Engineering of metabolism and membrane transport in Saccharomyces cerevisiae for improved industrial performance

Abstract

Nearly 200 parties signed and committed to the Paris agreement in 2017, which comprises the long-term goal to keep the average global temperature increase well below 2 degrees above pre-industrial levels. Virtually all possible scenarios drafted to reach this goal include a strongly increased use of biofuels for transport by land, sea and air. Bioethanol, whose production could, in principle and in contrast to fossil fuel production, involve a closed carbon cycle, is generated by microbial fermentation of sugars from plant-derived starch or agricultural waste. This liquid transport fuel provides a readily implementable alternative to fossil fuels as it combines the advantages of sustainable fuel production and compatibility with existing combustion engine technologies, without a requirement for time-consuming and expensive changes in our current infrastructure. To date, bioethanol is the largest volume product of industrial biotechnology. 99% of this ethanol is generated via 1st generation processes, largely derived by fermentation of hydrolysed sugar cane or corn starch by bakers’ yeast (Saccharomyces cerevisiae). So-called ‘2nd generation’ bioethanol, for which the first commercial-scale plants are now starting up, is made by fermentation of sugars present in lignocellulosic biomass, typically harvested from agricultural waste streams, such as wheat straw or sugar beet pulp. Whilst such feedstocks enable a “food and fuel” scenario, their industrial implementation brings along additional challenges for yeasts and biotechnologists. Hydrolysis of lignocellulosic biomass, in particular the cellulose and hemicellulose fractions, releases a mixture of different sugars as well as inhibiting compounds that impair growth and viability of S. cerevisiae. Whilst glucose is the most abundant fermentable carbon source, the pentose sugar d-xylose can cover up to 30% of the total sugar content. The fraction of the pentose l-arabinose typically varies between 2 – 20%, depending on the feedstock used. Although pentose sugars cannot be fermented to ethanol by wild-type S. cerevisiae strains, international research efforts over the past two decades yielded metabolic engineering strategies to enable anaerobic conversion of d-xylose and l-arabinose to ethanol by S. cerevisiae. The expression of heterologous, d-xylose- and l-arabinose-isomerase based pathways from fungi or bacteria, together with over-expression of genes of the non-oxidative pentose phosphate pathway (PPP) and deletion of the unspecific aldose-reductase gene GRE3 within S. cerevisiae allows this yeast to aerobically metabolize both sugars. The recent advances in metabolic engineering tools, such as CRISPR-Cas9-assisted genome editing, greatly advanced the construction and characterization of metabolically engineered S. cerevisiae strains with improved yields, kinetics and robustness in 2nd generation ethanol production processes.