Culture Optimization And Hydrophobin Gene Overexpression Study Of High-yield Hydrophobin From Pleurotus Tuber-regium

Pleurotus tuber-regium hydrophobin is a secreted non-catalytic fungal protein that acts on the interface and can self-assemble to form a "raincoat" on the fruiting body of Pleurotus tuber-regium.This characteristic makes hydrophobin play an important role in the growth and development of t Pleurotus tuber-regium.And Pleurotus tuber-regium hydrophobin has good foam stability and emulsifying properties,so it has great potential for commercial application in the food industry.But the production is too low to affect the large-scale commercialization of Pleurotus tuber-regium hydrophobin.Therefore,the aim of this study was to improve the production of hydrophobin from Pleurotus tuber-regium through two aspects of culture optimization and hydrophobin gene overexpression.The main findings are as follows:(1)Optimization of conditions for hydrophobin production by liquid cultured Pleurotus tuber-regium myceliumThrough the orthogonal test,it can be found that the optimal medium for the production of hydrophobin by liquid fermentation of Pleurotus tuber-regium is 20 g/L corn flour,5 g/L yeast powder,1 g/L magnesium sulfate,1 g/L potassium dihydrogen phosphate.The fermentation medium was prepared in this way and cultured for 8 d in a shaker with a temperature of 32 °C and a rotation speed of 120 r/min.Under this culture condition,the yield of dried mycelium of Pleurotus tuber-regium could reach 11 g/L,while the yield of hydrophobin was about 48 mg/L.(2)Cloning and bioinformatics analysis of hydrophobin gene Vmh3The hydrophobin gene Vmh3 of Pleurotus tuber-regium has a CDS sequence of 327 bp in full length,encoding 108 amino acids,a molecular weight of about 13 k Da,and a theoretical isoelectric point of 6.11.The full-length DNA sequence of Vmh3 gene is 452 bp.It contains 3 exons(1-242,242 bp;296-333,39 bp;406-452,47 bp)and two introns(243-295,53 bp;334-405,72 bp).The hydrophobin Vmh3 has no transmembrane helical region,and is probably an extracellular protein.And because only a very small amount of hydrophobin can be extracted from the fermentation broth,it is speculated that Vmh3 is a protein located on the cell wall.(3)Growth of transformants overexpressing hydrophobinThe overexpression vector was successfully constructed,and the transformants were obtained from the transformation of Agrobacterium tumefaciens for fluorescence microscopy and Southern blot verification,and three stably inherited overexpression transformants were obtained.Two of the transformants with better growth were analyzed by fluorescence quantitative PCR,and it was found that the gene expression of the transformants was significantly higher than that of the wild type,and the relative expression of the best hydrophobin gene reached 6 times that of the wild type.The transformant with the highest gene expression was cultured in liquid and extracted hydrophobin,and found that the yield was about 1.6 times that of the wild type.