Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production

The first commercial-scale plants for production of ethanol from lignocellulosic feedstocks, which are now coming on line, integrate the results of two decades of intensive metabolic engineering research. In my lecture, I will discuss metabolic and evolutionary engineering studies aimed at transforming Saccharomyces cerevisiae, the mainstay microorganism in ‘first generation’ ethanol production, into an efficient cell factory for converting lignocellulosic hydrolysates.

As part of a world-wide effort, our group in Delft initially focused on enabling anaerobic S. cerevisiae cultures to efficiently ferment the pentoses D-xylose and L-arabinose, which are important substrates in lignocellulosic hydrolysates. Functional expression of heterologous, isomerase-based pentose-dissimilation pathways, combined with the overexpression and deletion of native yeast genes, enabled high ethanol yields. Evolutionary engineering, using different selection strategies, plays a major role in improving the kinetics of pentose fermentation. Moreover, whole-genome resequencing of the resulting strains, followed by reverse engineering of the observed mutations in ‘naïve’ S. cerevisiae strains, continues to identify new mutations that further improve the capacity and affinity of pentose fermentation.

Lignocellulosic hydrolysates contain many compounds that inhibit growth and fermentation by S. cerevisiae. Inhibitor tolerance is therefore a crucial target for the development of robust industrial strains. Since acetyl moieties are an integral part of plant biomass, acetic acid is an almost inevitable inhibitor in lignocellulosic hydrolysates. Dynamic evolutionary engineering experiments, in which cells were alternately grown in the presence and absence of acetic acid stress, proved to be a powerful approach for selecting highly and constitutively acetic-acid tolerant strains. To improve process economics, we explore redox engineering strategies that use NADH, generated in biosynthetic reactions, for converting external electron acceptors such as carbon dioxide and acetic acid, into ethanol. These strategies have the potential to significantly increase ethanol yields in industrial processes and, in the case of acetic acid, to convert an inhibitor into additional product.