The Functional Study Of The Acetolactate Synthetase Gene AlsS In Acetobacter Pasteurianus

Besides the acetic acid and furfural,3-hydroxy butanone is the third highest volatile substance in the traditional solid-fermented vinegar in our country.3-hydroxy butanone is also the precursor of ligustrazine,which is a iconic hygienical component in vinegar.Ligustrazine is an important active pharmaceutical alkaloid in ligusticum wallichii.Acetobacter pasteurianus is a representative microorganism in vinegar brewing industry.There were no immediate reports that two acetolactate synthetase genes alsS exist in als operon of Acetobacter pasteurianus.It indicates that there might be a novel synthetic route of 3-hydroxy butanone in Acetobacter pasteurianus.We got a strain Acetobacter pasteurianus from China microbial species preservation management center,whose preservation number is CICC 22518.We analysised the function of acetolactate synthetase genes alsS of A.pasteurianus.First of all,two acetolactate synthetase genes were heterologously expressed.Escherichia coli BL21(pET28a-alsS1)can express the alsS1 gene with acetolactate synthetase activity.The specific activity of the purified protein AlsS1 can reach 129.5U/mg,when the E.coli cells were induced by 0.6 mM IPTG and grown at 37?for 7hours.The optimal temperature and pH of the AlsS1 protein are 55?and 6.5,respectively.AlsS1 is indistinctive by Na~+and K~+,but highly inhibited by EDTA.In addition,AlsS1 can be indistinctive to some enzyme cofactors,such as NADP,NADPH,NAD and NADH and some branched-chain amino acid,such as leucine,isoleucine,valine.We couldn't detect enzyme activity about protein AlsS2 after fumbling temperature and pH.Next,two acetolactate synthetase genes were deleted.We built three gene deleted strains,named A.pasteurianus-?alsS1,A.pasteurianus-?alsS2,A.pasteurianus-?(alsS1+alsS2),then explored their growing states,producing states about3-hydroxy butanone and L-lactic acid degradation.We found that biomass of deleted strains were all inferior to wild strains,indicating that deletion of genes can partly affect strains'growth.Deletion of acetolactate synthetase gene alsS1 can significantly influence on synthesis of 3-hydroxy butanone.The result indicates that this gene plays a primary part in synthesis of 3-hydroxy butanone,also gene alsS2 can play a minor role not primary role.We cant detect synthesis of 3-hydroxy butanone in fermentation of A.pasteurianus-?(alsS1+alsS2),indicating that deletion of both alsS1 and alsS2interdicts the forming path of 3-hydroxy butanone.The utiling rate of Acetobacter pasteurianus about L-lactic acid is the highest.The utiling rate of deleted strains are under wild strain.This study lay the theoretic foundation for enhancing 3-hydroxy butanone yield of fermentation strains.