attB位SIPI-3927-位点attB位， 分别。同时，分别获得了三个含有过表达糖基转移酶A，B和C基因的菌株SIPI-3927-C3，C4和C5。通过发酵分析，结果表明SIPI-3927-C1和C2可以提高A82846B的比例，其中SIPI-3927-C1具有更好的性能。此外，SIPI-3927-C3的滴度在三种菌株中最高。最后，通过整合orf10编码的卤化酶和orf11编码的糖基转移酶A构建SIPI-3927-C6菌株，其在摇瓶发酵中的A82846B的产量和比例分别达到1200mg / L和73.6％。此外，在5-L发酵罐中，SIPI-3927-C6中A82846B的产量和比例分别增长到2520 mg / L和86.5％。总之，过分表达orf10基因可以增加A82846B的比例，而过表达orf11基因也可以增加A82846B的效价。人工attB位点可有效插入新基因。