Coradini Alessandro L V, da Silveira Bezerra de Mello Fellipe, Furlan Monique, Maneira Carla, Carazzolle Marcelo F, Pereira Gonçalo Amarante Guimaraes, Teixeira Gleidson Silva
Department of Genetics, Evolution, Microbiology, and Immunology, Institute of Biology, University of Campinas, Rua Monteiro Lobato 255, Campinas, 13083-862, Brazil.
Molecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, CA, 90089-2910, USA.
Biotechnol Biofuels. 2021 Dec 16;14(1):239. doi: 10.1186/s13068-021-02079-6.
Saccharomyces cerevisiae is largely applied in many biotechnological processes, from traditional food and beverage industries to modern biofuel and biochemicals factories. During the fermentation process, yeast cells are usually challenged in different harsh conditions, which often impact productivity. Regarding bioethanol production, cell exposure to acidic environments is related to productivity loss on both first- and second-generation ethanol. In this scenario, indigenous strains traditionally used in fermentation stand out as a source of complex genetic architecture, mainly due to their highly robust background-including low pH tolerance.
In this work, we pioneer the use of QTL mapping to uncover the genetic basis that confers to the industrial strain Pedra-2 (PE-2) acidic tolerance during growth at low pH. First, we developed a fluorescence-based high-throughput approach to collect a large number of haploid cells using flow cytometry. Then, we were able to apply a bulk segregant analysis to solve the genetic basis of low pH resistance in PE-2, which uncovered a region in chromosome X as the major QTL associated with the evaluated phenotype. A reciprocal hemizygosity analysis revealed the allele GAS1, encoding a β-1,3-glucanosyltransferase, as the casual variant in this region. The GAS1 sequence alignment of distinct S. cerevisiae strains pointed out a non-synonymous mutation (A631G) prevalence in wild-type isolates, which is absent in laboratory strains. We further showcase that GAS1 allele swap between PE-2 and a low pH-susceptible strain can improve cell viability on the latter of up to 12% after a sulfuric acid wash process.
This work revealed GAS1 as one of the main causative genes associated with tolerance to growth at low pH in PE-2. We also showcase how GAS1 can improve acid resistance of a susceptible strain, suggesting that these findings can be a powerful foundation for the development of more robust and acid-tolerant strains. Our results collectively show the importance of tailored industrial isolated strains in discovering the genetic architecture of relevant traits and its implications over productivity.
酿酒酵母在许多生物技术过程中广泛应用，涵盖从传统食品和饮料行业到现代生物燃料及生化制品工厂等领域。在发酵过程中，酵母细胞通常会面临各种恶劣条件的挑战，这往往会影响生产效率。就生物乙醇生产而言，细胞暴露于酸性环境与第一代和第二代乙醇生产中的产量损失都有关。在这种情况下，传统用于发酵的本土菌株因其复杂的遗传结构脱颖而出，这主要归因于它们具有高度稳健的背景，包括对低pH的耐受性。
在这项工作中，我们率先使用数量性状基因座（QTL）定位来揭示工业菌株佩德拉-2（PE-2）在低pH条件下生长时赋予其耐酸性的遗传基础。首先，我们开发了一种基于荧光的高通量方法，利用流式细胞术收集大量单倍体细胞。然后，我们能够应用分离群体分析法来解析PE-2中耐低pH的遗传基础，该方法揭示了X染色体上的一个区域是与所评估表型相关的主要QTL。相互半合子分析表明，编码β-1,3-葡聚糖转移酶的等位基因GAS1是该区域的因果变异。不同酿酒酵母菌株的GAS1序列比对指出，野生型分离株中存在一个非同义突变（A631G），而实验室菌株中不存在该突变。我们进一步证明，在硫酸洗涤处理后，PE-2与低pH敏感菌株之间的GAS1等位基因交换可使后者的细胞活力提高多达12%。
这项工作揭示了GAS1是与PE-2耐低pH生长相关的主要致病基因之一。我们还展示了GAS1如何提高敏感菌株的耐酸性，这表明这些发现可为开发更强健、耐酸性更高的菌株提供有力基础。我们的结果共同表明了定制工业分离菌株在发现相关性状的遗传结构及其对生产效率的影响方面的重要性。
