Directed Evolution Of Alcohol Dehydrogenase From Acetobacter Pasteurianus Ab3 Based On Tat Translocation Expression

Acetic acid bacteria are widely used in the process of vinegar industrial production due to its outstanding performance of high yield acid and acid resistance.Alcohol dehydrogenase(PQQ-ADH)plays an important role in this process because of its ability to oxidize ethanol to produce acetaldehyde and,under the action of acetaldehyde dehydrogenase converted to acetic acid.In this paper,the functional site of the alcohol dehydrogenase large subunit(AdhA)which was cloned from Acetobacter pasteurianus Ab3,isolated from Zhejiang traditional rose vinegar by means of Tat translocation expression system.Based on the bioinformatics analysis method,in this paper we firstly analyzed the AdhA in different acetic acid bacteria,including the prediction of signal peptide,the secondary structure and the three-stage structure prediction.The sites prone to affecting the AdhA activity were suggested..On the basis of this,the Tat-bla expression system of AdhA was constructed,and the functional site of AdhA was optimized by the method of error-prone PCR.The results of bioinformatics showed that the amino acid sequences of AdhA in different acetic acid bacteria were highly conserved,meanswhile some differences occur.Almost of AdhA contains a unique signal peptide containing the bis-arginine sequence S-R-R-x-F-L-K,which can be recognized by the bacterial Tat translocating expression system which can achieve transmembrane transport of soluble and folding proteins.By comparing the amino acid sequences of AdhA,Ala30Thr,Va131Met,Pro32Ala,Ala33Ser,Asp36Asp,Glu43Glu,Glu53Gly,Lys262Gln,Pro266Pro,Lys267Thr,Ser289Ala,Ile311Lys,Gln393Val,Leu402Lys,Lys457Thr,Glu508Ala,Thr509Glu,Val510Ala,Trp511Trp,Gly559Gly,Asp639Asp,Met662Val,Thr697Gly,Asn731Gly changes were found in their secondary structures.On the basis of bioinformatics analysis,adhA gene and ampicillin resistance gene were successfully cloned,and the recombinant plasmid pADH-Bla was constructed to investigate the soluble expression of adhA gene in Escher-ichia coli.Using the unique advantage of E.coli Tat expression system combining with Bla resistance screening,the directed evolution of adhA gene was carried out by the error-prone PCR.Mutant strains M14,M21,M28,M44,M59 and M60 with improved ability of ampicillin were screened out.The adhA gene of M14 and M21 had three mutations A158G,C290G and A784C.The corresponding amino acid mutations were Glu53Gly,Ala97Gly and Lys262Gln.The adhA gene of M28 and M60 had two mutations,respectively,A158G,C1204G,the corresponding amino acid mutation to Glu53Gly,Leu402Lys.Where M14 and M28 have a common mutation site A158G.The adhA gene of M44 had 5 mutations,namely A129G,G643T,A1523C,C1677A and A1984G,corresponding amino acid mutations to Glu43Glu,Ala215Ser,Glu508Ala,Gly559Gly and Met662Val.The mutant sites of adhA gene of M59 were T473A,T1529C,and the corresponding amino acid mutations were Phe158Tyr and Va1510Ala.The development of this subject reveals some sites that affect the activity of alcohol dehydrogenase,which provides a theoretical basis for further orienting ADH and improving the activity of ADH.At the same time,the successful development of Tat system also provides the basis for the study of other key enzymes in acetic acid bacteria.