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Biological Characterizations of the Mutant Strain with HOR2 Gene Deleted in Sacchromyces Cerevisiae

Author: ChenYongJin
Tutor: HuangJianZhong
School: Fujian Normal University
Course: Microbiology
Keywords: Saccharomyces cerevisiae HOR2 gene Knockout Ethanol Glycerin
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 25
Quote: 0
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Abstract


Since the industrial revolution , the development of the world economy can not be separated from the development of energy , fossil fuels has been the anchor of the mainstream energy supply , economic development . However, with the development of science and technology , the progress of industry and population expansion , the demand for energy is more and more urgent . However, due to these fossil fuels are non-renewable fuels, in varying degrees, on the environment caused by pollution and destruction , and thus find a renewable fuel will be crucial. Biological is particularly important as the medium of renewable energy development , which highlights the renewable fuel ethanol . Wine yeast ( Saccharomyces cerevisiae ) as traditional eukaryotic model organisms bacteria typical ethanol -producing bacteria , has been widely used in experimental research and fermentative production of bio-ethanol . Glycerin is one of the byproducts of ethanol metabolism in Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) . Saccharomyces cerevisiae containing the the two genes ( HOR2 RHR2 ) encoding the glycerol metabolism 3 - phosphoglycerate enzymes . HOR2 osmotic pressure regulating type , under the conditions of high osmotic pressure , there is a relatively large amount of expression ; RHR2 compared to anaerobic regulator type , a higher amount of expression in hypoxic conditions . Accordingly, by knockout HOR2 , or RHR2 ( this study and select the knock except HOR2 ) gene under certain conditions can be blocking or partial blocking of glycerol metabolism , thereby reducing the production of glycerol , and finally to the carbon source used for transformation ethanol lay a foundation for the metabolic regulation of Saccharomyces cerevisiae . This thesis by design containing HOR2 ( GPP2 ) gene flanking sequences homologous long primer , plasmid PUG6 as template for PCR the Saccharomyces cerevisiae HOR2 knockout construct containing the Cre / loxP system components , transforming Saccharomyces yeast ( Saccharomyces cerevisiae ) YS2 the kanr was replaced by the loxP - Kan - loxP sequence component arising positive clones . The plasmid pSH65 then transferred to the positive clones induced the Cre enzyme resection selection marker , a loxP site reserved in the original ORF gene lost the plasmid after get HOR2 haploid deficient strain . Repeat into the knockout component to another allele knockout . The successfully obtained the Saccharomyces cerevisiae HOR2 gene deletion mutant , and is named YS2 - HOR2 . The fermentation experiments show that the mutant glycerol production decreased by 3.34% , ethanol production increased by 1.96% .

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