Metabolic engineering of non-model yeasts for polymer precursor production and cellulose degradation

Abstract

Methyl methacrylate (MMA) is a building block for polymethylmethacrylate (PMMA), which is a transparent material that has diverse applications in both industrial and medical area. Currently, MMA is produced by petrochemical feedstocks. Biobased routes using carboxylic acids as precursors to produce MMA are potential alternatives to petrochemical processes. Considering the bioprocess development and final chemical conversions, itaconic acid and citramalate are two promising intermediates. Since low pH fermentation using acid-tolerant microbes could decrease both downstream processing costs and environmental footprints in industry, we tried to engineer a non-model yeast Pichia kudriavzevii (also known as Issatchenkia orientalis) to produce itaconic acid and citramalate. P. kudriavzevii is an acid-tolerant yeast that have been engineered to produce various organic acids. We introduced genetic manipulation tools (such as vector, CRISPR-Cas system, and Transposon system) and performed pathway engineering with the developed tools to demonstrate the potential of using this species as a generic production platform for organic acids. In the end, about 1.8 g/L of itaconic acid and 5g/L of citramalate were produced at low pH in shake flasks. Cellulose is a linear polysaccharide that can be degrade into glucose by the synergistic function of three cellulases: endoglucanase, cellobiohydrolase and β-glucosidase. As the most abundant natural biopolymer on earth, engineering efforts have been done to convert cheap cellulose to valuable products via microbial cell factories. Pichia pastoris is known for high level expression of secreted proteins and has been engineered to produce high-valued proteins and chemicals. Here we also explored cellulose degradation in P. pastoris and reviewed the current advance in synthetic biology applications of Pichia species. Collectively, the work presented in this thesis introduced genetic manipulation tools for P. kudriavzevii, established organic acids production in P. kudriavzevii and studied cellulose degradation in P. pastoris. All these efforts will help advance the development of bioprocesses in non-conventional yeasts for industrial applications.