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Sulfate transport mutants affect hydrogen sulfide and sulfite production during alcoholic fermentation

机译：硫酸盐运输突变体在酒精发酵过程中影响硫化氢和亚硫酸盐

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Hydrogen sulfide is a common wine fault, with a rotten-egg odour, which is directly related to yeast metabolism in response to nitrogen and sulfur availability. In grape juice, sulfate is the most abundant inorganic sulfur compound, which is taken up by yeast through two high-affinity sulfate transporters, Sul1p and Sul2p, and a low affinity transporter, Soa1p. Sulfate contributes to H2S production under nitrogen limitation, by being reduced via the Sulfur Assimilation Pathway (SAP). Therefore, yeast strains with limited H2S are highly desirable. We report on the use of toxic analogues of sulfate following ethyl methane sulfate treatment, to isolate six wine yeast mutants that produce no or reduced H2S and SO2 during fermentation in synthetic and natural juice. Four amino acid substitutions (A99V, G380R, N588K and E856K) in Sul1p were found in all strains except D25-1 which had heterozygous alleles. Two changes were also identified in Sul2p (L268S and A470T). The Sul1p (G380R) and Sul2p (A470T) mutations were chosen for further investigation as these residues are conserved amongst SLC26 membrane proteins (including sulfate permeases). The mutations were introduced into EC1118 using Crispr cas9 technology and shown to reduce accumulation of H2S and do not result in increased SO2 production during fermentation of model medium (chemically defined grape juice) or Riesling juice. The Sul1p (G380R) and Sul2p (A470T) mutations are newly reported as causal mutations. Our findings contribute to knowledge of the genetic basis of H2S production as well as the potential use of these strains for winemaking and in yeast breeding programmes.
机译：硫化氢是一种常见的葡萄酒缺陷，带有臭鸡蛋的气味，这与酵母对氮和硫的代谢直接相关。在葡萄汁中，硫酸盐是最丰富的无机硫化合物，酵母通过两种高亲和力硫酸盐转运蛋白Sul1p和Sul2p以及一种低亲和力转运蛋白Soa1p吸收硫酸盐。硫酸盐通过硫同化途径（SAP）被还原，有助于氮限制下的H2S生成。因此，H2S含量有限的酵母菌株是非常理想的。我们报告了在甲烷硫酸乙酯处理后使用有毒的硫酸盐类似物，分离出六种葡萄酒酵母突变体，它们在合成和天然果汁发酵过程中不产生或减少H2S和SO2。除D25-1具有杂合等位基因外，在所有菌株中均发现Sul1p中存在四种氨基酸替换（A99V、G380R、N588K和E856K）。在Sul2p（L268S和A470T）中也发现了两种变化。选择Sul1p（G380R）和Sul2p（A470T）突变进行进一步研究，因为这些残基在SLC26膜蛋白（包括硫酸盐渗透）中是保守的。使用Crispr cas9技术将突变引入EC1118，结果表明，在模型培养基（化学定义的葡萄汁）或雷司令汁发酵期间，突变可以减少H2S的积累，不会导致SO2产量增加。Sul1p（G380R）和Sul2p（A470T）突变是新报告的因果突变。我们的发现有助于了解H2S产生的遗传基础，以及这些菌株在酿酒和酵母育种计划中的潜在用途。

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