Accelerated Evolution of lager yeast strains for improved flavour profiles
Roberto de la Cerda
Objectives: Screening of lager yeast strains for growth and flavour profiles. Generate novel lager yeast strains using thermal stress adaptive evolution (TSAE). High-throughput screening of adapted strains with enhanced survival in limited nutrients. Characterisation of genetic alterations in best performing adapted strains by RNAseq and genome sequencing. To examine the influence of gene copy number in determining flavour profiles.
Expected Results: Novel lager yeast strains with improved flavour profiles. Understanding of key genetic and protein networks altered in the improved strains. Understanding how gene copy number influences flavour compound production.
Influence of hybrid genes on flavour profiles in lager yeasts
Objectives: Establish a model system to study the role of lager yeast hybrid genes in flavour profiles. Expression of hybrid genes in model system. Co-expression of hybrid genes in model system. Determine if copy number of hybrid genes affects flavour profiles. Characterise the genetic networks affected by hybrid genes.
Expected Results: Understanding of role played by unique hybrid genes on flavour profiles in lager yeasts. Identification of key metabolic pathways influenced by hybrid genes. Understanding of how copy number of hybrid genes influence flavour compound production.
Selection of bespoke high-growth rate yeast strains in a selection of environments
Objectives: Creation of a large number of different yeast hybrids, both intraspecific and interspecific. Analysis of growth profiles at different temperatures, different sugars and presence of different stressors. Competition experiments in turbidostat (continuous culture which allow each strains to grow at his maximum growth rate) to select for faster growing strains. Fitness profiling on select strains to screen for improved biomass. Analysis of genomes, transcriptome, and metabolic pathways of the best strain performers via NGS, RNAseq, metabolic profiling. Synbio approaches to improve further specific metabolic pathways.
Expected Results: Selection of fast growing and high biomass producing yeast hybrid strains. Characterisation of the genetic loci linked to specific metabolic profiles.
Mining and modelling the genome of yeast industrial hybrids
Objectives: Construct genome scale models for strains belonging to the natural hybrid S. pastorianus yeast. Use Flux Balance Analysis and Thermodynamic Tools to predict modifications in metabolic pathways to optimise isoamylacetate production (important flavour compound) at different temperatures. Verify predictions in vivo via genome editing (loxP/Cre or CRISPR/Cas9). Fitness profiling at different temperatures and flavour analysis. Genome and transcriptome analysis of best performing strains. Integration of genome, transcriptome and fitness data to identify genetic features responsible for improved traits.
Expected Results: Creation of a genome scale model for hybrid genomes which can be applied to industrial relevant aneuploidy strains. Creation of hybrids strains with improved traits.
Designer Yeasts utilising Copy Number Variations to improve fermentation speed and flavour compound formation
Objectives: Compilation of lager, ale and wine yeast sequences for copy number changes and allelic divergence in glycolytic & flavour genes. Comparison of CNV in glycolytic genes between lager yeasts and human cancer cell lines. Use of conventional molecular yeast genetics to maximise lager yeast flavour production. Selection of mutants resistant to fluoro-amino acids for up regulation of Ehrlich pathway. Use of molecular yeast breeding to combine increase copy number of target genes in industrial yeasts. Use of genome editing technology (CRISPR/Cas9) to modify glycolytic & flavour pathways in industrial yeast for targeted proof of concept studies. HTP small scale & Lab scale fermentations with modified strains. Design/Build/Test/Analyse cycles for fermentation and flavour strain improvement.
Expected Results: Overview of lager yeast glycolytic & flavour gene Copy Number Variations. Comparison of lager yeast with cancer cell glucose metabolism. Detection and exploitation of synergies. Up regulation of Ehrlich pathway for increased flavour production, e.g. by removing feedback inhibitions. Combination of these traits to generate novel lager yeast strains with altered & improved fermentation characteristics concerning fermentation speed, increased or decreased ethanol or flavour production.
Co-fermentations as a means to generate low alcohol beverages with full flavour
Objectives: Compilation of yeast strains to be used for co-fermentations using yeast biodiversity studies. Flavour map of different yeasts to be used for co-fermentation. Establish lab scale co-fermentations procedures, e.g. co-fermentation at different pitching rates, parallel or sequential fermentations. Use of conventional molecular yeast breeding to generate non-GMO strains for flavour matching. Lab scale fermentations with modified strains, Flavour analyses. Up scaling to pilot scale.
Expected Results: Identification of suitable strains for co-fermentation with beneficial impact on flavour production. Generate new knowledge on industrially relevant procedures for co-fermentations. Establishment of novel procedures to reduce alcohol content in fermented beverages. Patenting of co-fermentation designs and methods. Delivery of strain pairs for pilot trials.
Identification of species-specific genetic determinants of flavour compounds by comparative genomic analysis
Objectives: Screening of Saccharomyces sp isolates for aroma compound production. Comparative genomic analysis of Saccharomyces sp and interspecies hybrids to identify divergence of genes involved in aroma compound production. Heterologous expression of identified genes in S. cerevisiae to examine their role in aroma synthesis. Transcriptome and fermentation analysis of best identified strains/isolates. Genetic improvement of Saccharomyces strains through inter/intra-species hybridisation.
Expected Results: Identification of species-specific gene variants involved in aroma compound synthesis. Determination of best combinations of gene variants for optimal aroma compounds synthesis. Generation of novel inter- and intra species hybrids producing optimal aroma compounds.
Flavour profiling, by GC-MS
Objectives: Development of GC/MS/MS analytical method for quantifying flavour compounds. Comprehensive assessment of flavour profiles of fermented beverages. Identification of all-important classes of flavour compounds by targeted metabolomics approaches. Development of GCxGC of TOF analytical method for untargeted analysis. Overall view of the different flavour compounds produced by platform strains and variants. Structural elucidation of unknown biomarkers with the aim to discover new “unknown” biomarkers influenced by different yeast strains.
Expected Results: Discovering new metabolite biomarkers produced by different species of yeasts. Better understanding of metabolic pathways controlling flavour compound production in yeasts. Flavour profiles of platform yeasts and newly bred yeast variants.
Identification of genes involved in variations in sulfur compounds formation by wine yeasts
Irene de Guidi
Objectives: Identification of genes and alleles involved in sulfur compounds formation in alcoholic fermentation by wine strains. Develop genetic tools to control of the production of sulfur compounds through breeding. To map genes involved in sulfur compound formation through a multiparent QTL strategy to simultaneously identify relevant genetic variations in several strains.
Expected Results: Generation of new strains of wine yeasts with altered flavour profiles. Understanding of gene alleles and networks controlling sulfur compound production in wine yeasts.
Control of the production of volatile sulfur compounds by Saccharomyces cerevisiae during fermentation to enhance aromatic profiles of wine
Rafael Jiménez Lorenzo
Objectives: Assess impact of environmental factors relevant for the winemaking process on the production of volatile sulfur compounds. Identify the metabolic pathways involved in the volatile sulfur compounds synthesis in wine yeasts. Examine the influence of environmental factors such temperature, nitrogen, oxygen and nutrients on sulfur compound production. Strategies to scale up ideal physiological/environmental conditions in pilot-scale fermentations using natural grape juice.
Expected Results: Detailed understanding of the regulation of the formation of sulfur compounds by environmental parameters. Improved fermentation management in order to reduce the synthesis of these off-flavours.
Innovative analytical platforms for screening the ability of micro-organisms to produce high impact aroma compounds in fermentative processes
Objectives: Develop original microfermentation strategy specifically suitable for the subsequent sensory, olfactometric and GC-MS quantitative screening of the odorants produced upon fermentation. Apply those systems to screen the ability of different strains of S. cerevisiae to produce powerful and distinctive aroma molecules in the fermentation of real musts. Identify major metabolic routes via transcriptomic studies.
Expected Results: Microfermentation systems able to trap and stabilise the aromas formed during fermentation. Ranking of multiple strains of S. cerevisiae attending to the sensory characteristics of the aromas produced. List of the aroma molecules responsible for the observed main sensory differences. Comprehensive analytical method for the quantitative determination of those odorants. List with the major metabolic routes and principal regulation systems in which those odorants are formed.
Screening of Saccharomyces and non-conventional yeasts for aroma production
Objectives: Screening of Saccharomyces sp and non-conventional yeasts from Lallemand and CSIC collections for aroma compound production. Generation of hybrids for aroma production. Develop yeast supplements with optimal nitrogen levels. Evaluation of volatile sulfur compounds and flavour profiles of co-fermented conventional and non-conventional yeasts. Industrial scale of of fermentations using best strains identified in the project.
Expected Results: Identification of yeasts (conventional and non-conventional) with optimal flavour profiles. Improved yeasts (hybrids) for aroma production. Identification of best combination of conventional and non-conventional yeasts from optimal flavour compound production. Industrial scale application and implementation of laboratory based research.
Quantification of vitality in novel heterozygotes S. cerevisiae industrial strains
Jose Enrique Cervera
Objectives: Screening of the existing collection of S. cerevisiae industrial strains for fitness in specific environmental contexts (using Singer HDA RoToR Robotics). Using high-content live cell imaging developed in Singer to determine yeast vitality such as the number and size of mitochondria and other organelles in different yeast strains and hybrids. Screening new heterozygotes genetic combination created in the consortium for specific fitness traits and for vitality (number and size of mitochondria/organelles). Integration of fitness data, cell-imaging (vitality) and flavour profiles to select strains with the most robust genetic combination for a particular industrial process.
Expected Results: Create a new library of heterozygotes strains to improve strain performance. Identify particular S. cerevisiae genetic background combination with robust fitness and vitality in responses to fermentations.
Tailor made substrates for increased flavour formation during yeast fermentation
Claire Lin Lin
Objectives: Compilation of potential proteases for treatment of juice, malt and adjuncts to release specific amino acids and peptides as precursors for flavour formation. Screening of selected protease candidates in small scale trials. Analysis of amino acid composition of respective liquids. Selection of the most promising candidates. Small scale fermentation of wort and must with control strains and a selection of low alcoholic flavour producers from the consortium. Flavour analysis by GC-MS. Identification of lead candidate of proteases and flavour producing yeast.
Expected Results: Comparison of proteases and their influence on amino acids composition in wort. Identification of lead candidates for tailor made wort. Wort production with increased availability of specific amino acids. Fermentation of wort with control strain and selection of novel strains from consortium. Aroma analysis of lead candidates.