The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae

Background: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these...

Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae

Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch...

Fermentation of glucose-xylose-arabinose mixtures by a synthetic consortium of single-sugar-fermenting Saccharomyces cerevisiae strains

D-Glucose, D-xylose and L-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three ‘specialist’ Saccharomyces cerevisiae strains. A D-glucose- and L-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an...

A CRISPR/Cas9-based exploration into the elusive mechanism for lactate export in Saccharomyces cerevisiae

CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25...

Laboratory evolution of a biotin-requiring Saccharomyces cerevisiae strain for full biotin prototrophy and identification of causal mutations

Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01...

Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae

Background: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD⁺-dependent acetate reduction has been shown to improve ethanol yields and...

Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism

Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated...

Saccharomyces cerevisiae strains tor second-generation ethanol production: from academie exploration to industrial implementation

The recent start-up of several full-scale ‘second generation’ ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains,...

A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations

Background: Acetic acid, released during hydrolysis of lignocellulosic feedstocks for second generation bioethanol production, inhibits yeast growth and alcoholic fermentation. Yeast biomass generated in a propagation step that precedes ethanol production should therefore express a high and constitutive level of acetic acid tolerance before...

Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase

In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory,...