Construction Of Expression Vectors In Acetobacter Pasteurianus And Its Application

Acetic Acid Bacteria(AAB)has been widely used in vinegar production.The key factors in restricting the production of high acidity vinegar are the AAB species and their ability in acetic acid tolerance.While the useful molecular tools play an important role in the study of acetic acid tolerance.In this study,Acetobacter pasteurianus Ab3 was used for the construction of expression vector that is suitable for AAB,including the selection,prediction and verification of promoters,combined with the adoption of green fluorescence protein as the marker.In this study,five important proteins' coding gene involved in acetic acid resistance,as well as 16s rDNA gene were chose for the promoter analysis.Promoter PadhA,PdnaK,and PgrpE were finally selected for the vector construction based on the prediction score produced by BPROM,a web-based tool.The length of these three promoters were about 200 bp,and after ligated into the plasmid pBBRA,three different plasmids comprising PadhA(pBBRAP1),PdnaK(pBBRAP2),and PgrpE(pBBRAP3)were successfully constructed.The green fluorescence protein(GFP)was firstly used in the verification of promoter in AAB.Basically,the GFP coding gene gfp was located downstream of three different promoters to produce plasmids pBBRAP1GFP,pBBRAP2GFP and pBBRAP3GFP.All of these plasmids were transformed to A.pasteurianus Ab3 to verify that whether they were active or not.The result showed that the fluorescence level was much higher in recombinant strains compared to wild-type strains,indicating that all of these three promoters are functional and can be used in protein expression.However,the expression level of gfp under different promoters was different and was influenced by surviving conditions,e.g.the promoter PadhA was more active than other two promoters during the whole period of cell growth.In addition,the promoter Pdnak and PgrpE seem to be more active after exponential growth phase implies that the activity of these promoters are growth dependent as well as stress related,which is in accordance with the results obtained from previous proteomic analysis.So the plasmid constructed in this study is a potential way to produce interested proteins in A.pasteurianus at designed time point by replacing the promoter with other genes in the future.Furthermore,it is also a way to study the time-dependent protein expression map of A.pasteurianus.We also constructed the cyclopropane fatty acid synthase expression plasmid named as pBBRAPlcfa.pBBRAP2cfa and pBBRAP3cfa to further explore the function of cyclopropane fatty acid in AAB.The construction of the expression vector in A.pasteurianus paves the way for the further elucidating of acetic acid resistance mechanisms in AAB,especially the study of interested proteins involved in stress response as well as their time-dependent expression levels.In addition,the results of this work provide clues for the research of other strains belong to AAB.These results will enable the selection and breeding of industrial AAB strains,as well as the optimization of vinegar fermentation conditions.