Quantitative Methods to Engineer Yeast Whole-Cell Biocatalysts for Lignocellulosic Biomass Transformation to Biofuels
Creators: name: Ma, Xiao Yin
Publisher: name: My University
Year: 2023
Subjects: subject: Whole Cell Biocatalysis
subject: Metabolic Engineering
subject: Synthetic Biology
subject: Lignocellulosic Biomass
subject: Biofuel
subject: Yeast
subject: Engineering (General)
subject: Molecular, Cellular and Developmental Biology
subject: Natural Resources and Environment
subject: Science (General)
subject: Engineering
subject: Science

description: As the most abundant renewable carbon source on Earth, lignocellulosic biomass represents a promising, low-cost feedstock for biofuel production. In recent years, the use of yeasts as whole-cell biocatalysts for lignocellulosic conversion has garnered significant research interest due to their generally regarded as safe (GRAS) status, easy gene manipulation, and high alcohol tolerance. However, the yeast whole-cell biocatalysts (yWCBs) available today cannot efficiently break down lignocellulose and ferment the hydrolysates. In this dissertation, we developed quantitative methods to guide the optimization of yeast as a biosynthetic host and applied these methods to tackle challenges impeding the valorization of lignocellulose. One major barrier to the development of biomass refineries is the recalcitrance of cellulose, the main component of lignocellulosic biomass, to enzyme degradation. To enhance cellulose hydrolysis, enzyme co-localization strategies have been explored by engineering yWCBs to display multi-enzyme assemblies on the cell surface. However, maximizing the yWCB performance poses engineering challenges associated with the complexity and heterogeneity of biological systems. Thus, we developed a quantitative single-cell approach to yWCB characterization and demonstrated its ability to elucidate cell-to-cell variations and aid yWCB engineering efforts. This approach revealed that the yWCB performance is limited in two major ways: at the population level by a catalytically inactive cell population and at the single-cell level by surface crowding. We demonstrated that the inactive cell population can be eliminated to form compositionally uniform yWCBs. Furthermore, quantitative evaluation of structure-function relationships guided the optimization of the multi-enzyme assembly, allowing us to overcome molecular crowding and maximize enzyme density at the single-cell level. As a result, we achieved the highest ethanol titer, productivity, and conversion reported to date from 1% phosphoric acid swollen cellulose as substrate. Another challenge hindering the widespread utilization of lignocellulose is that its most abundant pentose sugar, xylose, cannot be naturally consumed by many industrial yeast strains, including S. cerevisiae. By introducing a heterologous xylose assimilation pathway, researchers have endowed S. cerevisiae with the ability to ferment xylose, albeit inefficiently. To guide the strain optimization process, we devised a strategy that enables the rapid identification and removal of metabolic bottlenecks. Termed Segmentation and Evaluation of Pathway Module Efficiency (SEPME), this method allowed us to track the shifts in bottlenecks between different enzymatic steps of the xylose-to-ethanol pathway. More importantly, SEPME provided insights on engineering strategies that could overcome each rate-controlling step. After eliminating these bottlenecks one by one, we obtained a strain approaching the theoretical xylose-to-ethanol yield of 90%, which is among the highest reported to date. A critical component of the quantitative single-cell approach and SEPME is the elucidation of the relationship between the protein expression level and yWCB performance. To develop these relationships, a broad range of protein expression levels is needed. Thus, we explored potential targets in the secretory pathway for increasing the intracellular expression and surface display of a protein of interest (POI). We identified four secretory pathway proteins, whose overexpression improved the intracellular POI expression level by at least 1.5-fold. Future work will involve translating the improvements in intracellular protein expression to surface display. Overall, this dissertation presents methods that are not only useful for accelerating the development of yeast as a biosynthetic host for biofuel production, but are also broadly applicable for engineering microbes to enhance their biomanufacturing potential in the chemical, therapeutics, and vaccine industries.