The Knockout Of Nucleotide Repair Excinuclease Gene UvrA And Its Effect On Acetic Acid Torlerance In Acetobacter Pasteurianus

Acetic acid,as a common organic acid,which is widely used in food and pharmaceutical industries.Acetic acid bacteria(AAB)are involved in production of acetic acid by oxidize ethanol,the remarkable ability of production and high resistance to acetic acid of AAB make it widely used in acetic acid fermentation industry.In this paper,we focus on the relationship between the nucleotide repair excinuclease UvrA of Acetobacter pasteurianum AC2005 and the acetic acid tolerance in A.pasteurianum,and constructed the uvrA gene knockouted strains and recombinant expression strains by genetic engineering method.Which in order to illustrate the relationship between them by the effect of UvrA on cell growth and acetic acid fermentation.Furthermore,this research will improve the acetic acid tolerance mechanisms in AAB and can guide the AAB strains breeding in fermentation,as well as shows important application value and scientific significance.The growth of A.pasteurianum AC2005 can be inhibited by acetic acid,and the time of lag phase increase from 1 h to 8 h at 1%acetic acid.We used the agarose gel electrophoresis to analysis the genomic integrity of AC2005,and found that with the increasing concentration of acetic acid,the genome integrity of AC2005 decline.The expression of nucleotide repair related proteins have raised 54.8%in average by analysis the proteins expression in AC2005 at acetic acid condition.The UvrA transcription level have been analysised by RT-PCR method,and found that the transcription level of uvrA have increased 146%and 275%under the condition of 1%and 2%acetic acid treatment compared with the control.All this indicated that the acetic acid can cause the genome DNA broken and the proteins associated with nucleotide repair,like UvrA,would be expressed in this process,and the UvrA may be related to acid tolerance in AAB.We established the method of gene konckouted in A.pasteurianum by the suicide plasmid pSUP202,and the pSUP202-uvrA-1::Km which be used for uvrA knockout was constructed.So the uvrA gene knockouted strains AC2005-?uvrA were constructed via the principle of homologous recombination.The pMV24 which is the shuttle plasmid between acetic acid bacteria and Escherichia coli was used to construct the uvrA gene recombinant expression plasmid pMV24-uvr A,and we have got the uvrA gene recombinant expression strain AC2005(pMV24-uvrA)and control strain AC2005(pMV24).The growth of A.pasteurianusAC2005 was analyzed at 1%,2%and 3%acetic acid condition in initial.And found that the biomass of AC2005-?uvrA decreased by 84.85%and 47.13%when cultured 48h at 1%and 2%acetic acid compared with the control strain AC2005(pMV24),while the AC2005(pMV24-uvrA)increased by 17.1%and 24.2%.When cultured 72 h at 3%acetic acid concentration,the growth of AC2005(pMV24)and AC2005-DuvrA was significantly inhibited,however the AC2005(pMV24-uvrA)improved 66.1%compared with the control strain.Then we analyzed the impact of cell survival at 6%acetic acid,and the survival rate of AC2005-?uvrA which be treated at 6%of acetic acid 40 min was only 2.56 × 10-5,but the survival rate of AC2005(pMV24-uvrA)was 2-fold than AC2005(pMV24).We used the fermentation medium which containing 7%ethanol to analysis the acetic acid fermentation by different strains,and found that the AC2005(pMV24-uvrA)and AC2005(pMV24)have a same growth trend,but the production of acetic acid increased about 10%than the control strain.However the growth of AC2005-?uvrA was affected by the concentration of acetic acid in fermentation broth which reached 0.45 g/100mL and the biomass and production of acetic acid began to decline.Then we analyzed the activity of alcohol dehydrogenase(ADH)and aldehyde dehydrogenase(ALDH)in the fermentation process and found that the highest activity of ADH and ALDH in AC2005-?uvrA decreased by 66.8%and 66.6%compared with controls,while the activity of ADH and ALDH in AC2005(pMV24-uvrA)increased by 17%and 1.5%.In this paper,we found that the UvrA have direct effect to the acetic acid torlerance by analyzed the impact on the growth and acetic fermentation of A.pasteurianum AC2005.And the acetic acid tolerance significantly reduced by the UvrA gene deletion,however the acetic acid tolerance improved with the UvrA gene overexpression.So the reason for it is the proteins related to UvrA involved in the nucleotide repair mechanism.