Mo Wenjuan, Wang Mengzhu, Zhan Rongrong, Yu Yao, He Yungang, Lu Hong
1State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai, 200438 China.
Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai, 200438 China.
Biotechnol Biofuels. 2019 Mar 22;12:63. doi: 10.1186/s13068-019-1393-z. eCollection 2019.
, the known fastest-growing eukaryote on the earth, has remarkable thermotolerance and capacity to utilize various agricultural residues to produce low-cost bioethanol, and hence is industrially important to resolve the imminent energy shortage crisis. Currently, the poor ethanol tolerance hinders its operable application in the industry, and it is necessary to improve ' ethanol resistance and unravel the underlying systematical mechanisms. However, this has been seldom reported to date.
We carried out a wild-type haploid FIM1 in adaptive evolution in 6% (v/v) ethanol. After 100-day evolution, the KM-100d population was obtained; its ethanol tolerance increased up to 10% (v/v). Interestingly, DNA analysis and RNA-seq analysis showed that KM-100d yeasts' ethanol tolerance improvement was not due to ploidy change or meaningful mutations, but founded on transcriptional reprogramming in a genome-wide range. Even growth in an ethanol-free medium, many genes in KM-100d maintained their up-regulation. Especially, pathways of ethanol consumption, membrane lipid biosynthesis, anti-osmotic pressure, anti-oxidative stress, and protein folding were generally up-regulated in KM-100d to resist ethanol. Notably, enhancement of the secretory pathway may be the new strategy KM-100d developed to anti-osmotic pressure, instead of the traditional glycerol production way in . Inferred from the transcriptome data, besides ethanol tolerance, KM-100d may also develop the ability to resist osmotic, oxidative, and thermic stresses, and this was further confirmed by the cell viability test. Furthermore, under such environmental stresses, KM-100d greatly improved ethanol production than the original strain. In addition, we found that may adopt distinct routes to resist different ethanol concentrations. Trehalose biosynthesis was required for low ethanol, while sterol biosynthesis and the whole secretory pathway were activated for high ethanol.
This study reveals that ethanol-driven laboratory evolution could improve ' ethanol tolerance via significant up-regulation of multiple pathways including anti-osmotic, anti-oxidative, and anti-thermic processes, and indeed consequently raised ethanol yield in industrial high-temperature and high-ethanol circumstance. Our findings give genetic clues for further rational optimization of ' ethanol production, and also partly confirm the positively correlated relationship between yeast's ethanol tolerance and production.
[具体生物名称]是地球上已知生长最快的真核生物，具有显著的耐热性以及利用各种农业废弃物生产低成本生物乙醇的能力，因此对于解决迫在眉睫的能源短缺危机具有重要的工业意义。目前，其较差的乙醇耐受性阻碍了它在工业中的实际应用，有必要提高[具体生物名称]的乙醇抗性并揭示其潜在的系统机制。然而，迄今为止这方面的报道很少。
我们在6%（v/v）乙醇中对野生型单倍体[具体生物名称]进行适应性进化。经过100天的进化，获得了KM - 100d群体；其乙醇耐受性提高到了10%（v/v）。有趣的是，DNA分析和RNA测序分析表明，KM - 100d酵母乙醇耐受性的提高并非由于倍性变化或有意义的突变，而是基于全基因组范围的转录重编程。即使在无乙醇培养基中生长，KM - 100d中的许多基因也保持上调。特别是，KM - 100d中乙醇消耗、膜脂生物合成、抗渗透压、抗氧化应激和蛋白质折叠等途径普遍上调以抵抗乙醇。值得注意的是，分泌途径的增强可能是KM - 100d为抵抗渗透压而开发的新策略，而不是[具体生物名称]中传统的甘油产生方式。从转录组数据推断，除了乙醇耐受性外，KM - 100d可能还发展出了抵抗渗透压、氧化应激和热应激的能力，细胞活力测试进一步证实了这一点。此外，在这种环境压力下，KM - 100d的乙醇产量比原始菌株有了很大提高。另外，我们发现[具体生物名称]可能采用不同途径来抵抗不同浓度的乙醇。低乙醇浓度时需要海藻糖生物合成，而高乙醇浓度时甾醇生物合成和整个分泌途径被激活。
本研究表明，乙醇驱动的实验室进化可以通过显著上调包括抗渗透、抗氧化和抗热过程在内的多种途径来提高[具体生物名称]的乙醇耐受性，并且确实在工业高温和高乙醇环境下提高了乙醇产量。我们的发现为进一步合理优化[具体生物名称]的乙醇生产提供了遗传线索，也部分证实了酵母乙醇耐受性与产量之间的正相关关系。
