Analysis of cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation

Kim, Heejin; Lee, Won Heong; Galazka, Jonathan M.; Cate, J.H.D.; Jin, Yong Su

doi:10.1007/s00253-013-5339-2

Cited by 50 publications

(44 citation statements)

References 22 publications

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“…Despite the promising results from the cellobiose fermentation experiments, transient cellodextrin accumulation by the transglycosylation of ␤-glucosidase was observed in the medium during the processes (24). The accumulated cellodextrin might decrease the ethanol productivity because the cellodextrin transport and reconsumption might not be as rapid as the cellobiose fermentation (22,24). S. cerevisiae strain D452-2 expressing the cellodextrin transporter and ␤-glucosidase from episomal plasmids (DCDT-1G) accumulated 15.5 g/liter of cellodextrin in the fermentation experiments using YP medium with 80 g/liter cellobiose under oxygen-limited conditions.…”

Section: Discussionmentioning

confidence: 96%

“…These results indicated that not only the absolute increase of the copy numbers of cdt-1 and gh1-1 but also their ratio is important for the large improvement in cellobiose fermentation that we observed in the evolved strain EJ2. Despite the promising results from the cellobiose fermentation experiments, transient cellodextrin accumulation by the transglycosylation of ␤-glucosidase was observed in the medium during the processes (24). The accumulated cellodextrin might decrease the ethanol productivity because the cellodextrin transport and reconsumption might not be as rapid as the cellobiose fermentation (22,24).…”

Section: Discussionmentioning

confidence: 99%

“…There are two potential problems with cellobiose fermentation in prior studies (22,24). First, the engineered yeast contained the episomal plasmids that can result in plasmid loss in nonselective medium due to a metabolic burden on the host (25).…”

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confidence: 99%

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Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Skerker

Kim

et al. 2016

Appl Environ Microbiol

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Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ␤-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1. We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. IMPORTANCEIn order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ␤-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evol...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

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Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Skerker

Kim

et al. 2016

Appl Environ Microbiol

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“…Cellobiose Uptake, but Still Allow Cellulase Gene InductionWhen heterologously expressed in S. cerevisiae, CDT-1 and CDT-2 have been shown to be high-affinity transporters, with CDT-1 acting as a proton symporter and CDT-2 acting as a facilitator (33). To identify functionally important residues within the cellodextrin transporters, CDT-1 and CDT-2, several conserved amino acids were individually mutated to alanine.…”

Section: Point Mutants In Cdt-1 and Cdt-2 Significantly Decreasementioning

confidence: 99%

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Znameroski

Tsai

et al. 2014

Journal of Biological Chemistry

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“…S1A in the supplemental material) (5). We hypothesized that cellobiose consumption results in an ATP deficit in glycolysis relative to glucose, due to the fact that the cellodextrin transporter (CDT-1) in the cellobiose-utilizing pathway is a proton symporter, requiring ATP hydrolysis for cellobiose uptake (7). Moreover, under anaerobic conditions, ATP production is limited to substrate-level phosphorylation, further restricting ATP availability.…”

Section: Resultsmentioning

confidence: 99%

Cellobiose consumption uncouples extracellular glucose sensing and glucose metabolism inSaccharomyces cerevisiae

Chomvong

Benjamin

Nomura

et al. 2016

Preprint

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Glycolysis is central to energy metabolism in most organisms and is highly regulated to enable optimal growth. In the yeast Saccharomyces cerevisiae, feedback mechanisms that control flux through glycolysis span transcriptional control to metabolite levels in the cell. Using a cellobiose consumption pathway, we decoupled glucose sensing from carbon utilization, revealing new modular layers of control that induce ATP consumption to drive rapid carbon fermentation. Alterations of the beta subunit of phosphofructokinase-1 (PFK2), H ϩ -plasma membrane ATPase (PMA1), and glucose sensors (SNF3 and RGT2) revealed the importance of coupling extracellular glucose sensing to manage ATP levels in the cell. Controlling the upper bound of cellular ATP levels may be a general mechanism used to regulate energy levels in cells, via a regulatory network that can be uncoupled from ATP concentrations under perceived starvation conditions. IMPORTANCE Living cells are fine-tuned through evolution to thrive in their native environments. Genome alterations to create organisms for specific biotechnological applications may result in unexpected and undesired phenotypes. We used a minimal synthetic biological system in the yeast Saccharomyces cerevisiae as a platform to reveal novel connections between carbon sensing, starvation conditions, and energy homeostasis.KEYWORDS PMA1, cellobiose, glucose sensors, metabolomics M ost microorganisms favor glucose as their primary carbon source, as reflected in their genetic programs hard-wired for this preference. Central to carbon metabolism is glycolysis, which is finely tuned to the dynamic state of the cell due to the fact that glycolysis first consumes ATP before generating additional ATP equivalents. To avoid catastrophic depletion of ATP, the yeast Saccharomyces cerevisiae has evolved a transient ATP hydrolysis futile cycle coupled to gluconeogenesis (1). Glycolysis in yeast is also tightly coupled to glucose transport into the cell, entailing three extracellular glucose-sensing mechanisms and at least one intracellular glucose signaling pathway (2).Synthetic biology and metabolic engineering of yeast hold promise to convert this microorganism into a "cell factory" to produce a wide range of chemicals derived from renewable resources or those unattainable through traditional chemical routes. However, many applications require tapping into metabolites involved in central carbon metabolism, a daunting challenge as living cells have numerous layers of feedback regulation that fine-tune growth to changing environments. Cellular regulation evolved

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Analysis of cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation

Cited by 50 publications

References 22 publications

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Cellobiose consumption uncouples extracellular glucose sensing and glucose metabolism inSaccharomyces cerevisiae

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