Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast

Abstract

Domestication of plants and animals promoted humanity's transition from nomadic to sedentary lifestyles, demographic expansion, and the emergence of civilizations. In contrast to the well-documented successes of crop and livestock breeding, processes of microbe domestication remain obscure, despite the importance of microbes to the production of food, beverages, and biofuels. Lager-beer, first brewed in the 15th century, employs an allotetraploid hybrid yeast, Saccharomyces pastorianus (syn. Saccharomyces carlsbergensis), a domesticated species created by the fusion of a Saccharomyces cerevisiae ale-yeast with an unknown cryotolerant Saccharomyces species. We report the isolation of that species and designate it Saccharomyces eubayanus sp. nov. because of its resemblance to Saccharomyces bayanus (a complex hybrid of S. eubayanus, Saccharomyces uvarum, and S. cerevisiae found only in the brewing environment). Individuals from populations of S. eubayanus and its sister species, S. uvarum, exist in apparent sympatry in Nothofagus (Southern beech) forests in Patagonia, but are isolated genetically through intrinsic postzygotic barriers, and ecologically through host-preference. The draft genome sequence of S. eubayanus is 99.5% identical to the non-S. cerevisiae portion of the S. pastorianus genome sequence and suggests specific changes in sugar and sulfite metabolism that were crucial for domestication in the lager-brewing environment. This study shows that combining microbial ecology with comparative genomics facilitates the discovery and preservation of wild genetic stocks of domesticated microbes to trace their history, identify genetic changes, and suggest paths to further industrial improvement.

Fig. 1.

S. eubayanus sp. nov. is distinct from S. uvarum and donated its genome to S. pastorianus. All 16 S. uvarum chromosomes are shown as x axes, with hash marks every 100 kbp; divergence (uncorrected) relative to S. uvarum CBS 7001 (30) is plotted above each chromosome; divergence relative to S. eubayanus is plotted (inverted) below each chromosome. Strains are color-coded according to the key. Each strain appears above and below the chromosomes to represent both comparisons. S. uvarum CBS 395^T is only shown in Dataset S1; the Patagonian S. eubayanus reference strain is only shown above the chromosomes. To avoid overlaps of the comparisons, some strains are offset from the x axis as follows: S. eubayanus, 0.3%; NBRC 1948, 0.2%; CBS 380^T, 0.1%. CBS 380^T contains both S. uvarum and S. eubayanus alleles in several regions, so its percentage heterozygosity (of base pairs) is also shown in thin gray above the chromosomes. The divergence plots for a strain generally move in parallel above and below the chromosomes because nearly all regions of each strain are closely related to either the S. uvarum or the S. eubayanus reference (within 2% of the axis on one side and 6–8% on the other side). Quantitative differences reflect regions with different evolutionary rates, heterozygous regions (CBS 380^T), or regions that may harbor older alleles because of incomplete lineage sorting. For example, on the left arm of chromosome VI, CBS 380^T is heterozygous, but NBRC 1948 contains only S. uvarum alleles; moving right, CBS 380^T becomes homozygous S. uvarum, and NBRC 1948 contains a large section from S. eubayanus before returning to S. uvarum characteristics toward the telomere. Across the filtered draft genome sequences (∼80% of 1-kb windows), S. pastorianus is comprised of S. eubayanus alleles (>99.9%), and the Patagonian S. uvarum is closely related to the S. uvarum reference (>99.9%).

Fig. 2.

S. pastorianus and two triple hybrid strains associated with brewing share identical domestication alleles. (A) Sliding window analysis of synonymous site divergence (dS, Jukes-Cantor corrected) for subtelomeric coding regions with arrows marking direction of transcription and with the color scheme and y axis as in Fig. 1, except the dS shown is to S. cerevisiae (Scer), rather than to S. uvarum (Suva); the y-value offsets necessary for visualization are 10-fold larger than in Fig. 1 because of the different scale; 2,000 sites are plotted with a 100-site window, a step of one, and an arbitrary intergenic spacer of 200 sites. (B) Close-up of partial coding sequences with dots denoting identity. Note the breakpoint fusing the 3′ portion of S. eubayanus (Seub) ZUO1 (blue) to the 5′ portion of ScerZUO1 (brown) and distal genes, including ScerBIO2 and ScerIMA1. [S. pastorianus clearly possesses ScerIMA1 and the ZUO1 breakpoint, but there is a gap across ScerBIO2 in the published assembly (11)]. (C) Partial coding sequence and translation of SUL1 highlighting a 1-bp deletion in SpasSUL1 that causes an inactivating frame-shift mutation. S. pastorianus (Spas) also represents NBRC 1948 and CBS 380^T in B and C because all called base pairs are identical in the coding sequences of ERV29, ZUO1, BIO2, and SUL1 in those strains.

Fig. 3.

A model of the formation of S. pastorianus and the hybrid strains of S. bayanus. First, wild S. eubayanus and ale-type S. cerevisiae hybridized to form an allotetraploid that gave rise to S. pastorianus. Second, domestication imposed strong selective pressure for strains with the most desirable brewing properties. Third, in the brewing vats with high densities of S. pastorianus, cell lysis releases large DNA fragments that occasionally transform, fourth, contaminating wild strains of S. eubayanus because of the lack of pure culture techniques. Fifth, multiple hybridization events with wild strains of S. uvarum gave rise to CBS 380^T and NBRC 1948. This model does not exclude prior or parallel involvement of S. uvarum in brewing or contamination.