Overexpression of wysR gene enhances wuyiencin production in ΔwysR3 mutant strain of Streptomyces albulus var. wuyiensis strain CK-15.

Abstract

The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK-15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin. We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin-resistant overexpression strain was confirmed by PCR using the Apra-F and Apra-R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant. We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real-time RT-PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development. Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.