Cloning and expression of pkg1 gene from the GH55 family of mycoparasite Pestalotiopsis kenyana PG52

Mengling Yan^1,2*Wenjiing Sui^1,2Chen Chen^1,2Ruotian Gao^1,2Jinqiu Li^1,2Jing Li^1,2*

• ¹College of Biological Science and Food Engineering, Southwest Forestry University, Kunming, Yunnan, China
• ²Forest Resources Exploitation and Utilization Engineering Research Center for Grand Health of Yunnan Provincial Universities, Southwest Forestry University, Kunming, Yunnan, China

Abstract: β-1,3-glucanases are involved in degrading the cell wall of phytopathogenic fungi and can be used to control plant diseases. Our research group previously predicted that Pestalotiopsis kenyana PG52 has more GH55 family genes than Pestalotiopsis sp. CR013. Therefore, their identification and expression were analyzed to screen the glucanase genes that may be involved in mycoparasitism. With bioinformatics methods, the GH55 gene family has been identified and predicted in PG52 strain. According to the expression level of the gene induced by aeciospores, the GH55 family gene pkg1 that may be involved in mycoparasitism was screened for cloning and expression. The expressed protein was purified, and its activity and ability to destroy aeciospores were determined. There were seven GH55 family genes from the PG52 genome. A endo-β-1,3-glucanase gene pkg1 that may have a mycoparasitic effect was screened out. The pkg1 was 2304 bp and expressed a 784 aa stable extracellular protein in Escherichia coli Rosetta (DE3). Enzyme activity of the PKG1 was 4.88 U/mL with laminarin as the substrate. The optimum temperature of PKG1 was approximately 60℃, the highest activity was at pH 7.0~9.0, and it had destructive activity against aeciospore walls. The β-1, 3-glucanase gene from P. kenyana was successfully cloned that showed its activity against aeciospores which point to its probable role in mycoparasitic activity of P. kenyana, suggesting a new source of enzymes for biological control strategies that target fungal cell walls.