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.
Highlights
Oritavancin is a semi-synthetic antibiotic, which was recently approved by the Food and Drug Administration (FDA) (Brade et al 2016; Corey et al 2014) for treatment of gram-positive pathogens induced Acute Bacterial Skin and Skin Structure Infections (ABSSSIs) and Methicillinresistant Staphylococcus Aureus (MRSA) (Edelsberg et al 2014; Kaatz et al 1998; Rupp et al 2001)
The attB/P integration system was widely used in Streptomyces, which depends on homologous recombination between the attB site and the attP site mediated by the ΦC31 integrase (Patricia et al 2002)
We provide a shortcut for recombinant strains construction by inserting an artificial attB site into K. aridum chromosome via homologous recombination
Summary
It is first time to construct engineering strains of K. aridum for A82846B production. The attB-site has inserted into K. aridum SIPI-3927 by homologous recombination. The double-gene overexpressing engineered strain was constructed. The highest titer of A82846B was obtained in K. aridum SIPI-3927-C6
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