Strategy for Producing the High-Quality Glycopeptide Antibiotic A82846B in Amycolatopsis orientalis Based on the CRISPR-Cas12a System.

Oritavancin is a new-generation semisynthetic lipoglycopeptide antibiotic used to prevent the spread of vancomycin-resistant Gram-positive bacteria. The glycopeptide A82846B is the direct precursor of oritavancin. Considering the structural similarity between A82846B and vancomycin, the vancomycin producer Amycolatopsis orientalis was used as a chassis for the construction of a strain producing high-quality A82846B. To construct the A82846B synthetic pathway, we established a highly efficient CRISPR-Cas12a system by optimizing the conditions of conjugation and by screening the regulatory elements in the A. orientalis, which is difficult to be genetically manipulated. The efficiency of gene knockout was almost 100%. The glycosyltransferases module (gtfDE) and glycosyl synthesis module (vcaAEBD) in the vancomycin gene cluster were replaced with the corresponding glycosyltransferases module (gtfABC) and glycosyl synthesis module (evaAEBD) in the A82846B cluster, respectively. A82846B was successfully produced by the artificially constructed synthetic pathway. Moreover, the titer of A82846B was increased 80% by expressing the pathway-specific regulatory strR. This strategy has excellent potential for remodification of natural products to solve antibiotic resistance.