Constructing Low Isopentanol Saccharomyces Cerevisiae With Knockout Gene THI3 And LEU2 And Its Application

Distilled alcohol is a well-known and pop?lar alcoholic beverage,which is widely used and nutritional.Distilled liquor is produced by microbes and raw material containing starch and sugariness,and this raw material need to be brewed and distilled.In the fermantation process,with Saccharomyces cerevisiae metabolism and the decomposition of its raw materials,not only can produce some nutrients or flavoring substances,but also produce some substances that have a bad effect on people's health,such as isopentanol,etc.Excessive isopentanol content will make the drinker have a bad feeling called‘Shang Tou'and other discomfortable reactions.One part of the isopentanol in distilled liquor is produced by hydrolysis of raw materials,another is produced by Saccharomyces cerevisiae metabolism.There are two approaches for Saccharomyces cerevisiae to produce isopentanol:One is amino acid transfer,Erlich pathway;Another is glucose metabolism,Harris pathway.The THI3 gene encoding pyruvate decarboxylase is the key genes in the Erlich pathway;LEU2 gene encoding ?-isopropylmalate dehydrogenase is a key gene in the Harris pathway.This experiment uses a low methanol Saccharomyces cerevisiae strain S₁ as starting strain,which is stored in the Institute of biology,South China University of Technology.Using the Cre-Loxp recombinant enzyme system that is based on the PCR technology and combine yeast electroporation method,firstly,a Saccharomyces cerevisiae strain S₂ with complete deletion of THI3 gene has been successf?lly constructed.Let the strain S₁ and strain S₂ continuously fermented 6d with the concentration of malt juice is 13 °P,the amount of bacteria is 3%,and the temperature is 30?,The relative content of isopentanol in distilled liquor and alcohol content of 20? were determined by gas chromatography and alcohol meter: After fermentation,the relative isopentanol content of S₁ is 1.13g/L,the alcohol content at 20? is 10.0%vol;the relative isopentanol content of S₂ is 0.81g/L,the alcohol content at 20? is 10.5%vol.This shows that the strain S₂ with THI3 gene knockout has a 28.3% relative isopentanol content reduce than the strain S₁,there is an increase in the alcohol content and has a good fermentation property.On the foundation of the strain S₂,the LEU2 gene was successf?lly knocked out and has constructed a Saccharomyces cerevisiae strain S₃ with complete deletion of THI3 gene and LEU2 gene.And using the above identical conditions of fermentation,and has measured the relative isopentanol content of S₃ is 0.70 g/L,13.6% lower than that of strain S₂,38.1% lower than the yeast strain S₁;And the alcohol content of S₃ is 8.0%vol,compared to the strain S₁ and strain S₂ both decreased and presented a poor alcohol fermentation performance.Selecting strain S₂ as the optimum fermentation strain,conduct a study on liquid and semi solid fermentation process,the experimental res?lts show that: about liquid fermentation,when the concentration of malt juice is 15 °P,the fermentation time is 5d,the fermentation temperature is 30?,and the inoc?lation amount is 3%,the relative content of isopentanol in distilled liquor is the lowest;For semi solid state fermentation,when the fermented material is sorghum,the relative content of isopentanol is the lowest,which is 0.77g/L,the relative content of isopentanol is the highest when the fermentation materials are produced by the mixture of rice and sorghum,which is 0.85g/L,And when using unhusked rice as fermentation raw material,the relatively high alcohol content is the lowest,which is 1.49g/L;When the raw material including the mixture of rice and sorghum,the relative high alcohol content is the highest,which is 1.72g/L.