The overall goal of this thesis study is to use metabolic engineering and biotechnology tools for developing optimal yeast strains capable of utilizing various sugars derived from renewable biomass and produce valuable chemicals. Sugars derived from lignocellulosic biomass, mainly cellobiose and xylose, cannot be assimilated by the industrial microorganisms, such as yeast Saccharomyces cerevisiae. To utilize cellobiose or xylose for the fuels and chemicals production by S. cerevisiae strains, heterologous expression of cellobiose or xylose metabolizing genes are required. The first part of this dissertation focuses on developing optimal yeast strains for utilizing renewable sugars, cellobiose, xylose and galactose, and understanding underlying mechanism for improvement on lignocellulosic sugar utilization.
Initially, cellobiose fermenting S. cerevisiae was developed by expressing Neurospora crassa cellodextrin transporters (CDT-1 and CDT-2) and β-glucosidase (BGL) or cellobiose phosphorylase (CBP) to breakdown cellobiose into hexose units. In various cellobiose fermentation conditions, the strains expressing CDT-1 showed efficient fermentation compared to the strains expressing CDT-2. Also, strains expressing BGL had better cellobiose fermentation compared to CBP. However, I hypothesized expressing CDT-2 and CBP might have a beneficial effect under energy-limiting conditions (such as industrial fermentation environment) due to minimal ATP requirement compare to pairing with/or CDT-1 and BGL. Because the rate of cellobiose fermentation with CDT-2 and CBP were very inefficient, laboratory evolution approach was employed to enhance cellobiose utilization. I isolated an evolved strain that harbored one SNP change in CDT-2 that is responsible for enhanced cellobiose utilization. With evolved CDT-2 and CBP, cellobiose fermentation rate improved drastically.
Previous studies successfully developed xylose-utilizing S. cerevisiae strain. However, the overall fermentation efficiency of the strain is relatively poor. Additionally, using different strain backgrounds resulted in various patterns of the xylose utilization. This variation is presumably caused by the genotype differences among the strains. Thus, I hypothesized the xylose-utilizing trait is a complex trait which phenotypes result from a multiple genes interaction. Quantitative Trait Locus (QTL) mapping strategy was used to identify ‘hotspots’ in the genome responsible for enhanced xylose utilization. The identified QTLs reveal that enhanced xylose fermentations are likely very complex due to many detected QTL. Also, previously reported gene targets that may improve xylose fermentation was not detected. This result indicates complex genetic interactions are responsible for xylose fermentation. Although specific genetic targets were not identified, our results show genome shuffling of two highly variable strains is a very effective methodology to obtain an efficient xylose-fermenting strain with minimal genetic engineering.
Galactose is abundantly found in marine biomass and hemicellulose from plant cell wall. Although yeast can naturally metabolize galactose, consumption rate and overall ethanol yield are usually lower compared to glucose. While screening various gene targets related glucose sensing, regulation and metabolism, TPS1 deletion expressed with mutant HXK2 allowed enhanced glucose or galactose fermentation. Because HXK2 also has a role in glucose signaling and repression, a mutation caused a change in this property. By deleting TPS1 and expressing mutant HXK2, yeast effectively utilized glucose and galactose under mixed sugar condition whereas wild-type strain could not consume galactose in the presence of glucose.
Following renewable sugar utilization by engineered strain, the second part of this dissertation investigates on chemical production by engineered yeast. To replace current chemical production system, diversification of product formation by the microbial system is necessary. I identified 2-isopropylmalate (2-IPM), and intermediate of leucine biosynthesis, as a target chemical production in yeast. The dicarboxylic organic acids are favorable for production due to possible bio-based polymer synthesis. I observed 2-IPM accumulation by deleting LEU1 and further optimized the strain by removing nitrogen catabolite repression (∆URE2), leucine feedback inhibition (mutant LEU4) and increase pyruvate flux to leucine biosynthetic pathway (Bacillus subtilis AlsS). Also, fermentation media optimization was performed. From no accumulation of 2-IPM by wildtype yeast, 0.033 g/g yield was achieved by optimizing strain and fermentation condition. At scale-up fermentation, 2-IPM titer reached 25 g/L by glucose-limited fed-batch strategy. After 2-IPM overproduction had been achieved, various prospective applications were explored. First, polymer synthesis derived from 2-IPM was investigated. By converting 2-IPM to anhydride form then reaction with epoxide yielded biodegradable elastomer. Also, taking advantage of the organic acid identity of 2-IPM and skin-whitening effect by inhibiting melanin formation was investigated.
In summary, this research demonstrates effective metabolic strategy employed for developing yeast strains capable of fermenting lignocellulosic sugars and diversified chemical production by discovering new target chemical with many projected applications
