Short-term adaptation of S. cerevisiae to lignocellulosic inhibitors: Underlying metabolic and physiological changes

Doctoral thesis, 2021

The limited tolerance of Saccharomyces cerevisiae (budding yeast) to inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. Short-term adaptation of the yeast to lignocellulosic hydrolysates during cell propagation has been shown to improve its tolerance, and thus its performance in lignocellulose fermentation. The overall aim of this thesis was to identify molecular and physiological changes during short-term adaptation.

In order to facilitate testing of S. cerevisiae physiology in lignocellulosic hydrolysate, a high-throughput methodology for the analysis of yeast strains in dark medium was developed. This methodology allows for monitoring of both aerobic and anaerobic growth of yeast in medium containing different hydrolysates at high reproducibility. The effect that individual nutrient components during propagation, rather than fermentation, has on lignocellulose fermentation performance is lacking. A high-throughput screening of certain vitamins, trace metals and nitrogen sources was performed. It was found that adding a mixture of pyridoxine, thiamine, and biotin to unadapted propagation cultures improved cell growth and ethanol yields during fermentation in wheat straw hydrolysate. Supplementing the propagation medium with nutrients in combination with short-term adaptation was thus demonstrated to be a promising strategy to improve the efficiency of industrial lignocellulosic fermentation.

Different S. cerevisiae strain backgrounds are used in the production of a suitable second-generation bioethanol host. In order to facilitate application of results obtained in laboratory experiments it is important to know whether short-term adaptation affects different strains differently. The physiology of two industrial S. cerevisiae strains were investigated while being short-term adapted. During propagation, fed with a hydrolysate containing feed, ethanol accumulation was observed for strain CR01 but not for KE6-12. Additionally, a larger increase in specific ethanol productivity for CR01 was observed than for KE6-12. Thus, short-term adaptation was found to affect S. cerevisiae physiology differently depending on strain background. To gain a more complete insight into the metabolic changes that S. cerevisiae experiences during short‑term adaptation, RNA sequencing was performed on a time-series of samples taken from propagation cultures undergoing short-term adaptation. Expression data was compared to a non‑adapted control using differential gene expression analysis. Results demonstrate, among others, an interesting role for multidrug proton antiporters YHK8 and FLR1 in the process of short-term adaptation.