Engineering yeast strains for producing fuels and value-added chemicals from cellulosic biomass

Metabolic engineering;Saccharomyces cerevisiae;Cellulosic biomass

Oh, Eun Joong

关键词: Metabolic engineering;    Saccharomyces cerevisiae;    Cellulosic biomass;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/89225/OH-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The biotechnological production of fuels and value-added chemicals from cellulosic biomass is a sustainable and ecofriendly process. Pretreatment and hydrolysis of the biomass produce six (glucose) and five-carbon (xylose) sugars, and toxic fermentation inhibitors, such as acetic acid. The efficient conversion of the cellulosic sugars in hydrolyzates by fermentation microorganisms is one of the key steps for the economically feasible production of biofuels and chemicals. Most xylose-fermenting engineered yeast cannot utilize xylose until glucose is completely consumed. In addition, acetic acid derived from hemicellulose during the pretreatment negatively influences the fermentation performance by yeast. The overall goal of my thesis study is to develop metabolically engineered yeast strains to overcome the abovementioned challenges. Simultaneous co-utilization of cellobiose (a dimer of glucose) and xylose could bypass the inhibition of xylose uptake by glucose and improve ethanol production. To this end, heterologous genes coding for cellodextrin transporter (cdt-1) and β-glucosidase (gh1-1) were integrated into the genome of the yeast, and the expression cassettes were massively amplified through laboratory evolution, resulting in significantly improved cellobiose fermentation. Besides ethanol production, the simultaneous co-conversion of cellobiose and xylose strategy was successfully applied to improve the production of value-added products, such as xylitol. Furthermore, integration of cellobiose, xylose, and acetic acid utilizing pathways into one microbial platform enabled efficient bioconversion of the mixed substrates in a synergistic way. Next, cellodextrin transporter 2 (CDT-2), which has the potential to surpass the performance of CDT-1 because of its energetic benefits under stress conditions, was engineered to improve cellobiose uptake under acidic conditions. The mutant CDT-2 (I96N/T487A) was isolated through the directed evolution of CDT-2 followed by high-throughput screening using flow cytometry, and its cellobiose consumption rate under low pH conditions was much higher than that of the wild type CDT-2. Lastly, a genetic perturbation for enhancing the acetic acid tolerance has been identified via genomic library screening. Overexpression of RCK1 coding for protein kinase involved in the oxidative stress response in yeast significantly improved both glucose and xylose fermentations under acidic conditions, reducing intracellular reactive oxygen species (ROS) accumulation. We demonstrated that the rational and combinatorial metabolic engineering strategies enabled engineered yeast strains to overcome the challenges in fermentation for the conversion of cellulosic biomass. Moreover, this study provided valuable findings that can be applied to industrial fermentation processes.