CRISPR/Cas9: A molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their...

In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation

Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by...

Evolutionary engineering in chemostat cultures for improved maltotriose fermentation kinetics in saccharomyces pastorianus lager brewing yeast

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion ("attenuation") of maltotriose by industrial S. pastorianus strains is a key requirement for process...

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...

Engineering oxygen-independent biotin biosynthesis in Saccharomyces cerevisiae

An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation...

Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium...

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic...

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...

Structural, physiological and regulatory analysis of maltose transporter genes in Saccharomyces eubayanus CBS 12357^T

Saccharomyces pastorianus lager brewing yeasts are domesticated hybrids of Saccharomyces cerevisiae and cold-tolerant Saccharomyces eubayanus. To understand the contribution of both parental genomes to maltose metabolism in brewing wort, this study focuses on maltose transport in the S. eubayanus type strain CBS 12357^T/FM1318. To...

Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in Saccharomyces pastorianus

The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S...

Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae

Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which...

Adaptive Laboratory Evolution and Reverse Engineering of Single-Vitamin Prototrophies in Saccharomyces cerevisiae

Quantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in S. cerevisiae CEN. PK113-7D, requirements for biotin were eliminated by laboratory...

Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis

Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d-xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic...

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...

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains

Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also...

Selection of Pof^- saccharomyces eubayanus variants for the construction of S. cerevisiae × S. eubayanus hybrids with reduced 4-Vinyl guaiacol formation

Saccharomyces pastorianus is an interspecies hybrid between S. cerevisiae and S. eubayanus. The identification of the parental species of S. pastorianus enabled the de novo reconstruction of hybrids that could potentially combine a wide array of phenotypic traits. Lager yeasts are characterized by their inability to decarboxylate ferulic acid...

Laboratory Evolution of a Saccharomyces cerevisiae × S. eubayanus Hybrid Under Simulated Lager-Brewing Conditions

Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they...

Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast

Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high...