The biobased-economy aims to create a circular biotechnology ecosystem to transition from a fossil fuel-based to a sustainable industry based on biomass. For this, new microbial cell-factories are essential. We present the draft genome of the CEN.PK-derived Saccharomyces cerevisiae SpyCas9 expressing strain (IMX2600), that serve as chassis of new cell-factories.

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Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1^I306A and ScFAS2^G1250S significantly improved ethyl hexanoate production and ScFAS1^R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2^G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

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The hybrid yeast Saccharomyces pastorianus is the microbial ‘workhorse’ responsible for production of lager beer. With an annual production of 1.9 billion hectolitres, this type of beer is the most popular alcoholic beverage worldwide. S. pastorianus ferments sugars present in wort to ethanol and a variety of flavour molecules that contribute to the taste and perception of the beverage. This yeast has emerged from a natural hybridization event in a human-caused environment, which probably occurred in late medieval times, between the mesophilic ale yeast Saccharomyces cerevisiae and the cryotolerant yeast Saccharomyces eubayanus. As a consequence, S. pastorianus strains harbour chromosomes from both these parental species. In addition, prolonged domestication in lager brewing processes has led to many additional changes in S. pastorianus: individual chromosomes underwent extensive recombination and occur in different copy numbers that, moreover, vary between strains. The advent of whole genome sequencing technologies has contributed to insights into the complex genomic ‘blueprints’ of S. pastorianus strains.

The allo-aneupolyploid genomes of currently used brewing yeast strains were shaped in centuries of evolution, with industrial brewing conditions providing selective pressure. Today, targeted genetic engineering enables researchers to alter or add traits beyond what is achievable by solely applying selective pressure. The rapid development of CRISPR-Cas-based tools for genome editing have, particularly in baker’s yeast S. cerevisiae and to a lesser extent in non-conventional yeast species, greatly accelerated strain development. However, applying these tools in heterozygous alloaneupolyploid contexts such as those in S. pastorianus poses substantial technological challenges.

The aim of the research described in this thesis was to develop novel genome editing strategies for yeasts, improving the genetic accessibility of hybrid yeasts and subsequently applying these methodologies to generate novel lager brewing strains with enhanced brewing characteristics...@en

ErCas12a is a class 2 type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces cerevisiae. The optimal design parameters for ErCas12a editing in S. cerevisiae were defined as a 21-nt spacer flanked by 19 nt direct repeats expressed from either RNApolII or III promoters, achieving near 100% editing efficiencies in commonly targeted genomic locations. To be able to transfer the ErCas12a genome editing tool to different strain lineages, a transportable platform plasmid was constructed and evaluated for its genome editing efficiency. Using an identical crRNA expression design, the transportable ErCas12a genome editing tool showed lower efficiency when targeting the ADE2 gene. In contrast to genomic Ercas12a expression, episomal expression of Ercas12a decreases maximum specific growth rate on glucose, indicating ErCas12a toxicity at high expression levels. Moreover, ErCas12a processed a multispacer crRNA array using the RNA self-processing capability, which allowed for simultaneous editing of multiple chromosomal locations. ErCas12a is established as a valuable addition to the genetic toolbox for S. cerevisiae.

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Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR-induced double strand breaks is unpredictable and preferentially uses the homoeologous chromosome as template, preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeScCHRIII. The landing sites were systematically selected and evaluated for (i) absence of loss of heterozygosity upon CRISPR-editing, (ii) efficiency of the gRNA, and (iii) absence of effect on strain physiology. Successful examples of highly efficient single and double gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development.

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Even for the genetically accessible yeast Saccharomyces cerevisiae, the CRISPR-Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events, and diverse approaches for multiplex genome editing have been described in the literature by means of SpCas9 or FnCas12a endonucleases and their associated guide RNAs (gRNAs). The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native Pol II or Pol III RNA polymerases. These gRNA expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in Saccharomyces cerevisiae. Using FnCas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single and duplex DNA editing. These DNA fragments can be custom ordered, which makes this approach highly suitable for high-Throughput strain construction. This work expands the CRISPR toolbox for large-scale strain construction programs in S. cerevisiae and promises to be relevant for other less genetically accessible yeast species.

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