Construction Of Sacchromyces Cerevisiae Mutants Without SFA1/ADH3 Gene By Disruption

Many scientists are paying much attention to Saccharomyces cerevisiae which is animportant traditional model organism producing alcohol. Breeding S.cerevisiae bymolecular biology techniques is becoming a tendency in S.cerevisiae breeding. Thesuitable mutants of S.cerevisiae without the related genes are essential to researchmechanism on alcohol metabolism and signal transmission. PCR-based gene disruptionmethods play an important role in constructing S. cerevisiae mutants, which are valuablein breeding theory and industrial applications.In this dissertation, two mutants were constructed by gene disruption method. Themain results are as follows:1. The SFA1 gene disruption cassette was produced by PCR-mediated genedisruption method. Two linear disruption cassettes were transformed into S. cerevisiae YS₁to disrupted SFA1 two alleles. The diploid mutant YS₁-SFA1 was finally generated. Theproduction of ethanol in shaking flask fermentation was improved with 8.0% comparedwith the original strain YS₁.2. Two alleles of ADH3 in S.cerevisiae YS₃ were disrupted in turn by using thecassette with Cre/loxP system and Kan^r marker. The diploid mutant YS₃-ADH3 with highgenetic stability was generated. The mutant was different from the original strain YS₃ ingrowth behavior under anaerobic condition. It is a satisfactory mutant as test strain inresearching on mechanism of alcohol metabolism and signal transmission.3. According to gene sequence, there were remarkable differences in upstream anddownstream sequence of ADH3 and SFA1 gene in S. cerevisiae.YS₃ and YS₄ comparedwith the typical S. cerevisiae strain whose genome was first sequenced. The sequences ofupstream of ADH3 gene in strain YS₃ and YS₄ showed 97.9% and 89.1% homologyrespectively to the typical stain in GenBank, while their downstream sequences showed 98.8% and 90.0% homology respectively to the typical stain. Additionally, the sequencesof: upstream of SFA1 gene in strain YS₃ and YS₄ showed 98.5% and 91.0% homologyrespectively to the typical stain, while their downstream sequences showed 99.1% and86.9% homology respectively to the model strain.4. The sequence of 18S rDNA and phylogenetic analysis indicated S. cerevisiae.YS₄(GenBank Accession No.:EF153844) and YS₅ (GenBank Accession No.:EF153845) wereclosed to the known strain GK3 and SCZ75578 respectively.