Biology subjects

Krogerus, K.

Publications and source records attributed to Krogerus, K..

12 recordsLinked to original sources

bioRxiv2025

Influence of ploidy and genetic background on stress tolerance of intraspecific yeast hybrids

Hybrid vigor, or heterosis, is widely exploited in yeast strain improvement. Yet, how ploidy and genetic background jointly shape heterosis across industrially relevant stresses remains unclear. Here, we generated 1023 Saccharomyces cerevisiae intraspecific hybrids derived from 18 genetically diverse parents, using two different approaches, yielding sets of hybrids with variable ploidy for the same parental combinations. High-throughput growth assays in media with five stress conditions (14% ethanol, 1.5 M NaCl, 0.15 M lactic acid, 0.05 M acetic acid, 0.05 M HMF) revealed extensive heterosis across 6138 hybrid-condition combinations. Most combinations displayed mid-parent heterosis and over a third exceeded the best parent, with the strongest gains during growth in the presence of 14% ethanol and 1.5 M NaCl. Increasing ploidy was generally associated with reduced growth and reduced best-parent heterosis, whereas greater predicted hybrid heterozygosity or genetic distance between parents was positively associated with heterosis in the presence of 14% ethanol and 1.5 M NaCl. Domestication status also affected these trends, as crosses between two domesticated strains tended to perform better in the presence of ethanol and NaCl, while crosses between two wild strains grew best in control conditions and in the presence of acetic acid. Together, these results demonstrate condition-dependent contributions of ploidy and parentage to heterosis and provide targeted breeding strategies for the improvement of stress-tolerance in industrial yeasts.

microbiology↗

bioRxiv2024

European Farmhouse Brewing Yeasts Form a Distinct Genetic Group

The brewing industry is constantly evolving, driven by the quest for novel flavors and fermentation characteristics that cater to evolving consumer preferences. This study explores the genetic and phenotypic diversity of European farmhouse yeasts, traditionally used in rural brewing practices and maintained outside of pure culture industrial yeast selection. We isolated landrace brewing yeast strains from diverse geographical locations across Europe, including Norway, Lithuania, Latvia, Russia, and also included African farmhouse brewing strains from Ghana. Our genomic analysis using long-read and short-read whole genome sequencing uncovered a genetically distinct group that diverges from industrial brewing yeasts. This group, which is closely related to ale brewing strains, is preliminarily named the "European Farmhouse" group and shows greater predicted admixture from Asian fermentation strains. Through genomic and phenotypic analyses, including flavor metabolite analysis via HS-GC-MS, sugar metabolite analysis via HPLC, and wort fermentation analysis, we found a broad spectrum of fermentation capabilities, from rapid and efficient fermentation to unique aroma and flavor compound profiles, potentially offering novel traits for brewing applications. This study highlights the importance of preservation of brewing cultural heritage knowledge and resources including yeast cultures. Key PointsO_LIA large set of geographically diverse farmhouse brewing strains were characterized C_LIO_LINorwegian and Baltic farmhouse brewing strains form a distinct genetic group C_LIO_LIFarmhouse strains show considerable diversity in fermentation and flavour formation C_LI

microbiology↗

bioRxiv2022

A simple and rapid CRISPR-Cas12a-based detection test for diastatic Saccharomyces cerevisiae

Diastatic Saccharomyces cerevisiae is a common contaminant in the brewing industry. Currently available detection methods are either time-coansuming or require specialized equipment. The aim of this study was to develop a new rapid and simple assay for the detection of diastatic yeast from beer and yeast samples. More specifically, we aimed to develop a simple and rapid assay that requires minimal laboratory equipment or training, and ideally yields results as accurate as PCR-based methods. The developed assay consisted of three main steps: DNA extraction, pre-amplification of DNA, and CRISPR-Cas12a-based detection and visualisation. We compared different preamplification and visualisation techniques, and the final assay involved a one-pot reaction where LAMP and Cas12a were consecutively used to pre-amplify and detect a fragment from the STA1 gene in a single tube. These reactions only required a heat block, a pipette, and a centrifuge. The assay result was then visualised on a lateral flow strip. We used the developed assay to monitor an intentionally contaminated beer fermentation, and it was shown to yield results as accurate as PCR using previously published primers. Furthermore, the assay yielded results in approx. 75 minutes starting from a beer sample. The developed assay therefore offers reliable and rapid quality control for breweries of all sizes and can be performed without any expensive laboratory equipment. We believe the assay will be particularly useful for smaller breweries that dont already have well-equipped laboratories and are looking to implement better quality control.

microbiology↗

bioRxiv2022

Increased volatile thiol release during beer fermentation using constructed interspecies yeast hybrids

Interspecies hybridization has been shown to be a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. Thanks to the popularity of heavily hopped India Pale Ale-style beers, there is an increased demand from brewers for strains that can boost hop aroma. Here, we explored whether hybridization could be used to construct strains with an enhanced ability to release hop-derived flavours through {beta}-lyase activity, which releases desirable volatile thiols. Wild Saccharomyces strains were shown to possess high {beta}-lyase activity compared to brewing strains, however, they also produced phenolic off-flavours (POF) and showed poor attenuation. To overcome these limitations, interspecies hybrids were constructed by crossing pairs of one of three brewing and one of three wild Saccharomyces strains (S. uvarum and S. eubayanus). Hybrids were screened for fermentation ability and {beta}-lyase activity, and selected hybrids showed improved fermentation and formation of both volatile thiols (4MMP, 3MH and 3MH acetate) and aroma-active esters compared to the parent strains. Undesirable traits (e.g. POF) could be removed from the hybrid by sporulation. To conclude, it was possible to boost the release of desirable hop-derived thiols in brewing yeast by hybridization with wild yeast. This allows production of beer with boosted hop aroma with less hops (thus improving sustainability issues).

microbiology↗

bioRxiv2021

Unlocking the functional potential of polyploid yeasts

Breeding and domestication have generated widely exploited crops, animals and microbes. However, many Saccharomyces cerevisiae industrial strains have complex polyploid genomes and are sterile, preventing genetic improvement strategies based on breeding. Here, we present a novel strain improvement approach based on the budding yeasts property to promote genetic recombination when meiosis is interrupted and cells return-to-mitotic-growth (RTG). We demonstrated that two unrelated sterile industrial strains with complex triploid and tetraploid genomes were RTG-competent and developed a visual screening for easy and high-throughput identification of recombined RTG clones based on colony phenotypes. Sequencing of the evolved clones revealed unprecedented levels of RTG-induced genome-wide recombination. We generated and extensively phenotyped a RTG library and identified clones with superior biotechnological traits. Thus, we propose the RTG-framework as a fully non-GMO workflow to rapidly improve industrial yeasts that can be easily brought to the market.

genetics↗

bioRxiv2021

Repurposing brewery contaminant yeast as production strains for low-alcohol beer fermentation

A number of fungal isolates were recently obtained from a survey of the microbiota of multiple breweries and brewery products. Here, we sought to explore whether any of these brewery contaminants could be repurposed for beneficial use in beer fermentations, with particular focus on low-alcohol beer. 56 yeast strains were first screened for the utilization of different carbon sources, ability to ferment brewers wort, and formation of desirable aroma compounds. A number of strains appeared maltose-negative and produced desirable aromas without obvious off-flavours. These were selected for further scaled-up wort fermentations. The selected strains efficiently reduced wort aldehydes during fermentation, thus eliminating undesirable wort-like off-flavours, and produced a diverse volatile aroma profile. Sensory analysis of the beer samples using projective mapping identified two strains, Trigonopsis cantarellii and Candida sojae, that produced beers similar to a commercial reference lager beer. 30 L-scale wort fermentations were performed with these two strains together with a commercial Saccharomycodes ludwigii reference strain. Both strains performed comparably to the commercial reference, and the T. cantarellii strain in particular, produced low amounts of off-flavours and a significantly higher amount of the desirable monoterpene alcohol trans-geraniol. The strain was also sensitive to common food preservatives and antifungal compounds, and unable to grow at 37 {degrees}C, suggesting it is relatively easily controllable in the brewery, and appears to have low risk of pathogenicity. This study shows how the natural brewery microbiota can be exploited as a source of non-conventional yeasts for low-alcohol beer production. Take Away- Fungal isolates from brewery microbiota were screened for beer production - Numerous maltose-negative strains were tested for low-alcohol beer fermentation - Trigonopsis cantarellii showed promise compared to a commercial reference strain - T. cantarellii produced no off-flavours and higher levels of trans-geraniol

microbiology↗

bioRxiv2021

Kveik brewing yeasts demonstrate wide flexibility in beer fermentation temperature and flavour metabolite production and exhibit enhanced trehalose accumulation

AO_SCPLOWBSTRACTC_SCPLOWTraditional Norwegian Farmhouse ale yeasts, also known as kveik, have captured the attention of the brewing community in recent years. Kveik were recently reported as fast fermenting thermo- and ethanol tolerant yeasts with the capacity to produce a variety of interesting flavour metabolites. They are a genetically distinct group of domesticated beer yeasts of admixed origin with one parent from the "Beer 1" clade and the other unknown. While kveik are known to ferment wort efficiently at warmer temperatures, its range of fermentation temperatures and corresponding flavour metabolites produced, remain uncharacterized. In addition, the characteristics responsible for its increased thermotolerance remain largely unknown. Here we demonstrate variation in kveik strains at a wide range of fermentation temperatures and show not all kveik strains are equal in fermentation performance, flavour metabolite production and stress tolerance. Furthermore, we uncovered an increased capacity of kveik strains to accumulate intracellular trehalose, which likely contributes to its increased thermo- and ethanol tolerances. Taken together our results present a clearer picture of the future opportunities presented by Norwegian kveik yeasts and offer further insight into their applications in brewing.

microbiology↗

bioRxiv2021

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching

Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase {beta}-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high {beta}-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for {beta}-lyase activity. Selected phenolic off-flavour positive (POF+) hybrids were further sporulated to generate meiotic segregants with high {beta}-lyase activity, efficient wort fermentation and lack of POF; all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast. Key PointsO_LICRISPR/Cas9-based mating type switching was applied to industrial yeast strains C_LIO_LITransformed strains could be readily mated to form intraspecific hybrids C_LIO_LIHybrids exhibited heterosis for a number of brewing-relevant traits C_LI

microbiology↗

bioRxiv2021

Lager yeast design through meiotic segregation of a fertile Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae x Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15 {degrees}C, 15 {degrees}Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains. Contribution to the fieldLager beer fermentations have traditionally been carried out with natural S. cerevisiae x S. eubayanus hybrids. These strains possess both the ability to tolerate low temperatures and the ability to utilize efficiently wort sugars. However, being closely related, strains within the group exhibit limited phenotypic variability. Since the recent discovery of wild strains of S. eubayanus, it has been possible to generate lager yeast hybrids artificially, thereby increasing the genetic and phenotypic diversity of lager brewing strains. Here, to demonstrate the potential for further increased diversity, a constructed tetraploid hybrid was sporulated and spore clones derived from a single ascus were evaluated with respect to fermentation performance (sugar utilization, stress tolerance and volatile aroma synthesis). Meiosis introduced variability in a number of key parameters. One fertile spore clone from this F1 generation was sporulated to introduce further diversity and to demonstrate the potential of clone selection in steering phenotypes in a desirable direction. Genome instability of hybrids was observed, but this can be exploited to further increase diversity. This was demonstrated by assessing performance of variants isolated after ten consecutive rounds of fermentation. The approach allows for the introduction of phenotypic diversity without the need for targeted genetic modification.

microbiology↗

bioRxiv2021

Unique brewing-relevant properties of a strain of Saccharomyces jurei isolated from ash (Fraxinus excelsior)

The successful application of Saccharomyces eubayanus and Saccharomyces paradoxus in brewery fermentations has highlighted the potential of wild yeast for brewing, and prompted investigation into the application potential of other members of the genus. Here, we evaluate, for the first time, the brewing potential of Saccharomyces jurei. The newly isolated strain from an ash tree (Fraxinus excelsior) in Upper Bavaria, Germany, close to the river Isar, was used to ferment a 12{degrees}P wort at 15{degrees}C. Performance was compared directly with that of a reference lager strain (TUM 34/70) and the S. eubayanus type strain. Both wild yeast rapidly depleted simple sugars and thereafter exhibited a lag phase before maltose utilization. This phase lasted for 4 and 10 days for S. eubayanus and S. jurei, respectively. S. eubayanus utilized fully the available maltose but, consistent with previous reports, did not use maltotriose. S. jurei, in contrast, utilized approx. 50% of the maltotriose available, making this the first report of maltotriose utilization in a wild Saccharomyces species. Maltotriose use was directly related to alcohol yield with 5.5, 4.9, and 4.5 % ABV produced by S. pastorianus, S. jurei and S. eubayanus. Beers also differed with respect to aroma volatiles, with a high level (0.4 mg/L) of the apple/aniseed aroma ethyl hexanoate in S. jurei beers, while S. eubayanus beers had a high level of phenylethanol (100 mg/L). A trained panel rated all beers as being of high quality, but noted clear differences. A phenolic spice/clove note was prominent in S. jurei beer. This was less pronounced in the S. eubayanus beers, despite analytical levels of 4-vinylguaiacol being similar. Tropical fruit notes were pronounced in S. jurei beers, possibly resulting from the high level of ethyl hexanoate. Herein, we present the successful results of the first intentional application of S. jurei as a yeast for beer fermentation known to us and compare its fermentation performance to other species of the genus. Results indicate considerable potential for S. jurei application in brewing, with clear advantages compared to other wild Saccharomyces species.

microbiology↗

bioRxiv2019

A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains

Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162 bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent in among the Beer 2/Mosaic Beer brewing strains. These strains utilize maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of maltotriose through STA1 appears to be the dominant mechanism enabling maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewers wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer, and can be exploited for strain development where maltotriose use is desired.

microbiology↗

bioRxiv2017

Enhanced wort fermentation with de novo lager hybrids adapted to high ethanol environments

Interspecific hybridization is a valuable tool for developing and improving brewing yeast in a number of industry-relevant aspects. However, the genomes of newly formed hybrids can be unstable. Here, we exploited this trait by adapting four brewing yeast strains, three of which were de novo interspecific lager hybrids with different ploidy levels, to high ethanol concentrations in an attempt to generate variant strains with improved fermentation performance in high-gravity wort. Through a batch fermentation-based adaptation process and selection based on a two-step screening process, we obtained eight variant strains which we compared to the wild-type strains in 2L-scale wort fermentations replicating industrial conditions. The results revealed that the adapted variants outperformed the strains from which they were derived, and the majority also possessed several desirable brewing-relevant traits, such as increased ester formation and ethanol tolerance, as well as decreased diacetyl formation. The variants obtained from the polyploid hybrids appeared to show greater improvements in fermentation performance. Interestingly, it was not only the hybrid strains, but also the S. cerevisiae parent strain, that appeared to adapt and showed considerable changes in genome size. Genome sequencing and ploidy analysis revealed that changes had occurred both at chromosome and single nucleotide level in all variants. Our study demonstrates the possibility of improving de novo lager yeast hybrids through adaptive evolution by generating stable and superior variants that possess traits relevant to industrial lager beer fermentation.\n\nImportanceRecent studies have shown that hybridization is a valuable tool for creating new and diverse strains of lager yeast. Adaptive evolution is another strain development tool that can be applied in order to improve upon desirable traits. Here we apply adaptive evolution to newly created lager yeast hybrids by subjecting them to environments containing high ethanol levels. We isolate and characterize a number of adapted variants, which possess improved fermentation properties and ethanol tolerance. Genome analysis revealed substantial changes in the variants compared to the original strains. These improved variants strains were produced without any genetic modification, and are suitable for industrial lager beer fermentations.

microbiology↗