Construction of Saccharomyces cerevisiae strains with enhanced ethanol tolerance by mutagenesis of the TATA‐binding protein gene and identification of novel genes associated with ethanol tolerance

Abstract

Since elevated ethanol is a major stress during ethanol fermentation, yeast strains tolerant to ethanol are highly desirable for the industrial scale ethanol production. A technology called global transcriptional machinery engineering (gTME), which exploits a mutant library of SPT15 encoding the TATA-binding protein of Saccharomyces cerevisiae (Alper et al., 2006; Science 314: 1565 Science 314: -1568, seems to a powerful tool for creating ethanol-tolerant strains. However, the ability of created strains to tolerate high ethanol on rich media remains unproven. In this study, a similar strategy was used to obtain five strains with enhanced ethanol tolerance (ETS1-5) of S. cerevisiae. Comparing global transcriptional profiles of two selected strains ETS2 and ETS3 with that of the control identified 42 genes that were commonly regulated with twofold change. Out of 34 deletion mutants available from a gene knockout library, 18 were ethanol sensitive, suggesting that these genes were closely associated with ethanol tolerance. Eight of them were novel with most being functionally unknown. To establish a basis for future industrial applications, strains iETS2 and iETS3 were created by integrating the SPT15 mutant alleles of ETS2 and ETS3 into the chromosomes, which also exhibited enhanced ethanol tolerance and survival upon ethanol shock on a rich medium. Fermentation with 20% glucose for 24 h in a bioreactor revealed that iETS2 and iETS3 grew better and produced approximately 25% more ethanol than a control strain. The ethanol yield and productivity were also substantially enhanced: 0.31 g/g and 2.6 g/L/h, respectively, for control and 0.39 g/g and 3.2 g/L/h, respectively, for iETS2 and iETS3. Thus, our study demonstrates the utility of gTME in generating strains with enhanced ethanol tolerance that resulted in increase of ethanol production. Strains with enhanced tolerance to other stresses such as heat, fermentation inhibitors, osmotic pressure, and so on, may be further created by using gTME.

Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

“…As a screening result, only BTN2 was confirmed as the gene whose protein levels were significantly increased under severe ethanol stress so far. BTN2 encodes a v-SNARE binding protein and is associated with ethanol tolerance (Chattopadhyay and Pearce, 2002; Kama et al, 2007; Espinazo-Romeu et al, 2008; Yang et al, 2011). We first examined BTN2 mRNA level in yeast cells under severe ethanol stress (9 or 10% ethanol).…”

“…In the present study, we focused on BTN2 whose deficiency induces hypersensitivity to ethanol (Espinazo-Romeu et al, 2008; Yang et al, 2011). BTN2 encodes a v-SNARE binding protein that is involved in intracellular protein trafficking (Kama et al, 2007) and plays a role in protein deposition in the nucleus (Miller et al, 2015).…”

“…BTN2 encodes a v-SNARE binding protein that is involved in intracellular protein trafficking (Kama et al, 2007) and plays a role in protein deposition in the nucleus (Miller et al, 2015). Because Btn2 is important for the correct localization of various proteins, btn2 Δ cells show pleiotropic phenotypes, including decreased resistance to ethanol, acidic pH, hydrostatic pressure, and L -canavanine (Chattopadhyay et al, 2000; Chattopadhyay and Pearce, 2002; Kim et al, 2005; Espinazo-Romeu et al, 2008; Yang et al, 2011). We found that BTN2 mRNA was efficiently translated under severe ethanol stress and that Btn2 protein levels decreased after ethanol elimination.…”

Insertion of transposon in the vicinity of SSK2 confers enhanced tolerance to furfural in Saccharomyces cerevisiae

“…The entire open reading frame (ORF) of SSK2 (4,740 bp) was cloned into the BamHI/SalI site of pRS316 TDH3 , a pRS316-derived plasmid harboring the TDH3 promoter (formerly described as pRS316-GCYH2gR in Yang et al 2011). However, the SSK2 ORF contains three internal BamHI sites at nucleotide (nt) 2389, 3025, and 4662 counting from the initiation codon, which should be removed without changing the reading frame.…”

A Transcriptional Regulator Sll0794 Regulates Tolerance to Biofuel Ethanol in Photosynthetic Synechocystis sp. PCC 6803

“…The technology progresses contributed significantly to the recent increase of worldwide ethanol production, from 17.0 billion liters in 2000 to more than 84.6 billion liters in 2011 (2). One of the successful strain improvement strategies was to obtain ethanol-tolerant strains from either mutant selection or more directed metabolic and genome engineering approaches and to apply them directly in industry, as ethanol is known to be highly toxic to cells (3)(4)(5)(6)(7)(8). In addition to engineering an individual gene or enzyme for better tolerance, increasing studies were recently conducted using regulatory genes or proteins as targets for ethanoltolerance improvements, as more evidences suggested that microbes tend to employ multiple resistance mechanisms in dealing with stress of single biofuel product (9,10), and the manipulation of regulatory genes could provide a route to complex phenotypes that are not readily accessible by traditional methods of targeting some number of metabolic genes (11,12).…”