Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering. Biotechnol Biofuels 3:13

Department of Applied Microbiology, Lund University, PO Box 124, SE-22100 Lund, Sweden. .
Biotechnology for Biofuels (Impact Factor: 6.04). 06/2010; 3(1):13. DOI: 10.1186/1754-6834-3-13
Source: PubMed


Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced.
Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways.
To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.

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Available from: João R. M. Almeida
    • "In addition to the aforementioned rational approach, laboratory evolution and random mutagenesis have been performed to overcome unknown limiting factors of xylose metabolism (Kim et al., 2013c; Kuyper et al., 2005b; Liu and Hu, 2010; Ni et al., 2007; Peng et al., 2012; Sanchez et al., 2010; Sonderegger and Sauer, 2003; Tomitaka et al., 2013; Wisselink et al., 2009). At least two independent studies observed that evolved mutants capable of metabolizing xylose efficiently acquired a mutation in the PHO13 gene, which encodes an-alkaline phosphatase (Kim et al., 2013c; Van Vleet et al., 2008). "
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    ABSTRACT: The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.
    No preview · Article · Dec 2015 · Metabolic Engineering
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    • "Economic feasibility of biosynthetic fuel and chemical production depends on optimization of these biocatalysts to achieve high yields of the desired products. Saccharomyces cerevisiae is currently the most employed microbial catalyst in the biotechnology industry, but this yeast is limited in its range of substrates for producing fuel ethanol, and although genetic engineering has improved its utilization of the constituent pentose sugars of lignocellulosic materials, development of a recombinant S. cerevisiae strain capable of efficient pentose utilization remains a challenge (Casey et al. 2013; Garcia Sanchez et al. 2010; Hughes et al. 2009a, b; Kim et al. 2013a, b; Matsushika et al. 2014; Nielsen et al. 2013; Oreb et al. 2012; Zhou et al. 2012). Other microbial catalysts are being investigated for the production of biofuels and value-added bioproducts. "
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    ABSTRACT: Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.
    Full-text · Article · Aug 2015 · Applied Microbiology and Biotechnology
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    • "However, independently of the xylose utilizing pathway used, the uptake of xylose across the yeast plasma membrane occurs through a transport system that has been reported to have significantly lower affinities for xylose, when compared to glucose, exhibiting substantial metabolic flux control especially when the intracellular pathway is optimized [15] [16] [17] [18] [19] [20] [21] [22]. Furthermore, mutant or evolved yeast strains showing improved xylose fermenting performance have also improved xylose transport kinetics [23] [24] [25] [26]. Thus, it is clear that xylose transport limits pathway flux, and improvements of intracellular metabolism will only exacerbate the transport bottleneck [27]. "
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    ABSTRACT: Since the uptake of xylose is believed to be one of the rate-limiting steps for xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) with an integrative plasmid to overexpress the genes for xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using xylose, glucose, or xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose/glucose co-fermentations, but was incapable to allow xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient xylose fermentation, but during xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing xylose/glucose blends, whereas the HXT7 permease would be a better choice for xylose-enriched sugar streams
    Full-text · Article · Sep 2014 · Enzyme and Microbial Technology
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