The Influence Of Knocking-out GPD2on Ethanol Fermentation In An Industrial Strain Of Saccharomyces Cerevisiae

Saccharomyces cerevisiae is the traditional micro-organism for industrial ethanolproduction. It is estimated that about4%-10%of the total carbon source maybe converted toglycerol during anaerobic ethanol fermentation by Saccharomyces cerevisiae. If all of the carbonsource that converted to glycerol was fermentated into ethanol, addidtional1.3billion liters ofethanol wolud be produced annually without increasing the cost, which would yield substantialeconomic benefits. Therefore, it is of great interest to reduce glycerol yield and increase theproductivity of ethanol at the same time.In this study, in order to increase the convertion rate from glucose to ethanol, the glycerolsythesis pathway was partly disrupted by knocking-out the GPD2gene from an industrial strainof Saccharomyces cerevisiae AS2.489. To completely delete both loci of GPD2in the diploidyeast, it is critical to prepare the haploid strains of AS2.489, because there is only one copy ofgene in haploid so that it is easily to silence a gene when the yeast is haploid. The yeast wasfirstly cultured on on suitable sporulation medium plate to produce haploid spores. Haploidstrains were screen from the spores using the method of bulk sergeant of spores. The mating typeof the selected haploid strains were determined by hybridization with W303-1A (MATa), whosemating type was already known. In total, five haploid strains, one MATα and four MATa, wereselected. Two out of the five strains (one MATα and one MATa) were choosed for the followinggene knock-out experiments. The two haploid strains were named as AS-1(MATa) and AS-2(MATα).The GPD2gene was knocked out from the yeast genome by homologous recombination.The disruption cassette consisted of a G418-resisted gene flanked by two39-mer homologoussequence of GPD2was constructed by PCR. It was transformed into the haploid strains AS-1and AS-2. The gpd2Δ mutant strains named as AS-3(MATa gpd2Δ) and AS-4(MATα gpd2Δ)respectively, were screened by G418resistance and confirmed by PCR. The diploid strain ofyeast of double deletion of GPD2was obtained by mating AS-3and AS-4, the diploid mutantwas screened and confirmed by sporulation and PCR and named as AS-5(MATa/αgpd2Δ/gpd2Δ). To examine the influence of deletion of GPD2on the growth characteristics, the productionof ethanol and glycerol, batch fermentation cultivations were carried out between wild andmutant yeasts in100ml culture medium containing20g/L glucose. The result of anaerobicfermentation showed that both the growth rate and glucose consumption rate of gpd2Δ haploidmutant strains AS-4and AS-3were slower than their wild haploid strains AS-2and AS-1, whilethe glycerol conversion rate of AS-4and AS-3decreased by29.4%and23.01%, the ethanolproduction rate of them increased by2.8%and2.1%respectively. In contrast to wild diploidstrain AS2.489, the growth rate and glucose consumption rate of the double deletion strain AS-5also became slow. The glycerol production rate decreased31.1%and ethanol production rateincreased by3.3%for AS-5. Meanwhile, dramatic reduction in the yield of acetate was observedin gpd2Δ haploid and diploid mutants when compared to their wild type strains.In this study, GPD2gene was knocked out from both haploid and diploid strains ofS.cerevisiae. Compared to the original strains, the yields of glycerol and acetate really decreasedmeawhile the ethanol production rates increased. However, the growth conditions of the gpd2Δmutants were obviously limited. This leads to slower glucose consumption rate and longerfermatation period. To improve the growth of the mutant strain, introduction of extraheterologous genes (such as glutamate synthase, GLT1) may be a feasible choice in futurestudies.