Lilly M, Lambrechts M G, Pretorius I S
Institute for Wine Biotechnology and Department of Viticulture and Oenology, University of Stellenbosch, ZA-7600 Stellenbosch, South Africa.
Appl Environ Microbiol. 2000 Feb;66(2):744-53. doi: 10.1128/AEM.66.2.744-753.2000.
The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.
葡萄酒、白兰地及其他葡萄衍生酒精饮料的独特风味受多种化合物影响，包括酒精发酵过程中产生的酯类。发酵香气中的特征性果香主要归因于乙酸己酯、己酸乙酯（苹果样香气）、乙酸异戊酯（香蕉样香气）、辛酸乙酯（苹果样香气）和乙酸苯乙酯（带有蜂蜜味的果香、花香）的混合物。本研究的目的是通过过表达一种控制发酵过程中乙酸酯生成的内源性酵母基因，来探究改善葡萄酒和蒸馏酒香气的可行性。葡萄酒酵母酿酒酵母在发酵过程中乙酸酯的合成至少归因于三种乙酰转移酶活性，即乙醇乙酰转移酶（AAT）、乙醇乙酰转移酶和异戊醇AAT。为了研究AAT活性增加对白诗南葡萄酒和哥伦巴基酒蒸馏酒感官品质的影响，我们过表达了酿酒酵母的乙醇乙酰转移酶基因（ATF1）。位于第十五号染色体上的ATF1基因是从广泛使用的商业葡萄酒酵母菌株酿酒酵母VIN13中克隆出来的，并置于组成型酵母磷酸甘油酸激酶基因（PGK1）启动子和终止子的控制之下。染色体印迹分析证实了修饰后的ATF1拷贝整合到了三种商业葡萄酒酵母菌株（VIN7、VIN13和WE228）的基因组中。Northern印迹分析表明，在这些酵母转化体中ATF1呈高水平组成型表达。根据发酵温度、品种和所用酵母菌株的不同，乙酸乙酯、乙酸异戊酯和乙酸苯乙酯的含量分别增加了3至10倍、3.8至12倍和2至10倍。癸酸乙酯、辛酸乙酯和乙酸己酯的浓度仅显示出微小变化，而乙酸浓度下降了一半以上。葡萄酒和蒸馏酒成分的这些变化对最终蒸馏酒的溶剂或化学香气（与乙酸乙酯和乙酸异戊酯相关）以及草本和酒头相关香气，以及对白诗南葡萄酒的溶剂或化学香气和果香或花香特征产生了显著影响。本研究确立了这样一个概念，即过表达诸如ATF1等乙酰转移酶基因可深刻影响香气不足的葡萄酒和蒸馏酒的风味特征，从而为生产在装瓶后较长时间内保持更浓郁果香的产品铺平了道路。
