Enhancing A82846B production by artificial attB-assisted overexpression of orf10–orf11 genes in Kibdelosporangium aridum SIPI-3927

Abstract

AbstractA82846B, 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.