The Construction Of A GPD1 Overexpression Mutant Of Saccharomyces Cerevisiae And Study Of Its Biological Characteristics

With the rapid development of China’s dairy industry,the proportion of cows nutritional and metabolic diseases is growing,which can effectively alleviate the negative energy balance.During the perinatal period,especially the high-producing dairy cows,energy demands of fetal growth and lactation are increased,but the dry matter intake(DMI)may decrease.Thus,dairy cows frequently enter a state of negative energy balance,in which energy intake can not meet the energy requirements.To compensate for this energy deficiency,dairy cows increase the mobilization of body fat.However,excessive lipolysis will result in many energy metabolic diseases.Because of ruminal metabolism characteristic,glucose is directly broken down by ruminal microorganisms but not absorbed across the ruminal wall.Ninety percent of glucose needed for the ruminants depended on the gluconeogenesis.Glycerol,an important glucogenic precursor,is closely related with glucose metabolism,and can effectively solve the problems of animal energy metabolism by feeding to ruminants.With a widespread attention,produced glycerol by fermentation has many advantages,like clean ang safety ect..Production of glycerol yeast NAU-ZH-GY1 have independent intellectual property rights owned by my lab,Saccharomyces cerevisiae is also allowed as probiotics additives by the Ministry of Agriculture.Saccharomyces cerevisiae NAU-ZH-GY1 which our laboratory possessed the independent intellectual property,was a kind of probiotics permitted for feed by laws.Gene modification could contribute to glycerol production of Saccharomyces cerevisiae NAU-ZH-GY1.The yeast culture from fermentation given to transition period fairy cows as feed additive,may alleviate the negative energy balance,meanwhile play a role of probiotics.In this experiment,GPD1 gene overexpression mutant strains of Saccharomyces cerevisiae was constructed from NDE1 gene deletion mutant strains of Saccharomyces cerevisiae.This experiment provided a good opportunity for developing a kind of microbial ecological agent preventing and treating energy metabolism disorder.Experiment 1 The construction of PGC vectorFirstly,the GPD1 gene of glyceraldehyde 3-phosphate dehydrogenase and its promoter fragment in Saccharomyces cerevisiae was amplified,then the fragment was inserted into plasmid pUG6 which has G418 resistance gene and LoxP sites and cytochrome c(CYC1)terminator fragment was inserted at downstream of cytochrome c(CYC1).Finally,the integration of the expression vector PGC was constructed successfully.Experiment 2 The construction of a gpd1 overexpression mutant of Saccharomyces cerevisiaeIn our lab,the stain of NAU-ZH-GY1-ndel△ was construction successfully,starting with strain NAU-ZH-GY1-ndel△,The vector PGC was linearized by restricted endonuclease BspT1041,then was transformed into NAU-ZH-GY1-ndel△.Basing on the mechanism of homologous recombination in S.cerevisiae,With the help of Cre/loxP recombination system,a gene copy was added.At last,a nde1△ and GPD1 gene overexpression mutant strain of Saccharomyces cerevisiae was constructed successfully.Experiment 3 Study of biological characteristics of a gpdl overexpression mutant of Saccharomyces cerevisiaeAerobic fermentation of NAU-ZH-GY1-ndel △-gpd1,NAU-ZH-GY1-nde1△ and NAU-ZH-GY1 were performed,meanwhile their growth conditions,genetic stability,production of glycerol,viable count biomass and were monitored in the experiments.The experimental results showed that the growth conditions,biomass and viable count NAU-ZH-GY1-nde1△-gpd1 were similar to NAU-ZH-GY1-nde1△ and NAU-ZH-GY1.Strain NAU-ZH-GYl-ndel《had stable genetical characters.The glycerol yield of NAU-ZH-GY1-ndel△-gpd1 was 28.04g.L-1,increased 21.12%against the wild yeast and increased 15.20%against the strain NAU-ZH-GY1-ndel △.So,the glycerol yield of NAU-ZH-GY1-ndel △-gpdl impoved significantly.