Genomic Evolution of Saccharomyces cerevisiae under Chinese Rice Wine Fermentation

Yudong Li 2 Weiping Zhang 1 Daoqiong Zheng 0 Zhan Zhou 0 Wenwen Yu 2 Lei Zhang 2 Lifang Feng 2 Xinle Liang 2 Wenjun Guan 0 Jingwen Zhou 1 Jian Chen 1 Zhenguo Lin 3 4 0 College of Life Sciences, Zhejiang University , Hangzhou, China 1 Key Laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University , Wuxi, China 2 Department of Bioengineering, School of Food Sciences and Biotechnology, Zhejiang Gongshang University , Hangzhou, China 3 Department of Ecology and Evolutionary Biology, Rice University 4 Department of Biology, Saint Louis University Rice wine fermentation represents a unique environment for the evolution of the budding yeast, Saccharomyces cerevisiae. To understand how the selection pressure shaped the yeast genome and gene regulation, we determined the genome sequence and transcriptome of a S. cerevisiae strain YHJ7 isolated from Chinese rice wine (Huangjiu), a popular traditional alcoholic beverage in China. By comparing the genome of YHJ7 to the lab strain S288c, a Japanese sake strain K7, and a Chinese industrial bioethanol strain YJSH1, we identified many genomic sequence and structural variations in YHJ7, which are mainly located in subtelomeric regions, suggesting that these regions play an important role in genomic evolution between strains. In addition, our comparative transcriptome analysis between YHJ7 and S288c revealed a set of differentially expressed genes, including those involved in glucose transport (e.g., HXT2, HXT7) and oxidoredutase activity (e.g., AAD10, ADH7). Interestingly, many of these genomic and transcriptional variations are directly or indirectly associated with the adaptation of YHJ7 strain to its specific niches. Our molecular evolution analysis suggested that Japanese sake strains (K7/UC5) were derived from Chinese rice wine strains (YHJ7) at least approximately 2,300 years ago, providing the first molecular evidence elucidating the origin of Japanese sake strains. Our results depict interesting insights regarding the evolution of yeast during rice wine fermentation, and provided a valuable resource for genetic engineering to improve industrial wine-making strains. - As one of the oldest alcoholic beverages in human history, the Chinese rice wine (Huangjiu) has been brewed and consumed for more than 5,000 years (McGovern et al. 2004). Huangjiu is typically fermented from rice with wheat Qu (koji in sake) and the budding yeast Saccharomyces cerevisiae, which is the most dominant microorganism in rice wine fermentation processes. The wheat Qu contains many molds (fungi), such as Aspergillus oryzae, which break down starches to sugars and digest proteins to peptides or amino acids. The sugars are further fermented by yeasts to produce alcohols. The combination of progressive saccharification of starches and alcoholic fermentation is called parallel fermentation (supplementary fig. S1, Supplementary Material online). The parallel fermentation process avoids exposure of yeast cells to high glucose content and results in high ethanol production, which can be >20% (v/v) in the final fermentation must (Chen and Xu 2010; Akao et al. 2011). Moreover, the formation of sensory characteristics of Chinese rice wine is influenced by different S. cerevisiae strains that produce different flavor compounds, such as higher alcohols, acetates, ethyl esters, and aldehydes (Chen and Xu 2010). To control the quality of wine product, different S. cerevisiae strains have been selected as starter cultures by the wine-maker to manipulate the influence of yeasts. The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Huangjiu has been regarded as a alcohol beverage with high nutritional and pharmacological values (Xie 2008). However, fermentation of Huangjiu may also produce some undesired byproducts, including higher alcohols and ethyl carbamate (Zhao et al. 2013). Higher alcohols (e.g., isoamyl alcohol, phenylethyl alcohol) may trigger a headache in people after consumption of Huangjiu, whereas ethyl carbamate is possibly carcinogenic to humans. A better understanding of genetic basis responsible for the metabolism of these undesired byproducts is necessary to reduce their production. Furthermore, as industrial S. cerevisiae strains have been adapted to the specific wine brewing environmental conditions, their genomes might have been subjected to strong selective pressures (Querol et al. 2003). A complete sequenced genome of Huangjiu strain may provide a better understanding of the genetic basis of the strain for adaptation to specific fermentation environments. However, although the genomes of many S. cerevisiae strains have been completely sequenced, including a Japanese sake strain K7 (Akao et al. 2011; Borneman et al. 2011, 2012; Babrzadeh et al. 2012; Nijkamp et al. 2012; Brown et al. 2013; Treu et al. 2014), the genome sequences of Huangjiu strain have not yet been determined. In addition, although the brewing processes of Japanese sake and Huangjiu are similar (supplementary fig. S1, Supplementary Material online), their sensory characteristics and nutrients are quite different, and the genetic basis leading to such differences remains unexamined. Comparative studies of genomes and transcriptomes are indispensable to unravel the underlying genetic variations responsible for the unique sensory characteristics and nutrients of Huangjiu, which will pave the way for genetic manipulation of yeast strains to improve their product quality. In this study, we sequenced the genome and transcriptome of a Huangjiu strain YHJ7, and compared it with the laboratory strain S288c, Japanese sake strain K7, and a Chinese industrial bioethanol strain YJSH1. We identified many single nucleotide polymorphisms (SNPs)/InDels, gene loss and gains, and differentially expressed genes. In addition, many of these genomic variations are likely associated with the adaption to Huangjiu fermentation environment. Furthermore, our molecular phylogeny analysis suggested that the Japanese sake strains might have originated from Huangjiu strains about 2,300 years ago, which is consistent with the historical record about the ancient cultural interactions between the two countries. Materials and Methods Strains and Growth Conditions The haploid strain S. cerevisiae YHJ7 was generated by sporulation from a strain isolated from Huangjiu fermentation sample (Li et al. 2013). Strains were routinely grown in YPD medium (1% yeast extract, 1% peptone, and 1% glucose) at 28 C and shaken at 200 rpm. The genomic DNA of YHJ7 strain was extracted from cells in midexponential phase (~18 h), using yeast DNA extraction kits a (...truncated)