Development of bottom-fermenting saccharomyces strains that produce high SO2 levels, using integrated metabolome and transcriptome analysis

Abstract

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO(2) is desirable, it is complicated by the fact that undesirable H(2)S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO(2)-producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO(2) and H(2)S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO(2), resulting in high SO(2) and low H(2)S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO(2) than the parent but produced parental levels of H(2)S.

FIG. 1.

Schematic of the reductive sulfate assimilation pathway in yeast. YNL311C encodes an F-box protein (13). Ynl311c negatively regulates Met14 and degrades it via a ubiquitin-related pathway.

FIG. 2.

Changes in metabolite pools related to reductive sulfate assimilation in bottom-fermenting and baker's yeasts. Extracellular SO₂ and H₂S levels are concentrations in the spent media. Concentrations of the other metabolites (pmol) are expressed per unit yeast biomass (an amount of cells equivalent to an optical density [OD] at 600 nm of 1). The data are means of three independent experiments, with the error bars indicating standard deviations. Diamonds and squares indicate baker's yeast (S288C) and bottom-fermenting yeast (KBY011), respectively (experiment 1). SAH, S-adenosylhomocysteine; PAPS, 3′-phospho-5′-adenylylsulfate.

FIG. 3.

Changes in expression levels of genes involved in reductive sulfate assimilation in bottom-fermenting and baker's yeasts. Diamonds, squares, and triangles indicate baker's yeast, bottom-fermenting yeast S. cerevisiae (Sc)-type, and bottom-fermenting yeast Lg-type genes, respectively (experiment 1). The y axis is the expression ratio relative to the zero time point. These microarray data are means of analyses taken from two independent fermentation experiments with very similar results (experiment 1).

FIG. 4.

Changes in metabolite pools related to reductive sulfate assimilation in baker's yeast with or without threonine. Concentrations of metabolites are indicated as described in the Fig. 2 legend. The data represent mean values from three independent experiments, with error bars showing standard deviations. Squares and diamonds indicate baker's yeast with and without the addition of threonine, respectively (experiment 2). SAH, S-adenosylhomocysteine; PAPS, 3′-phospho-5′-adenylylsulfate; OD, optical density.

FIG. 5.

Effect of OAH on H₂S production in bottom-fermenting yeast. All compounds shown were added to YPDL plates at a final concentration of 1 mM. Cells of bottom-fermenting yeast strain KBY011 were incubated at 25°C on YPDL plates for 4 days.

FIG. 6.

SO₂ and H₂S production in the spontaneous mutant. (A) Strategy for isolating mutant strain YMO106 from parental strain KBY011. Yeast cells were incubated at 20°C on YPDL plates for 5 days. (B) Extracellular SO₂ and H₂S concentrations in media are shown. Diamonds and triangles indicate parental strain KBY011 and mutant strain YMO106, respectively (experiment 5).