Laboratory genetic modified yeasts with lower ethanol production

CUELLO R.A.; FLORES MONTERO K.J.; COMBINA M.; MERCADO L.; CIKLIC I.

Recife

Jornada; V Jornadas Latinoamericanas de Biología y Biotecnología de Levaduras; 2015

Red Latinoamericana de Levaduras

Up to day wine industry is concern about the high amount of alcohol in wines and tries to find a way to solve it. Our research center has tried to address this concern through genetic engineering of Saccharomyces cerevisiae laboratory strains. We developed a strategy based on introduction of deletions with the help of homologous recombination, to construct yeast with capacity to reduce ethanol content in wine. First, we designed a set of functional strategic mutations in both termini of the PDC2 gene, which encodes a transcriptional factor that regulates the availability of the pyruvate decarboxylase enzyme (PDC1). Then, after obtaining different mutant variants we developed a series of laboratory-scale fermentations. Δ519 is a BY4743 diploid strain yeast, with 519 (pdc2Δ519) amino acids less than the complete sequence of 925 in the N-terminal region. It has a gene copy where only the DNA-binding site is conserved (pdc2Δ519), while the other gene copy still remains intact. Only this strain could achieve the goals previously established, reducing almost 8 % of ethanol, with no negative side effects in other qualitative characteristics as acetic acid or residual sugar. Also, there?s no difference in the biomass production and fermentation kinetics comparing to the wild type strain. In fact, the mutated strain showed an interesting yield behavior: more sugar was necessary for 1 % v/v alcohol production, with significant difference. This characteristic is a proof of a real metabolism change in the way of sugar consumption. The next step is the quantification of other biosynthetic metabolites as acetoin, diacetyl and 2,3-butanediol with HPLC analysis, to know where the metabolism is driving to. After that there?s planned to transfer the mutation to a commercial enology strain. Parallel experiments in that we called ?micro-domains? sites in the C-terminal region, are also in progress. There is previous data about the structural importance of these amino acids in the DNA-binding site union, so we decided to substitute them. After the design and synthesis of seven specific micro-domains substitutions we could clone one fragment in the plasmid pIC01. The plasmid was transformed into yeasts but there was no viability of these in several experiments. There are two other combinations to test in which we hope observe a phenotypic reduced-ethanol behavior similar but enhanced to deletions on N-terminal region of PDC2 gene.