Abstract
BackgroundLow ethanol tolerance of Kluyveromyces marxianus limits its application in high-temperature ethanol fermentation. As a complex phenotype, ethanol tolerance involves synergistic actions of many genes that are widely distributed throughout the genome, thereby being difficult to engineer. TATA-binding protein is the most common target of global transcription machinery engineering for improvement of complex phenotypes.ResultsA random mutagenesis library of K. marxianus TATA-binding protein Spt15 was constructed and subjected to screening under ethanol stress. Two mutant strains with improved ethanol tolerance were identified, one of which (denoted as M2) exhibited increased ethanol productivity. The mutant of Spt15 in strain M2 (denoted as Spt15-M2) has a single amino acid substitution at position 31 (Lys → Glu). RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from Spt15-M2. Spt15-M2 caused changes in transcriptional level of most of the genes in the central carbon metabolism network. Compared with control strain, 444 differentially expressed genes (DEGs) were identified in strain M2 (fold change > 2, Padj < 0.05), including 48 up-regulated and 396 down-regulated. The up-regulated DEGs are involved in amino acid transport, long-chain fatty acid biosynthesis and MAPK signaling pathway, while the down-regulated DEGs are related to ribosome biogenesis, translation and protein synthesis. Five candidate genes (GAP1, GNP1, FAR1, STE2 and TEC1), which were found to be up-regulated in M2 strain, were overexpressed for a gain-of-function assay. However, the overexpression of no single gene helped improve ethanol tolerance as SPT15-M2 did.ConclusionsThis work demonstrates that ethanol tolerance of K. marxianus can be improved by engineering its TATA-binding protein. A single amino acid substitution (K31E) of TATA-binding protein Spt15 is able to bring differential expression of hundreds of genes that acted as an interconnected network for the phenotype of ethanol tolerance. Future perspectives of this technique in K. marxianus were discussed.
Highlights
Low ethanol tolerance of Kluyveromyces marxianus limits its application in high-temperature ethanol fermentation
ribonucleic acid sequencing (RNA-Seq)-based transcriptomic analysis revealed cellular transcription profile changes resulting from Spt15M2: Spt15-M2 caused changes in transcriptional level of most of the genes in the central carbon metabolism network; genes associated with amino acid transport, long-chain fatty acid biosynthesis and MAPK signaling pathway were up-regulated, while genes related to ribosome biogenesis, translation and protein synthesis were down-regulated
The overexpression of no single gene helped improve ethanol tolerance as SPT15-M2 did, indicating that the differentially expressed gene (DEG) acted as an interconnected network for the phenotype of ethanol tolerance
Summary
Low ethanol tolerance of Kluyveromyces marxianus limits its application in high-temperature ethanol fermentation. To produce bioethanol costly and effectively, we have developed a novel advanced solid-state fermentation (ASSF) technology to produce ethanol using crushed sweet sorghum stalks, which is equipped with an optimized and redesigned rotary drum fermenter and a proprietary yeast strain [1–4]. Low efficiencies of mass and heat transfer limit the industrial application of solid-state fermentation (SSF) [1, 5]. The rotary drum fermenter of the ASSF system improves the mass and heat transfer efficiencies and can thereby increase the ethanol productivity from sweet sorghum to a great extent [6], first demonstrating that SSF can be applied at industrial scale for ethanol production [2]. Like other pentose-utilizing yeast species, K. marxianus has lower ethanol tolerance compared with Saccharomyces cerevisiae [11], which limits its application in ethanol fermentation
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