Mapping Phenolics Metabolism and Metabolic Engineering of Saccharomyces cerevisiae for Increased Endogenous Catabolism of Phenolic Compounds

Doctoral thesis, 2016

Abstract Sustainable, biotechnological utilization of non-food, plant biomass has been demonstrated to be a viable means of producing energy, fuels, materials and chemicals, representing a paradigm shift from fossil-derived sources. However, the presence of chemicals that inhibit fermentation by microorganisms such as Saccharomyces cerevisiae, commonly used for bioconversion, causes a bottleneck in such processes. Phenolic compounds are aromatic compounds that serve as building blocks of lignin in plants. During the deconstruction of plant biomass, phenolic compounds are released as degradation products from the lignin component of wood into the hydrolysates, inhibiting fermentation. The aim of the work presented in this thesis was to explore approaches for the development of strains of Saccharomyces cerevisiae that have improved tolerance to phenolic compounds, and to better understand its endogenous metabolism of phenolic compounds. A study was performed on the interaction between the yeast and phenolic compounds using single phenolic compounds in defined growth medium. The toxicity of thirteen phenolic compounds was determined. The concentrations at which each compound completely inhibited the growth of S. cerevisiae was found to differ among the compounds, and three distinct physiological responses were observed. The influence of the structure and the presence of the methyl, aldehyde, carboxylic acid and hydroxyl functional side groups that often decorate phenolic compounds were studied in coniferyl aldehyde, ferulic acid and p-coumaric acid. The conversion of these compounds into less toxic phenolic compounds was observed. Based on the product profile, a conversion route was hypothesized for the catabolism of phenolic compounds in S. cerevisiae. Finally, two strains of S. cerevisiae, B_CALD and APT_1, were engineered. B_CALD was metabolically engineered to exhibit increased endogenous conversion of coniferyl aldehyde, while APT_1 was metabolically engineered to exhibit increased endogenous conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid, and to test the hypothesized conversion pathway. The engineering of both B_CALD and APT_1 was successful.