Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of multiple pathways
Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of multiple pathways
复制标题
马克斯克鲁维酵母在适应性进化过程中发展出乙醇耐受性，并显着改善了多种途径
DOI：
10.1186/s13068-019-1393-z
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发表时间：
2019-03-22
影响因子：
6.3
通讯作者：
Lu, Hong
中科院分区：
文献类型：
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作者：
Mo, Wenjuan;Wang, Mengzhu;Lu, Hong
研究背景马克斯克鲁维酵母（Kluyveromycesmarxianus）是地球上已知生长最快的真核生物，具有显著的耐热性和利用各种农业废弃物生产低成本生物乙醇的能力，对解决迫在眉睫的能源危机具有重要的工业意义。目前，乙醇耐受性差，阻碍了其在工业上的可操作性应用，有必要提高K。marxianus的乙醇抗性，并阐明了潜在的系统机制。然而，这已经很少报道，到目前为止。marxianus FIM 1在6%（v/v）乙醇中的适应性进化。经过100天的进化，获得了KM-100d群体，其乙醇耐受性提高到10%（v/v）。有趣的是，DNA分析和RNA-seq分析表明，KM-100d酵母的乙醇耐受性改善不是由于倍性变化或有意义的突变，而是基于全基因组范围内的转录重编程。即使在无乙醇培养基中生长，KM-100d中的许多基因仍保持其上调。特别是，KM-100d的乙醇消耗，膜脂合成，抗渗透压，抗氧化应激和蛋白质折叠的途径普遍上调，以抵抗乙醇。值得注意的是，分泌途径的增强可能是KM-100d抗渗透压的新策略，而不是S.啤酒。从转录组数据推断，除了乙醇耐受性之外，KM-100d还可以发展抵抗渗透、氧化和热应激的能力，并且这通过细胞活力测试进一步证实。此外，在这样的环境压力下，KM-100d大大提高了乙醇产量比原始菌株。此外，我们还发现K.马克思主义者可能会采取不同的路线来抵抗不同的乙醇浓度。低浓度乙醇需要海藻糖的生物合成，而高浓度乙醇则激活甾醇的生物合成和整个分泌途径。marxianus通过显著上调抗渗透、抗氧化和抗热等多种途径提高了乙醇耐受性，从而提高了工业高温高乙醇环境下的乙醇产量。我们的研究结果提供了遗传线索，进一步合理优化K。marxianus的乙醇产量，也部分证实了酵母的乙醇耐受性与产量之间的正相关关系。
BackgroundKluyveromyces marxianus, 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 K. marxianus’ ethanol resistance and unravel the underlying systematical mechanisms. However, this has been seldom reported to date.ResultsWe carried out a wild-type haploid K. marxianus 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 S. cerevisiae. 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 K. marxianus 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.ConclusionsThis study reveals that ethanol-driven laboratory evolution could improve K. marxianus’ 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 K. marxianus’ ethanol production, and also partly confirm the positively correlated relationship between yeast’s ethanol tolerance and production.