Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered
            <i>Saccharomyces cerevisiae</i>
            Strain

Lu, Chengfeng; Jeffries, Thomas W.

doi:10.1128/aem.00955-07

Cited by 88 publications

(65 citation statements)

References 42 publications

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“…As an attempt to address these issues, combinatorial transcriptional engineering coupled with efficient gene assembly tools has offered tremendous opportunities for customized optimization of multi-gene pathways 14 . Excellent examples include creation of yeast xylose pathways by promoter shuffling 15 , heterologous production of anticancer taxol precursors in E. coli 16 and rapid assembly and screening of multi-gene mutant pathway libraries in E. coli and yeast 17,18 .…”

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confidence: 99%

Modular optimization of multi-gene pathways for fatty acids production in E. coli

et al. 2013

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Modular optimization of multi-gene pathways for fatty acids production in E. coli

et al. 2013

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“…Balancing of the pathway flux by modulating transcriptional expression was shown to be a critical element in pathway construction for efficient xylose utilization by Saccharomyces cerevisiae and isoprenoid pathway optimization for paclitaxel precursor overproduction in Escherichia coli (1,2). In previous studies, the expression levels of the genes constituting the pathways were varied by manually shuffling a small number of promoters of different strengths, which significantly limited the ranges of enzyme expression and their combinations.…”

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confidence: 99%

“…oordinating the intracellular activities of multiple enzymes in a heterologous metabolic pathway is crucial in order to maximize the flux through the pathway without the accumulation of undesirable intermediates (1)(2)(3). The intracellular enzyme activity can be modulated by changing enzyme expression at transcriptional (promoter-engineering) or translational (engineering of the ribosomal binding sites) levels (2,4,5).…”

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confidence: 99%

“…The intracellular enzyme activity can be modulated by changing enzyme expression at transcriptional (promoter-engineering) or translational (engineering of the ribosomal binding sites) levels (2,4,5). The method of tunable intergenic regions (TIGRs) exploits the prokaryotic operon system to coordinate the expression of multiple genes to regulate several genes simultaneously.…”

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confidence: 99%

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Combinatorial Design of a Highly Efficient Xylose-Utilizing Pathway in Saccharomyces cerevisiae for the Production of Cellulosic Biofuels

Kim

Eriksen

et al. 2013

Appl Environ Microbiol

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Balancing the flux of a heterologous metabolic pathway by tuning the expression and properties of the pathway enzymes is difficult, but it is critical to realizing the full potential of microbial biotechnology. One prominent example is the metabolic engineering of a Saccharomyces cerevisiae strain harboring a heterologous xylose-utilizing pathway for cellulosic-biofuel production, which remains a challenge even after decades of research. Here, we developed a combinatorial pathway-engineering approach to rapidly create a highly efficient xylose-utilizing pathway for ethanol production by exploring various combinations of enzyme homologues with different properties. A library of more than 8,000 xylose utilization pathways was generated using DNA assembler, followed by multitiered screening, which led to the identification of a number of strain-specific combinations of the enzymes for efficient conversion of xylose to ethanol. The balancing of metabolic flux through the xylose utilization pathway was demonstrated by a complete reversal of the major product from xylitol to ethanol with a similar yield and total by-product formation as low as 0.06 g/g xylose without compromising cell growth. The results also suggested that an optimal enzyme combination depends on not only the genotype/phenotype of the host strain, but also the sugar composition of the fermentation medium. This combinatorial approach should be applicable to any heterologous pathway and will be instrumental in the optimization of industrial production of value-added products.

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“…Expressing the XI pathway can avoid the cofactor imbalance problem under anaerobic conditions, but xylitol accumulation has also been observed in strains expressing XI (17,18,20), because the nonspecific aldose reductase encoded by the GRE3 gene can produce xylitol from xylose (27). Various rational approaches have been used to reduce xylitol accumulation and improve xylose utilization, such as optimizing the expression levels of xylose-assimilating reactions (26), engineering the cofactor preference of XR/XDH enzymes (28)(29)(30)(31)(32)(33), perturbing the pentose phosphate pathway by gene knockout or overexpression (34)(35)(36)(37)(38)(39), or deleting GRE3 in strains expressing the XI pathway (21,40,41). While extensive previous efforts focused on manipulating intracellular metabolic reactions to improve xylose utilization and reduce by-product (e.g., xylitol) accumulation, controlling the xylitol export process might also be a meaningful strategy for reducing its formation and increasing carbon flux toward target products.…”

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Deletion of FPS1 , Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Wei

Kim

et al. 2013

Appl Environ Microbiol

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Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD ؉ / NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

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Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered Saccharomyces cerevisiae Strain

Cited by 88 publications

References 42 publications

Modular optimization of multi-gene pathways for fatty acids production in E. coli

Modular optimization of multi-gene pathways for fatty acids production in E. coli

Combinatorial Design of a Highly Efficient Xylose-Utilizing Pathway in Saccharomyces cerevisiae for the Production of Cellulosic Biofuels

Deletion of FPS1 , Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

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