Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of multiple pathways

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s13068-019-1393-z) contains supplementary material, which is available to authorized users.

Kluyveromyces marxianus: Current State of Omics Studies, Strain Improvement Strategy and Potential Industrial Implementation

“…The thermotolerant yeast K. marxianus has many good traits to be used as cell factory in food and biotechnology [3]. These advantages include the fastest growth rate (with the maximum growth rate of 0.80 h −1 ) among any eukaryotic microbes [4,5], the ability to assimilate a wide range of sugars (e.g., glucose, lactose, galactose, xylose, inulin, and arabinose), thermo (up to 52 • C) and toxin (furaldehyde) tolerance, a wide range of pH values (pH 2.5-9), high ethanol yield at elevated temperatures, production of value-added aromatic chemicals (e.g., 2-phenylethylethanol and 2-phenylethyl acetate), and secretion of lytic enzymes [6][7][8].…”

Kluyveromyces marxianus: Current State of Omics Studies, Strain Improvement Strategy and Potential Industrial Implementation

“…The thermotolerant yeast K. marxianus has many good traits to be used as cell factory in food and biotechnology [10]. These advantages include the fastest growth rate (with the maximum growth rate of 0.80 h -1 ) among any eukaryotic microbes [11,12], the ability to assimilate a wide range of sugars (e.g., glucose, lactose, galactose, xylose, inulin, and arabinose), thermo-(up to 52 o C) and toxin (furaldehyde) tolerance, wide range of pH values (pH 2.5-9), high ethanol yield at elevated temperatures, production of valueadded aromatic chemicals (e.g., 2-phenylethylethanol and 2-phenylethyl acetate), and secretion of lytic enzymes exposure. Similarly, in the study of Wang et al [18], various genes encoding fatty acid and ergosterol metabolism were downregulated under multiple inhibitors stress such as phenols, furfural, HMF, and acetic acid.…”

Comparative genomic analysis reveals metabolic mechanisms for Kluyveromyces marxianus’ fast growth during evolution

“…Within the last decade, K. marxianus as one of 'non-conventional' yeasts, has the traits of food safety, utilization of multiple carbon sources, thermotolerance, and fast growth [1,2]. It has been successfully applied in ethanol fermentation [3,4] and heterologous protein production [5]. The faster growth rate guarantees the superior result of fermentation course [6].…”

“…Transcriptome studies for K. marxianus have focused on its physiological properties, such as xylose catabolism [12,14], high temperature resistance [12], and ethanol tolerance [3]. These current studies mainly analyzed K. marxianus' gene expression changes in different environments.…”