Biotechnol. J. 2008, 3, 1355–1367
normally to produce wine while simultaneously de- grading viscous polymers thus significantly en- hancing processing steps such as filtration. Yeast engineered to degrade polysaccharides to improve filtration are also able to increase the ‘fruity’ bou- quet of the wine. Wine yeast strains produce many of the aroma and flavour compounds that define the individual character of specific wines . These compounds include in particular esters, higher alcohols and various sulphites, as well as acids, glycerol and terpenoids. Genetic and meta- bolic engineering strategies are able to modify the metabolic flux in a particular pathway in such a way as to favour the production of desirable com- pounds while concomitantly reducing undesirable ones. The power of a metabolic engineering ap- proach is evident when considering that the over- expression of single genes are able to significantly modulate the levels of over a dozen important aro- ma compounds in yeast simultaneously [41–44]. Similar data have been generated for many genes, allowing a better understanding of the interactions between many metabolic pathways involved in aro- ma compound production [43, 44]. In addition to aroma compounds, yeast has the potential to pro- duce compounds of human medical importance. Grape-derived compounds, such as the phenolic compound resveratrol, is linked to the health ben- efits of moderate wine consumption . Resvera- trol is positively correlated epidemiologically with reduced cardiovascular disease and so the IWBT was interested in increasing the amounts present in wine using a designer yeast strategy . Two genes from the metabolic pathway needed to pro- duce resveratrol (a coenzyme A ligase-encoding gene, 4CL216, from hybrid poplar and the grapevine resveratrol synthase gene, vst1) were cloned into a laboratory strain of S. cerevisiae . The results obtained by analytical liquid chro- matography-coupled mass spectrometry demon- strated that the yeast transformants were able to produce piceid, which is the glucose-bound form of resveratrol . These yeasts therefore have the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the final product. Although mi- crobes, such as the resveratrol-producing yeast strain, impart positive attributes to wine, a common cause of wine spoilage is the growth of micro-or- ganisms such as bacteria that have the potential to produce off-flavours . A common practise to prevent such spoilage is the addition of sulphur dioxide as a preservative. While sulphur dioxide is satisfactory in this regard, and in addition also acts as an antioxidant, alternatives are desired and ac- tively encouraged because of the potentially nega-
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tive influence of the compound on aroma and health, a significant percentage of the population being hypersensitive to sulphur dioxide.The IWBT has generated yeast strains that control the growth of spoilage organisms by secreting peptides or en- zymes that specifically inhibit the growth of un- wanted organisms . An example being the use of bacteriocin genes cloned into a laboratory strain of yeast . Two bacteriocin-encoding genes, pe- diocin PA-1 (pedA) produced by Pediococcus acidi- lactici and leucocin B (lcaB) from Leuconostoc carnosum, were cloned into a multicopy episomal plasmid under the control of the alcohol dehydro- genase 1 promoter and terminator, and the yeast mating pheromone _-factor secretion signal . Transformed yeast strains were shown to produce active forms of pediocin and leucocin . These bactericidal yeast strains were not only able to con- duct fermentations in wine, but also act as a biolog- ical control agent by inhibiting the growth of spoilage bacteria . Although certain bacteria are able to cause wine spoilage, some bacteria im- part beneficial properties to wine and are thus added to wine formulations to encourage growth.
Lactic acid bacteria biotechnology
Lactic acid bacteria (LAB) have historically been associated with food and beverage fermentations as they occur naturally in the starting materials used . Lactic acid bacteria also occur in must and wine and perform the secondary fermentation, known as malolactic fermentation (MLF). The process of MLF includes a reduction in acidity, re- sulting from the degradation of L-malic acid to L- lactic acid with the concomitant release of carbon dioxide. LAB isolated from grapes and wines are from the genera Lactobacillus, Leuconostoc, Oeno- coccus and Pediococcus. Oenococcus oeni is used commercially in malolactic starter cultures, as they are the LAB best adapted to wine conditions. Al- though MLF is primarily performed to reduce wine acidity, especially in cooler climate regions, it is also considered beneficial to the wine’s sensory quality due to flavour modification. Additionally MLF pro- vides microbial stability, since malic acid, which can serve as a carbon source to support the growth of potential spoilage LAB, is degraded . In addi- tion, LAB produce antimicrobial agents that pro- tects the finished product by inhibiting spoilage bacterial growth . These bacteria are able to compete for nutrients during fermentation and can produce antimicrobial compounds such as organic acids, ethanol, hydrogen peroxide and bacteriocins [46, 50]. A wide range of LAB have the ability to