Engineering yeast for the production of optimal levels of volatile phenols in wine

[摘要] ENGLISH ABSTRACT:Phenolic acids (principally p-coumaric and ferulic acids), which are generally esterified withtartaric acid, are natural constituents of grape must and wine, and can be released as freeacids during the winemaking process by certain cinnamoyl esterase activities. Free phenolicacids can be metabolised into 4-vinyl and 4-ethyl derivatives by several microorganismspresent in wine. These volatile phenols contribute to the aroma of the wine.The Bretfanomyces yeasts are well known for their ability to form volatile phenols inwine. However, these species are associated with the more unpleasant and odorousformation of the ethylphenols and the formation of high concentrations of volatile phenols.Other organisms, including some bacterial species, are responsible for the formation ofvolatile phenols at low concentrations, especially the 4-vinylphenols, and this enhances theorganoleptic properties of the wine.The enzymes responsible for the decarboxylation of phenolic acids are called phenolicacid decarboxylases; and several bacteria and fungi have been found to contain the genesencoding these enzymes. The following genes have been characterised: PAD1 fromSaccharomyces cerevisiae, fdc from Bacillus pumilus, pdc from Lactobacillus plantarum andpadc from Bacillus subtilis. PadA from Pediococcus pentosaceus was also identified.S. cerevisiae contains the PAD1 (phenyl acrylic acid decarboxylase) gene, which issteadily transcribed in yeast. The activity of the PAD1-encoded enzyme is low. Phenolicacid decarboxylase from B. subtilis, as well as p-coumaric acid decarboxylase fromL. plantarum displays substrate inducible decarboxylating activity with phenolic acids. Boththe p-coumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) geneswere cloned into PGK1 PT expression cassette. The PGK1 PT expression cassette consistedof the promoter (PGK1 p) and terminator (PGK1 T) sequence of the yeastphosphoglyceratekinase I gene (PGK1). Episomal and yeast integration plasmids wereconstructed for the PAD1 gene under the control of the PGK1 PT for overexpresion in yeast.Industrial strains with the PAD1 gene disrupted were also made. Overexpression of pcoumaricacid decarboxylase (pdc) and phenolic acid decarboxylase (padc) in S. cerevisiaeshowed high enzyme activity in laboratory strains. The overexpressed PAD1 gene did notshow any higher enzyme activity than the control strain. Both bacterial genes, under thecontrol of the PGK1 PT cassette, were also cloned into a yeast-integrating plasmid, with theSMR1 gene as selective marker. The cloning and transformation of pdc and padc intoindustrial wine yeast strains can therefore be used to detect the effect of phenolic aciddecarboxylase genes in the winemaking process for the possible improvement of winearoma. Wine was made with all three strains (the bacterial genes overexpressed and PAD1disrupted). The effect of these genes in wine was determined through GC analysis. The results showed that the bacterial genes could effectively produce higher levels of volatilephenols in the wine. The manipulated strains also produced enzymes capable of producinglarge amounts of favourable monoterpenes in the wine.This study paves the way for the development of wine yeast starter culture strains forthe production of optimal levels of volatile phenols, thereby improving the sensorial quality ofwine.

[发布机构] Stellenbosch University