Recombinant Expression Of PQQ Synthetic Protein And Its Effect On Acetic Acid Fermentation Of Acetobacter Pasteurianus

Acetic acid bacteria(AAB)were widely used in vinegar industry owing to their remarkable acetic acid-producing ability and high resistance to acetic acid.PQQ-alcohol dehydrogenase(PQQ-ADH)has an important effect on ethanol oxidation and acetic acid tolerance in AAB.In this thesis,recombinant expression vectors of Acetobacter pasteurianum were constructed,then recombinant expression of PQQ biosynthetic protein was realized.The relationship between PQQ and acetic acid tolerance and acetic acid fermentation in A.pasteurianum was further analyzed.Furthermore,the research will complete the acetic acid tolerance mechanisms in AAB,and it shows important scientific value and application value.The pqq gene cluster was amplified by using the A.pasteurianum AC2005 genome as template,recombinant expression of the pqq gene cluster in E.coli,then the PQQ was detected in E.coli(pMV24-pqq),while the control strain not be detected,indicating that the pqq gene cluster of A.pasteurianum can biosynthetic coenzyme PQQ.The shuttle vector pMV24-pl13 was selected of E.coli and A.pasteurianum by using pMV24 as the vector and gfp as the reporter gene and the recombinant strain AC2005(pMV24-pl13-pqq)was constructed and the PQQ concentration reached 1.85 ?g/L,which increased by 153.00%compared with the control strain,and the catalytic activity of ADH and aldehyde dehydrogenase(ALDH)increased by 52.92%and 67.04%,respectively.The growth and acetic acid tolerance of A.pasteurianum were analyzed.And found that the growth of recombinant strain was no different compared with the control under 1%acetic acid,while the biomass of the recombinant strain increased by 182.50%when cultured 48 h at 2%acetic acid compared with the control strain AC2005(pMV24),and the inhibition effect was enhanced when the acetic acid concentration was more than 3%,however the recombinant strain showed better acetic acid tolerance than the control,and the biomass increased by 23.40%and 13.80%at 3%and 4%acetic acid concentrations,respectively.The survival rate of the recombinant strain which be treated at 4%and 6%of acetic acid 40 min,respectively,were increased by 30.70%and 76.20%compared with the control.The effect of recombinant expression of PQQ synthetic protein on acetic acid fermentation in A.pasteurianum was analyzed.The acid production of recombinant strain increased by 16.96%compared with the control,under 7%initial ethanol concentration,The effect of recombinant expression of PQQ synthetic protein on the ethanol oxidation and the acetic acid tolerance-related proteins was analyzed by RT-PCR,the results showed that it is no significant effect on ADH,ALDH,citrate synthase(CS)and molecular chaperone DnaK proteins expression.Fermentation experiments were carried out using a 5 L autotrophic fermentor,the fermentation rate ofthe recombinant strain was improved to 1.57 g/(L·h),a 16.96%increase compared with the control under the condition of 8%initial ethanol concentration.It was proved that recombinant expression of PQQ synthetic protein mainly increased the concentration of coenzyme PQQ,thus improved the catalytic activity ofADH,then the acetic acid tolerance and the fermentation efficiency were increased.