Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1I306A and ScFAS2G1250S significantly improved ethyl hexanoate production and ScFAS1R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

中文翻译：

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通过酵母菌的代谢工程解锁贮藏啤酒的风味调色板，以增强乙酯的产生


尽管乙酯以微量存在，但在拉格啤酒中作为风味化合物起着至关重要的作用。在酵母中，己酸乙酯、辛酸乙酯和癸酸乙酯负责果味和花香味调，由脂肪酸合成酶复合物在脂肪酸生物合成过程中释放的有毒中链酰基辅酶 A 中间体合成，作为保护机制。本研究的目的是通过改善中链酰基辅酶 A 前体的供应来提高杂交贮藏啤酒酿造酵母 Saccharomyces pastorianus 中乙酯的产生。通过基于 CRISPR-Cas9 的基因工程，在脂肪酸合成复合物结构域中携带突变的特定 FAS1 和 FAS2 基因以单一和组合方法过表达。ScFAS 基因中的这些突变导致相应乙酯的特异性过生产：ScFAS1I306A 和 ScFAS2G1250S 的过表达显着改善了己酸乙酯的产生，而 ScFAS1R1834K 促进了辛酸乙酯的产生。ScFAS1 突变基因与 ScFAS2G1250S 的组合可预测地大大提高了在全麦芽麦芽汁上生长的培养物中的最终乙酯浓度，但也导致游离中链脂肪酸水平增加，从而导致风味特征发生变化。最后，通过过表达酯酶 ScEEB1，升高的中链脂肪酸库被引导至乙酯。转基因 S. pastorianus 菌株被用于拉格啤酒生产，所得饮料表现出显着改变的风味特征，从而大大扩展了拉格啤酒风味调色板的可能性。