Metabolic engineering of polyploid Saccharomyces cerevisiae for production of ethanol and 2,3-butanediol

Abstract

Industrial polyploid Saccharomyces cerevisiae is a versatile yeast strain for large-scale fermentations due to rapid growth rate, high sugar consumption rate and high tolerance to ethanol and inhibitors. Despite these advantageous traits, however, industrial strains have not been widely used and studied as a host for genetic and metabolic engineering because of some drawbacks including aneuploidy or polyploidy, unstable mating-type and no auxotrophic markers. Generally, the desired phenotypes of industrial strains have been obtained through random mutagenesis or evolutionary adaptation rather than rational genetic engineering. This thesis has focused on using an industrial JHS200 strain as a host for metabolic engineering approaches in order to produce ethanol and 2,3-butanediol with high productivity and yield. <br/> First of all, an industrial strain of S. cerevisiae JHS200 that was isolated from Korean Nuruk and identified to be a polyploidy (4n) was characterized for fermentation properties. The industrial JHS200 strain showed a higher maximum specific growth rate (0.547 hr-1) than haploid S. cerevisiae strains such as CEN.PK2-1D (0.452 hr-1), D452-2 (0.445 hr-1), L2612 (0.413 hr-1) and BY4742 (0.478 hr-1). In addition, the JHS200 exhibited superior properties in terms of sugar consumption rate, ethanol tolerance and resistance to fermentation inhibitors. For further metabolic engineering, the auxotrophic mutants of polyploidy JHS200 were constructed with the modified Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 genome editing tool. A stop codon (TAA) was introduced into the middle of each gene (URA3, TRP1, HIS3, LEU2) through a point mutation. Finally, four auxotrophic mutants of the polyploidy JHS200 (JHS200ura-, JHS200ura-trp-, JHS200ura-trp-his-, JHS200ura-trp-his-leu-) were constructed to possess single or multiple auxotrophic traits. The auxotrophic mutants kept the excellent fermentation properties without significant growth defects. <br/> Second, in order to produce cellulosic ethanol, the xylose-assimilating pathway was introduced into the auxotrophic industrial strain. Plasmid pRS306_XYL123 harboring the genes coding for xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) from Scheffersomyces stipitis with strong and constitutive promoters was integrated into the JHS200ura-trp- strain, in which the URA3 and TRP1genes were previously disrupted. The resulting strain of JX123 produced 45.6 g/L ethanol with 0.73 g/L·hr of xylose consumption rate. As 7.25 g/L of xylitol accumulated as a major by-product in the JX123 strain, the heterologous noxE gene from Lactococcus lactis which reduces surplus intracellular NADH generated from the xylose metabolism was additionally introduced. As a result, the JX123_noxE strain expressing the noxE gene produced 47.0 g/L ethanol with decreased xylitol accumulation (2.26 g/L). Additionally, the JX123_noxE strain was successfully cultivated by using silver grass hydrolysates as a sole carbon source. It produced 55.5 g/L ethanol with 1.63 g/L·hr of productivity and 0.43 g/g of yield from lignocellulose hydrolysates. Even both furfural and HMF inhibitors were present in the hydrolysates, the engineered JX123_noxE industrial strain showed a high xylose consumption rate of 0.90 g/L·hr. <br/> Third, the auxotrophic polyploidy strain (JHS200ura-trp-his-leu-, 4-JHS200) was used to produce 2,3-butanediol (2,3-BDO). In order to block the production of ethanol as a major metabolite, the genes coding for pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) were partially disrupted through the CRISPR-Cas9 method. To overcome the growth defect caused by complete deletion of all isozymes encoded by the PDC and ADH genes, a strategy of partial disruption of PDC1, PDC6 or ADH1 was employed to engineer the 4-JHS200 strain. The biosynthetic pathway of 2,3-BDO was introduced by transformation of plasmid p413_SDB harboring the genes coding for α-acetolactate synthase (alsS) and α-acetolactate decarboxylase (alsD) from Bacillus subtilis and 2,3-butanediol dehydrogenase (BDH1) from S. cerevisiae. And the heterologous NADH oxidase (noxE) from L. lactis was additionally expressed for modulating redox balance. The resulting strain of YG01_SDBN was able to produce 178.6 g/L of 2,3-BDO with a maximum productivity of 2.64 g/L·hr. In an optimized fermentation using cassava hydrolysates as carbon sources, 132.7 g/L of 2,3-BDO was produced with a productivity of 1.92 g/L·hr. On the other hand, glycerol accumulation in the 2,3-BDO-producing strains causes several problems such as low yield of 2,3-BDO and high cost of separation. To increase 2,3-BDO yield by elimination of glycerol accumulation, the genes coding for glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) in the glycerol synthetic pathway were disrupted in the YG01_SDBN strain. As excess NADH was oxidized by NoxE with an aid of oxygen molecule in the host strain, the Gpd-deficient polyploid strain (YG01_SDBN_dGPD1GPD2) could be obtained without severe redox imbalance. The YG01_SDBN_dGPD1GPD2 strain lost a glycerol producing ability completely, and hence produced 151.5 g/L of 2,3-BDO with 0.41 g/g yield. Additionally, this strain could produce 120.6 g/L of 2,3-BDO with 0.42 g/g yield using the cassava hydrolysates. The maximum 2,3-BDO yield in this optimized fed-batch fermentations using cassava hydrolysates was comparable to those for engineered bacterial systems. <br/> In conclusion, the polyploidy yeast of S. cerevisiae JHS200 was metabolically engineered by using the CRISPR-Cas9 genome editing tool. The engineered JHS200 variant with auxotrophic markers and various genes affecting metabolic phenotypes could produce ethanol and 2,3-BDO with high productivity, yield and concentration in optimized fed-batch fermentation processes. The engineered polyploid yeast strains could be used as a platform for economic production of valuable chemicals from various carbohydrate hydrolysates.<br/>