Study On Synthesis Regulation Of Acetyl-CoA In Saccharomyces Cerevisiae

Acetyl-CoA in Saccharomyces cerevisiae is an important precursor of esters.As the important flavor compounds in Chinese liquor,esters endow Chinese liquor rich flavors.This study mainly put emphasis on improving acetyl-CoA content and determining ethyl acetate to show the change of acetyl-CoA.The effects of acetyl-CoA were investigated by deleting ACH1(encoding acetate:succinyl-CoA transferase gene)and ovexpressing ALD6(encoding aldehyde dehydrogenase gene)and ACS 1/2(encoding acetyl-CoA synthetase gene).The main contents and conclusions were as follows:(1)?5?ACH1 strain was obtained by knocking out ACH1 gene with the long primer method.Under shaker condition and fermentation condition,the contents of acetyl-CoA by the ?5?ACH1 strain were increased by 33.24%and 17.70%,comprared with the control strain a5.However,the content of ethyl acetate did not change obviously.(2)The plasmid Yep-PAK6 was constructed to overexpress ALD6 gene.The strain?5-ALD6 was obtained by transferring the fragment AA-PGK1P-ALD6-PGK1T-KanMX-AB to ?5.Under shaker condition and fermentation condition,the contents of acetyl-CoA were increased by 41.59%and 24.62%,comprared with the control strain ?5.While after the fermentation,ethyl acetate content was decreased by 21.26%,and n-propanol,isobutanol alcohol and amyl alcohol were decreased by 33.91%,10.53%and 23.88%,seprately.(3)The effects of acetyl-CoA by ovexpressing ACS1 and ACS2 were examined.Firstly,the strains ?5-ACS1 and ?5-ACS2 were constructed to overexpress ACS1 and ACS2.Under shaker condition and fermentation condition,the contents of acetyl-CoA by ?5-ACS1 were increased by 44.34%and 56.34%,respectively and the contents of acetyl-CoA by ?5-ACS2 were increased by 42.60%and 30.94%,comprared with the control strain a5.Correspondingly,ethyl acetate contents were increased by 15%and 10.34%.Secondly,the strains A1-ATF1 and A2-ATF1 were obtained by overexpressing ATF1(encoding alcohol acetyltransferase gene)in ?5-ACS1 and?5-ACS2.The contents of ethyl acetate produced by the strains A1-ATF1 and A2-ATF1 were 2.04 times and 1.25 times by the control strain ?5-ATF1.Lastly,the co-expression strain AY-ACS1L707P was obtained by transferring the site-directed plasmid Yep-ACS1L707P to the diploid AY 15 and the integrated site-directed strain?5-ACS1L107P was obtained by transferring the amplified fragment AA-PGK1P-ACS1L707P-PGK1T-KanMX-AB into the ?5 strain.Acetyl-CoA content by the AY-ACS1L701p strain was increased by 32.35%,and the ethyl acetate was increased by 7.60%,comprared with the control strain ?5.The acetyl-CoA content by?5-ACS1L707p was increased by 1.04 times and the ethyl acetate content was increased by 20.25%,while the acetic acid content is decreased.(4)The free plasmid Yep-ABLS was constructed.By transferring the plasmid to AY 15 strain,the free co-expression strain AY-1 was obtained.The acetyl-CoA was increased to 63.70%,the acetic acid was decreased by 14.67%,and the ethyl acetate content was increased by 10.26%,comprared with the AY 15 strain.The integrated co-expression strain ?5-1 was obtained by transferring the co-expressed fragment to the ?5 strain.The acetyl-CoA was increased by 1.5 times,and the acetic acid content was also increased by 1.96 times.But the ethyl acetate was decreased by 28.24%.