| In the industrial vinegar fermentation,the fermentation performance of acetic acid bacteria,which is tightly related to fermentation efficiency,acetic acid productivity and production cost,and is affected by many environmental factors such as the concentration of ethanol,acetic acidand adverse temperature.This study attempts to obtain engineering strains through the modern molecular biology.And the engineering strain obtained was conducted for fermentation performance testing and metabolomics study,providing evidencefor the futher study on the ethanol tolerance mechanism of Acetobacter pasteurianus and the strain improvement.The hisl gene coding histidinol-phosphate aminotransferase correlated with ethanol tolerance according to related literature.According to the published gene sequence information of hisl designed primers,the purpose fragment was amplified based on the template of genomic DNA of Acetobacter pasteurianus HN101 and the recombinant plasmid was constructed.The engineering strain T1 was successfully constructed.The strain T1 could tolerate high concentrations of ethanol 9%(v/v)and produced high concentrations of acetic acid(37.66+1.51g/L),which increased than the production of A.pasteurianus by 191.07%.The metabolomics analysis revealed changes of intracellular metabolites in A.pasteurianus under high concentrations of ethanol based on gas chromatography-mass spectrometer(GC-MS).And the possible ethanol tolerance mechanism was preliminarily studied.The study showed that ethanol stress mainly caused the changes of metabolites involved in carbohydrates,lipids and amino acids.EMP and TCA were inhibited under ethanol stress condition.As the result,the carbon metabolic flow of T1 was turned to ethanol oxidation,so that the cell could hold the high activity of ADH and ALDH and have enough energy to protect itself from environmental stress.The synthesis of protective substances such as proline,glutamic acid and trehalose might play an important role to the ethanol tolerance of T1.The synthesis of saturated fatty acids were repressed and the unsaturated fatty acids were increased under high ethanol concentrations,as the result,the SFA/UFA decreased.This result indicated that high ethanol concentrations could change the cell membrane fluidity.The strain T1 could effectively adjust its fatty acid synthesis to decrease the increase of membrane fluidity under the stress of ethanol.The changes of fatty acids mainly involved hexadecanoic acid,octadecanoic acid and oleic acid.In conclusion,this study laid a foundation for the futher study on directional transformation strain. |
