The Construction Of A NDE1 Mutant Of Saccharomyces Cerevisiae And Investigation Of Its Bilolgical Function

The transition period of a dairy cow is defined as the period from 2-3 weeks prior to calving to 2-3 weeks after calving. This period had an enormous effect on the health and production performance of dairy cows, characterized by negative energy balance. Ketone disease and fatty liver which were based on metabolic disorder of energy were common diseases of transition period. So, glycerol could alleviate negative energy balance, because glycerol was glucogenic, independent of rate-limiting enzyme catalyzing. Glycerol production by fermentation which has less pollution and low poison attracted extensive attention. Saccharomyces cerevisiae NAU-ZH-GY1 that 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 probioticsIn this thesis, basing on the principle of metabolic engineering we construction a nde1△ mutant Saccharomyces cerevisiae. This strain was partially cut off the mitochondrial oxidation of cytosolic NADH. Since the transmitting from cytosolic NADH to mitochondrial respiratory chain had been reduced, more cytosolic NADH could turn into glycerol metabolic pathways and more glycerol could be produced. This thesis provided a good opportunity for developing a kind of microbial ecological agent preventing and treating energy metabolism disorder.Experiment 1 The construction of a ndel △ mutant of Saccharomyces cerevisiaeStarting with strain NAU-ZH-GY1, a diploid that our laboratory possessed the independent intellectual property, we used Cre/loxP system gene disruption method disrupting NADH dehydrogenase 1 gene (NDE1) and constructed a strain mutating an allele ofNDE1, only leaving a single loxP site at the chromosomal locus ofNDE1 ORF. This gene disruption method could reduce the biohazard of exogenous gene and be more simpler, precise and cost-effective than traditional method. Using competent cells during the metaphase of logarithmic growth which pretreated by DTT Buffer for 30 minutes, under the conditions of 400 ng·μL-1 foreign DNA,1.5 kV/cm electric field strength,25μF capacitance and 200 Q. resistance, the electric conversion efficiency was best. After the use of yeast allele separation and crossing technology, we successfully got a ndel△ mutant Saccharomyces cerevisiae named NAU-ZH-GYl-ndel△. Strain NAU-ZH-GYl-ndel △ had no resistance maker gene, met the security requirement of probiotics. The method used to constructe the strain was lowcost and high efficiency.Experiment 2 The biological function of a ndel△ mutant of Saccharomyces cerevisiaeAerobic fermentation of NAU-ZH-GYl-ndel △ and NAU-ZH-GY1 were performed, meanwhile their growth conditions, genetic stability, production of glycerol, biomass and viable count were monitored in the experiments. The experimental results showed that the growth conditions and viable count in NAU-ZH-GY1-ndel △ were similar to NAU-ZH-GY1. Strain NAU-ZH-GYl-ndel △ had stable genetical characters. Because of gene knock-out, the biomass of NAU-ZH-GY1-ndel △ reduced by 17.1%(p<0.05), which was compared with NAU-ZH-GY1.The glycerol yield of NAU-ZH-GYl-ndel△ was 26.61 g·L-1 and increased 12.9% against the glycerol output of the wild yeast(p<0.05).