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

Abstract

Aim: 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 DwysR3 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. Methods and Results: We developed a method to create a wysR overexpression strain based on the DwysR3 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 DwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycinresistant 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 DwysR3 mutant. Conclusion: We can conclude that overexpression of wysR gene in DwysR3 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. Significance and Impact of the Study: Overexpression of wysR gene in the DwysR3 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.

Effect of PAS-LuxR Family Regulators on the Secondary Metabolism of Streptomyces

“…CFA-L-Ile is an amino acid analogue, and the CFA-L-Ile gene cluster consists of two PKS genes ( cfa6 and cfa7 ), one acyl carrier protein (ACP) gene ( cfa1 ), two acyl coenzyme A ligase genes ( cfa5 and cfal ), one oxidoreductase gene ( scab79681 ), a cytochrome P450 monooxygenase gene ( sabc79691 ), two reductase genes ( cfa8 and sabc79721 ), a hydroxybutyryl coenzyme A dehydrogenase gene ( sabc79711 ), and two regulatory genes ( orf1 and cfaR , which encode a PAS-LuxR family protein) [ 14 ]. Wuyiencin is an aminoglycoside antibiotic [ 15 , 16 ] that was mainly studied in biocontrol. The gene cluster has only been partially sequenced and it contains PAS-LuxR gene wysR .…”

“…The transcript levels of wysE (thioesterase), wysRI (LAL family regulator), and wysRIII (DeoR family regulator) were analyzed in the wuyiencin biosynthetic gene cluster, and the results show that the transcript levels of wysRIII and wysE are remarkably lower in wysR deletion strain than that in the wild-type strain, while the transcript levels of wysRI remarkably increased compared with wild-type strains [ 16 ]. In the wysR overexpressing strain, the transcript levels of wysRIII and wysE remarkably increased, whereas the transcript levels of wysRI are significantly reduced compared with the wild-type strain [ 15 ]. Therefore, WysR directly or indirectly regulates the expression of other potential regulatory genes such as wysRI and wysRIII to affect the transcription of early wuyiencin PKS genes and regulates wysE to affect the late biosynthetic enzymes in wuyiencin production [ 16 ].…”

Wuyiencin produced by Streptomyces albulus CK–15 displays biocontrol activities against cucumber powdery mildew

“…Wuyiencin was separated and purified by Sephadex G-25 and ODS column chromatography and semipreparative high-performance liquid chromatography. These procedures were performed according to the methods of Wei et al (2020).…”

Characterization and engineering of Streptomyces griseofuscus DSM 40191 as a potential host for heterologous expression of biosynthetic gene clusters

“…In order to unravel the biosynthetic richness of Actinobacteria, multiple genetic engineering methods, e.g. induction of BGCs by co-expression of regulatory genes 7 , 8 , use of transcription factor decoys 9 , refactoring of the BGCs 10 , 11 or knockouts of the core genes of “constitutively” produced compounds 12 have been explored. Recently, broader attention has been brought to the development of heterologous expression hosts for the discovery and characterization of novel metabolites.…”