Polygenic analysis of phenylethyl acetate production in yeast for aroma production improvement in alcoholic beverages

Flavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side-activities of enzymes having their main function in other parts of metabolism. We have applied polygenic analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc), a flavor compound of great importance in alcoholic beverages, imparting rose and honey-like aromas. We have successfully applied pooled-segregant whole-genome sequence analysis to segregants of two non-selected parents, without significant difference in the trait of interest. This suggests that many more strains than anticipated from phenotypic profiling may harbor interesting superior alleles for traits of interest. The QTLs responsible for high production of 2-PEAc showed linkage to either one of the two parents. The causative genes in two QTLs linked to one parent were identified by reciprocal allele swapping into both parents using CRISPR/Cas9. The superior allele of the first major causative gene, FAS2, contained two unique SNPs responsible for high 2-PEAc production, not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex confirming that regular metabolic enzymes have side activities responsible for production of flavor compounds. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation of growth. It may affect flavor production indirectly. Exchange of both superior alleles in the inferior parent strain increased 2-PEAc production with 70%, i.e. nearly to the same level as in the best superior segregant. Our results suggest that polygenic analysis combined with CRISPR/Cas9 mediated site-directed genetic modification may evolve into a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages.List of Figures v List of Abbreviations vii Abstract ix Samenvatting xi Chapter I - Literature review 1 1. Saccharomyces cerevisiae and its applications 1 2. Flavor of fermented beverages 2 3. Ester production by S. cerevisiae 5 4. Factors affecting ester production 8 4.1. Suspended solids and fatty acid addition 8 4.2. Wort/must aeration 8 4.3. Fermentation temperature 8 4.4. Inoculum size (pitching rate) 9 4.5. Fermentation pressure 10 4.6. Amino acid composition of the fermentation medium 10 5. Increasing ester production by genetic modifications 12 LEU4: release from leucine-feedback inhibition leads to enhanced isoamyl alcohol and isoamyl acetate production. 12 5.1. TYR1 and ARO4: resistance to phenylalanine analogs correlates with increased 2-phenylethanol and 2-phenylethyl acetate production. 14 5.2. FAS2: release from cerulenin-inhibition leads to increased production of ethyl esters, especially ethyl hexanoate. 16 5.3. ATF1 and ATF2: Overexpression of the alcohol acetyl transferases Atf1 and Atf2 leads to increased acetate esters production. 17 5.4. BAT1 and BAT2: Overexpression of the aminotransferases BAT1 and BAT2 results in increased isobutanol and isobutyl acetate levels 18 6. Phenylethyl acetate production in fermented beverages 18 7. Improving yeasts by using naturally occurring “superior alleles” – QTL mapping 19 8. CRISPR/Cas9, a powerful tool for genetic engineering 22 9. Research objectives 27 Chapter II - QTL mapping of high 2-phenylethyl acetate production by using random parent strains 29 1. Introduction 29 2. Results 30 2.1. Flavor profile of industrial yeast and random parent selection 30 2.2. Pooled-segregant whole-genome sequence analysis and QTL mapping 39 3. Discussion 42 Chapter III - Identification of the causative genes conferring high phenylethyl acetate production by using CRISPR/Cas9 approach 45 1. Introduction 45 2. Results 47 2.1. Identification of TOR1 as a causative gene on chromosome X 47 2.2. Identification of FAS2 as causative gene on chromosome XVI 55 2.3. Achieving the superior phenotype by replacing TOR1 and FAS2 alleles in ER18 parent. 59 2.4. Production of the other flavor compounds in the ER18 strains engineered for high 2-PEAc production 61 3. Discussion 65 Chapter IV - Final remarks, conclusions and perspectives 71 Limitations of increasing 2-PEAc production by alteration in fermentation parameters 71 Genetic improvement of industrial yeast strains 73 Genetic tools 74 QTL mapping 74 CRISPR/Cas9 75 Perspectives 77 Materials and methods 79 Microorganisms and cultivation media 79 Fermentation experiments 81 Headspace GC-FID analysis 81 Sporulation and tetrad dissection 82 Mating type 83 Deletion of HO gene 83 Crossing of haploid strains 83 Molecular Biology methods 83 Genomic DNA extraction and whole genome sequence analysis 85 Allele-specific PCR 86 CRISPR/Cas9 experiments 89 Cas9 plasmid 89 gRNA plasmids 89 Design guide RNA targets 92 Donor DNA 92 Appendix 1 95 References 105status: publishe