Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in .

The lager-brewing yeast is a hybrid between and with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing.

Chromosome level assembly and comparative genome analysis confirm lager-brewing yeasts originated from a single hybridization.

Background: The lager brewing yeast, S. pastorianus, is a hybrid between S. cerevisiae and S.

Lager-brewing yeasts in the era of modern genetics.

The yeast Saccharomyces pastorianus is responsible for the annual worldwide production of almost 200 billion liters of lager-type beer. S. pastorianus is a hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been studied for well over a century.

Himalayan Genome Sequences Reveal Genetic Markers Explaining Heterotic Maltotriose Consumption by Saccharomyces pastorianus Hybrids.

strains are hybrids of and that have been domesticated for centuries in lager beer brewing environments. As sequences and structures of genomes are being resolved, molecular mechanisms and evolutionary origins of several industrially relevant phenotypes remain unknown. This study investigates how maltotriose metabolism, a key feature in brewing, may have arisen in early × hybrids.

Phenotype-Independent Isolation of Interspecies Hybrids by Dual-Dye Fluorescent Staining and Fluorescence-Activated Cell Sorting.

Interspecies hybrids of species are found in a variety of industrial environments and often outperform their parental strains in industrial fermentation processes. Interspecies hybridization is therefore increasingly considered as an approach for improvement and diversification of yeast strains for industrial application. However, current hybridization methods are limited by their reliance on pre-existing or introduced selectable phenotypes.

Laboratory Evolution of a × Hybrid Under Simulated Lager-Brewing Conditions.

lager-brewing yeasts are domesticated hybrids of x 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 contribute to their industrial performance. Here, an allodiploid laboratory hybrid of and was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors.

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.

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 degrees of heterozygosity are less amenable to genetic modification.

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed.

CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus.

Background: The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S.

Nanopore sequencing enables near-complete de novo assembly of Saccharomyces cerevisiae reference strain CEN.PK113-7D.

The haploid Saccharomyces cerevisiae strain CEN.PK113-7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C.

Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts.

Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV.

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 introduction into lignocellulosic hydrolysates. However, earlier laboratory evolution strategies for increasing acetic acid tolerance of Saccharomyces cerevisiae, based on prolonged cultivation in the presence of acetic acid, selected for inducible rather than constitutive tolerance to this inhibitor.

Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting.

High-throughput antibody repertoire sequencing (Ig-seq) provides quantitative molecular information on humoral immunity. However, Ig-seq is compromised by biases and errors introduced during library preparation and sequencing. By using synthetic antibody spike-in genes, we determined that primer bias from multiplex polymerase chain reaction (PCR) library preparation resulted in antibody frequencies with only 42 to 62% accuracy.