Engineering Yeast Transcription Machinery for Improved Ethanol Tolerance and Production

Department of Chemical Engineering, Massachusetts Institute of Technology, Room 56-469, Cambridge, MA 02139, USA.
Science (Impact Factor: 33.61). 01/2007; 314(5805):1565-8. DOI: 10.1126/science.1131969
Source: PubMed


Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes
important for technological applications. Here we show the application of gTME to Saccharomyces cerevisiae for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p
and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol.
The desired phenotype results from the combined effect of three separate mutations in the SPT15 gene [serine substituted for phenylalanine (Phe177Ser) and, similarly, Tyr195His, and Lys218Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods.

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Available from: Elke Nevoigt, Jun 20, 2014
    • "Up to now, laboratory evolution experiments of mainly fitness-linked phenotypes have been performed by exposing microorganisms to sequentially increasing levels of environmental stress (Eckdahl et al., 2015; Lee et al., 2013; Marietou et al., 2014; Oide et al., 2015; Reyes et al., 2014). Especially in the case of the yeast Saccharomyces cerevisiae, adaptation to an improved ethanol tolerance has been proven useful for increasing product formation (Alper et al., 2006; Jiménez and Benítez, 1987; Liu, 2006). "
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    ABSTRACT: Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188⁎) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains.
    Metabolic Engineering 10/2015; 32. DOI:10.1016/j.ymben.2015.09.017 · 6.77 Impact Factor
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    • "Meanwhile, it becomes clear that toxicity of the end-product biofuels to cyanobacterial cells may represent a major hurdle for further improving the efficiency and productivity of the processes. For rational construction of high-tolerant chassis (Alper et al., 2006), the knowledge on molecular mechanisms responsive to biofuels stress is necessary (Baer et al., 1987; Atsumi et al., 2010). To seek a better understanding of the biofuel-tolerance mechanisms, in this study, using the proteomic datasets collected from several previous studies (Liu et al., 2012; Qiao et al., 2012, 2013; Huang et al., 2013; Tian et al., 2013), we applied network-based methodologies to compare the stress responses induced by three biofuels stress and two environmental perturbations in Synechocystis, as the network-based strategy has the advantages of identifying low abundance or small changes and stress-specific response proteins (Singh et al., 2010; McDermott et al., 2011; Wang et al., 2013b). "
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    ABSTRACT: Although recognized as a promising microbial cell factory for producing biofuels, current productivity in cyanobacterial systems is low. To make the processes economically feasible, one of the hurdles, which need to be overcome is the low tolerance of hosts to toxic biofuels. Meanwhile, little information is available regarding the cellular responses to biofuels stress in cyanobacteria, which makes it challenging for tolerance engineering. Using large proteomic datasets of Synechocystis under various biofuels stress and environmental perturbation, a protein co-expression network was first constructed and then combined with the experimentally determined protein-protein interaction network. Proteins with statistically higher topological overlap in the integrated network were identified as common responsive proteins to both biofuels stress and environmental perturbations. In addition, a weighted gene co-expression network analysis was performed to distinguish unique responses to biofuels from those to environmental perturbations and to uncover metabolic modules and proteins uniquely associated with biofuels stress. The results showed that biofuel-specific proteins and modules were enriched in several functional categories, including photosynthesis, carbon fixation, and amino acid metabolism, which may represent potential key signatures for biofuels stress responses in Synechocystis. Network-based analysis allowed determination of the responses specifically related to biofuels stress, and the results constituted an important knowledge foundation for tolerance engineering against biofuels in Synechocystis.
    Frontiers in Bioengineering and Biotechnology 11/2014; 2:48. DOI:10.3389/fbioe.2014.00048
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    • "Recent genome-level studies have showed that microbes tend to employ multiple resistance mechanisms in dealing with the stress of a single biofuel product [15-17], and it is thus very challenging to achieve tolerance improvement by sequential multigene modifications. To address the issue, approaches have been proposed to focus on various regulatory systems for biofuel tolerance improvement [18]. For example, overexpression of the spo0A gene, encoding a regulator of stationary-phase events and required for transcription of solvent formation genes in butanol-producing Clostridium acetobutylicum, was shown to enhance the butanol tolerance [19]. "
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    ABSTRACT: Background Butanol production directly from CO2 in photosynthetic cyanobacteria is restricted by the high toxicity of butanol to the hosts. In previous studies, we have found that a few two-component signal transduction systems (TCSTSs) were differentially regulated in Synechocystis sp. PCC 6803 after butanol treatment. Results To explore regulatory mechanisms of butanol tolerance, in this work, by constructing gene knockout mutants of the butanol-responsive TCSTS genes and conducting tolerance analysis, we uncovered that an orphan slr1037 gene encoding a novel response regulator was involved in butanol tolerance in Synechocystis. Interestingly, the ∆slr1037 mutant grew similarly to the wild type under several other stress conditions tested, which suggests that its regulation on butanol tolerance is specific. Using a quantitative iTRAQ LC-MS/MS proteomics approach coupled with real-time reverse transcription PCR, we further determined the possible butanol-tolerance regulon regulated by Slr1037. The results showed that, after slr1037 deletion, proteins involved in photosynthesis and glycolysis/gluconeogenesis of central metabolic processes, and glutaredoxin, peptide methionine sulfoxide reductase and glucosylglycerol-phosphate synthase with stress-responsive functions were down-regulated, suggesting that Slr1037 may exhibit regulation to a wide range of cellular functions in combating butanol stress. Conclusions The study provided a proteomic description of the putative butanol-tolerance regulon regulated by the slr1037 gene. As the first signal transduction protein identified directly related to butanol tolerance, response regulator Slr1037 could be a natural candidate for transcriptional engineering to improve butanol tolerance in Synechocystis.
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