The Research Of ADHII Gene Disruption In Saccharomyces Cerevisiae

Saccharomyces cerevisiae is a traditional strain of alcohol fermentation, of which thefermenting characteristics is critical for enterprise efficiency. In order to improve the yield rateof ethanol, we attempt to block the branch of ethanol catabolism by deleting the ADHII gene. Inthis study, an industrial strain of S. cerevisiae, AS2.489, was used as by combining sporulationand the following five methods:1) comparison of the morphological characteristics of diploidand haploid;2) cross breeding;3) specific PCR;4) concentration analysis of genomic DNA; and5) stress tolerance of ethanol and glucose osmotic pressure, three stable haplotype strains （twoMATa, AS-5and AS-21, and one MATα AS-7） from the original strain the original strain.The ADHII gene was deleted from the genome of the haplotype strains by homologousrecombination using the method of long homology flanking-PCR （LHF-PCR）. The left and rightflanking sequences （649bp and665bp, respectively）, homology to the upstream anddownstream of ADHII, were amplified from the yeast genome by PCR. The ADHII genetargeting cassettes was constructed by cloning the flanking sequences into a vector that carriesthe G418resistance gene and amylase or endoglucanase2The bi-functional gene knockoutplasmids, pScIKP-adh2∷amy and pScIKP-adh2∷egII were then digested with Apa I andtransformed into the haplotype strains (W303^-1A, AS-7and AS-21). Five mutants have beenselected and the exogenous genes （i.e. EGII or AMY） were successfully expressed in the mutantcells.The changes of fermentation characteristics of the mutant haploid strains were identify bybatched culturing the origin and mutated haploid strains with10g L^-1glucose anaerobically.The results were as follows:W303^-1A was detected reaching its highest ethanol concentration after96h of cultivationand the sugar and alcohol conversion rate was78.86%; as for AS-7and AS-21, the ethanolconcentrations reached the maximum value within24h after inoculation and the conversion rateswere91.19%and92.17%respectively. The fermentation performance of W303^-1A issignificantly inefficient when compared to the haploid strains derived from the industrial strainof yeast.The difference between the original and mutated strains was also compared. The ethanolyield and concentration of W303^-1A were40.3%and3.974g L^-1respectively, while those of the mutant strain W303^-1A-2were39.5%and3.949g L^-1.Strain AS-21was detected reaching the highest ethanol concentration of4.699g L^-1after24h of cultivation. However, the ethanol concentration of the mutant strain AS-21-2reached themaximum value of4.675g L^-1after72h of cultivation. The yield of ethanol and the conversionrate of sugar to ethanol of the mutant strain were48.5%and94.9%respectively, compared tothose of AS-21were47.1%and92.17%. The results also indicates that the decrease of ethanolconcentration of the mutant strain during the period of72to144h was less than the parentalstrain.Both AS-7and AS-7-2were detected reaching the maximum ethanol concentrations (i.e.4.637g L^-1vs.4.563g L^-1) at24h, but a higher value of ethanol yield was found for the originalstrain AS-7than mutant strain （i.e.46.6%vs.45.8%）. However, the variations of the ethanolconcentration of AS-7and AS-7-2were similar during the period of24h to144h.The mutant diploid strains were constructed by cross-mating of the original and mutanthaploid strains. Ten diploid mutants with single or double deletion of ADHII gene were obtained.The fermentation features of the constructed of the diploid strains were accessed by anaerobicfermentation in20g L^-1glucose and the results were as follows:A double deletion diploid mutant strain （AS-7-2/AS-21-2） was compared to the wild typediploid （AS-7/AS-21）. The ethanol concentration and the conversion rate of sugar to ethanolwere9.443g L^-1and94.5%for the mutant strain and9.574g L^-1and93.9%for the wild type.During the fermentation period from32to120h, the decrease of the ethanol concentration of thestrain AS-7-2/AS-21-2was0.497g L^-1, which is less than that of the wild type strainAS-7/AS-21(1.134g L^-1).Compared to the double deletion and wild type strains, the two single deletion strainsAS-7/AS-21-2and AS-7-2/AS-21showed lower maximum sugar to ethanol conversion rates（92.76%and90.4%, respectively）. However, the decrease of the ethanol concentrations of bothsingle deletion strains during the fermentation period of32to120h were less than the wild typediploid (0.3or0.08g L^-1vs.1.134g L^-1), which suggested that the fermentation performance ofsingle-gene deletion strains were better than the wild type.Above of all, the phenomenon of strain variation was observed in haploid strains. Deletionof ADHII gene resulted in improvement of ethanol yield for the mutant strain derived from AS-21, but there were no distinct differences of alcohol catabolism has been found in themutants derived from AS-7or W303^-1A. However, double deletion of ADHII gene exhibitedhigher ethanol productivity than single deletion and wild diploid. The degree of reutilization ofethanol in single deletion strain was less than the wild strain. In conclusion, this study indicatesthat deletion of ADHII gene may be an effective way to decrease the oxidation of ethanol toacetaldehyde in anaerobic batch fermentation and help to increase the ethanol productivity ofS.cerevisiae.