The Ester-Producing Mechanism Of Pichia Pastoris Y₁₄EHT1 Gene And Its Application In Fruit Vinegar

In this study,we investigated the regulation mechanism of Pichia pastoris EHT1 gene and its application in jujube vinegar,and systematically demonstrated that EHT1 gene plays an important role in regulating the production of volatile fatty acids in Pichia pastoris fermentation products.To provide a theoretical basis for further study of the biological function of Pichia pastoris EHT1 gene.The main research contents and research results are as follows:(1)Molecular biological identification of different species of yeast revealed that CY1-CY25,CY27-CY29,CY31-CY67,CY69,CY72-CY74 were Saccharomyces cerevisiae;CY26 was Pichia scaptomyzae;CY30,CY71 is Candida humilis;CY68 is Rhodotorula sp.;CY70 is Pichia membranifaciens.(2)Y14(Pichia manshurica)has the highest content of esters in the fermentation products.These esters are beneficial to the formation of vinegar aroma.The use of Y2 and Y18(Candida alcoholica)can also increase the formation of volatile compounds and contribute to the complexity of the aroma of date vinegar.The fermentation test confirmed that the esters in the finished vinegar added with Y14 were richer in flavor and richer in flavor,and it was expected to be developed into a flavored starter.(3)The EHT1-encoded protein consists of 452 amino acids with a mass unit of 5,068.63 Da,an isoelectric point of 5.89,a total number of acidic amino acids(Asp+Glu)of 50,and a basic amino acid(Arg+Lys)of 40.One,theoretically assumed amino acid formula C2344H3509N593O678S7,half-life: 30 h,absorbance(A)value at a wavelength of 280 nm: 1.585,instability factor: 32.58.The secondary structure of EHT1: ?-helix 29.2%,? turn 12.17%,random coils 35.18%;no signal peptide and transmembrane structure,containing hydrophobic regions.Found 2 possible N-glycosylation sites,5 possible O-glycosylation sites for EHT1;28 serine(Ser),14 threonine(Thr),and 12 tyrosines(Tyr),there is a potential phosphorylation site.(4)EHT1 positive clones and protein identification results showed that the p PIC9K-EHT1 vector was successfully constructed and can be used for the preparation of recombinant bacterial agents.When the concentration of methanol was 2% at p H 6,the activity reached 98.2 U/m L at a temperature of 25 °C,which was significantly higher than that of other EHT1 activity-influencing factors(P < 0.01).(5)EHT1 knockout and overexpression strains were successfully constructed.EHT1 gene plays an important role in regulating the production of volatile fatty acids in Pichia pastoris fermentation products.Overexpression of EHT1 gene Pichia pastoris fermentation products,ethyl heptanoate,methyl octanoate,ethyl octanoate,methyl decanoate,methyl decanoate,ethyl decanoate,methyl laurate,lauric acid The relative percentages of ethyl ester and ethyl myristate were significantly higher than those of the control group.(6)EHT1 recombinant Pichia pastoris can significantly increase the content of esters in jujube vinegar(P<0.05).Comprehensive sensory evaluation,the use of EHT1 recombinant bacteria can improve the taste of jujube vinegar.