| Saccharomyces cerevisiae produces a small amount of aromatic esters in the fermentation process.Although the content of these aromatic esters is very low,they are very important for wine.Flavor esters in fermented beverages produced by Saccharomyces cerevisiae can be classified into two categories:acetate and ethyl ester.Although the content of ethyl ester(C6-C10)is low,it is essential to give the aroma of fermented drinks.Because the synthesis of ethyl ester is low and the metabolic mechanism is complex,there are few related studies.Therefore,in order to improve the content of ethyl ester,the speed-limiting step of fatty acid synthesis,the synthesis of malonyl-CoA biosensor,was studied.Firstly,a biosensor specifically responding to the synthesis of malonyl-CoA was constructed.On this basis,the synthesis of malonyl-CoA was enhanced,and the yield of ethyl ester was increased.The main research contents and conclusions are as follows:(1)Firstly,a malonyl-CoA biosensor was established for the detection of intracellular malonyl-CoA.The biosensor contains manipulator gene and reporter gene fapO-eGFP,repressor gene and nucleation-inducing gene FapR-SV40.When repressor protein FapR specifically binds to malonate monoacyl coenzyme A and separates from manipulator fapO,RNA Polymerase is allowed to be transcribed in vitro.The specific fluorescence intensity of the reporter protein eGFP in the biosensor was positively correlated with the concentration of intracellular malonate monoacyl coenzyme A.A single-copy overexpression manipulator gene and reporter gene fapO-eGFP strain?-OGs were constructed.The fluorescence intensity radio of the strain was 43.0%higher than that of the original strain AY12a.A strain of a-OGm with multiple copies of manipulator gene and reporter gene fapO-eGFP was constructed,and recombinant strains with different expression intensity were obtained.Three recombinant strains,?-OGm12,?-OGm18 and ?-OGm28,were screened from strong to weak,and their fluorescence intensity radio increased by 254.4%,75.6%and 50.0%respectively compared with the original strain AY12a.On this basis,a single copy strains ?-OGm12-Rs,?-OGm18-Rs,?-OGm28-Rs overexpressing the repressor gene FapR-SV40 was constructed,and the results showed that their fluorescence intensity radio was higher than that of the corresponding original strain ?-OGm12,?-OGm18 and ?-OGm28 decreased by 52.7%,31.7%and 26.3%,indicating that the repressor gene FapR-SV40 has a certain repressive effect.In order to obtain a strain with more obvious inhibitory contact effect,a recombinant strain,?-OGm12-Rd,was constructed with a double-copy overexpression repression gene FapR-SV40.The fluorescence intensity radio of the recombinant strain was 63.9%lower than that of the corresponding original strain,?-OGm12.Finally,a strain of biosensor(?-OGm12-Rd)was obtained.The fluorescence intensity radio of the strain specifically responded to the synthesis of malonyl-CoA,which provided a specific,sensitive and simple detection method for the later enhancement of the synthesis of malonyl-CoA.(2)Verification of malonyl-CoA biosensor.strains overexpressing ACC1(?OR-ACC1)and strains overexpressing phosphorylated mutated ACC1ser(659,1157)were constructed(?OR-ACC1*,?OR-yACC1-*).The results showed that the fluorescence intensity radio of strains of ?OR-ACC1*,?OR-ACC1*,and ?OR-yACC1*increased by 20.1%,94.7%and 98.9%respectively compared with the original strain of ?-OGm12-Rd.The results show that the biosensor constructed in this experiment can be used for the detection of malonyl-CoA.(3)The biosensor was constructed to enhance the synthesis of malonyl-CoA.The PMP1/TPI1 strain(?ORA1*-PMP1,?ORA1*-TPI1),was constructed based on the integrated overexpression of ACC1ser(659,1157)The results showed that the fluorescence intensity radio of ?ORA1*-PMP1 and ?ORA1*-TPI1 was 29.1%and 2.6%higher than that of the original strain ?OR-ACC1*respectively.This experiment successfully enhanced the synthesis of malonyl-CoA in the fatty acid synthesis pathway of Saccharomyces cerevisiae. |
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