Construction of low-ethanol producing yeasts though partial deletions and base pair substitutions of the Saccharomyces cerevisiae PDC2 gene

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

Mendoza

Congreso; 37° Congreso Mundial de la Vid y el Vino; 2014

Organizacion Internacional de la Vid y el Vino (OIV)

Global warming together with current viticultural practices of late grape harvesting, contribute to the present tendency of obtaining wines with high ethanol concentration. This could have a negative impact on the exportation market, considering the increasing demand for soft wines. There are at least three different approaches to address this problem; the viticultural approach, which implies the use of alternative vineyard management, the physical approach, through the application of dealcoholization techniques, and the biotechnological approach, which is the one used in the present work. During winemaking, alcoholic fermentation is carried out mostly by Saccharomyces cerevisiae. This microorganism can be genetically manipulated with great ease allowing the construction of yeast strains with specific oenological features. The aim of our work is to construct a low-ethanol producing S. cerevisiae strain, which preserves its desirable oenological properties. For this purpose, we designed a set of functional strategic mutations in both termini of the PDC2 gene, which encodes a transcription factor that regulates the availability of the pyruvate decarboxylase enzyme (PDC1). With these mutations, we hope to obtain a different version of the Pdc2p transcription factor with a reduced positive regulatory activity of PDC1. This would cause a reduction of the enzymatic activity and consequently a reduction in ethanol production. We used different methodologies to generate the mutations. Through homologous recombination of the KanMX resistance cassette and the incorporation of a stop codon, we made the two planed deletions for the N-terminal region of PDC2, obtaining two truncated proteins with 344 (pdc2Δ344) y 519 (pdc2Δ519) amino acids less than the complete sequence of 925. In the case of pdc2Δ344 the DNA-binding site and transactivation site are both intact, whereas in pdc2Δ519 only the DNA-binding site is conserved. Although it is known that the DNA-binding site alone conserves some DNA binding activity, considering the length of the deletions, both deletions could present severe phenotypes. For this reason, they will be tested in homozygosis and in heterozygosis. For the mutations in the DNA-binding site of the C-terminal region, we designed a series of substitutions in seven specific micro-domains in order to destabilize the protein-DNA union. These micro-domains have been selected based on coincidences founded in the crystal structure of the homologous CENP-B protein. Due to the design?s complexity, we employed a DNA synthesis service to simplify the construction of the mutants. The ethanol production of the mutant strains will be evaluated in lab scale fermentations, determining other critical parameters such as acetic acid production, glycerol and residual sugars. Referring to the results, we have already performed the lab scale fermentations with the homozygous and heterozygous pdc2Δ519 strain. The heterozygous pdc2Δ519 strain is the one that showed the most promising results, with ethanol concentration reductions of around 7 %, which corresponds to one degree of alcohol less for a wine with a 15 % v/v alcohol potential. Interestingly, this ethanol reduction did not increase the volatile acidity, and the sugar consumption was also similar to the control strain. In next experiments, we will continue with lab scale fermentations of the other mutants, choosing the ones with the most desirable phenotypes. Selected mutations will be inserted in the INTA MZA native strain for definitive pilot scale assays. Keywords: Saccharomyces cerevisiae, biotechnology, PDC2 gene, fermentation, ethanol.