Identification of the First Fungal NADP-GAPDH from <i>Kluyveromyces lactis</i>

Verho, Ritva; Richard, Peter; Jonson, Per Harald; Sundqvist, Lena; Londesborough, John; Penttilä, Merja

doi:10.1021/bi0265325

Cited by 60 publications

(45 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…GDP1 of Kluyveromyces lactis is a phosphorylating GAPDH, unique among nonplant eukaryotes, that can rescue the lethal phenotype of the PGI1 deletion in S. cerevisiae (17). When growing on pentoses, K. lactis avoids carbon exhaust by inducing the expression of this enzyme; GDP1 interferes with the interplay of glycolysis and the PPP by switching the glycolytic redox-cofactor from NAD(H) to NADP(H) (17,18).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A catabolic block does not sufficiently explain how 2-deoxy-d-glucose inhibits cell growth

Ralser

Wamelink

Struys

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

149

160

View full text Add to dashboard Cite

The glucose analogue 2-deoxy-D-glucose (2-DG) restrains growth of normal and malignant cells, prolongs the lifespan of C. elegans, and is widely used as a glycolytic inhibitor to study metabolic activity with regard to cancer, neurodegeneration, calorie restriction, and aging. Here, we report that separating glycolysis and the pentose phosphate pathway highly increases cellular tolerance to 2-DG. This finding indicates that 2-DG does not block cell growth solely by preventing glucose catabolism. In addition, 2-DG provoked similar concentration changes of sugar-phosphate intermediates in wild-type and 2-DG-resistant yeast strains and in human primary fibroblasts. Finally, a genome-wide analysis revealed 19 2-DG-resistant yeast knockouts of genes implicated in carbohydrate metabolism and mitochondrial homeostasis, as well as ribosome biogenesis, mRNA decay, transcriptional regulation, and cell cycle. Thus, processes beyond the metabolic block are essential for the biological properties of 2-DG.cell growth inhibition ͉ glycolysis ͉ off-target effect ͉ pentose phosphate pathway ͉ carbohydrate metabolism 2 -Deoxy-D-glucose (2-DG) is a stable glucose analogue that is actively taken up by the hexose transporters and phosphor ylated but cannot be fully metabolized. 2-DG-6-phosphate accumulates in the cell and interferes with carbohydrate metabolism by inhibiting glycolytic enzymes. 2-DG-6-phosphate inhibits phosphoglucose isomerase (PGI) in a competitive, and hexokinase (HXK) in a noncompetitive, manner (1-3). On the cellular level, 2-DG provokes rapid growth inhibition and results in altered glycosylation that is highly dependent on catabolic glucose intermediates (4, 5).2-DG has been used in numerous studies focused on the effects of reduced metabolic rates. Recently, 2-DG has been used in this study of glycolytic inhibition with respect to calorie restriction and aging. 2-DG increases the lifespan of C. elegans, an effect that is reversed by antioxidants (6, 7). Moreover, 2-DG mitigates disease progression in a murine model of temporal lobe epilepsy (8), possibly due to the repression of the BDNF promoter. 2-DG shows possibilities as an antiviral drug, as it targets gene expression in papillomavirus (9). Finally, 2-DG has been thoroughly studied in regard to cancer biology. Malignant cells exhibit increased glucose metabolism compared with surrounding tissue (10) and, therefore, increased 2-DG uptake. 2-DG efficiently inhibits tumor progression and is a focus of clinical trials (reviewed in ref. 11).In general, it is assumed that the biological effects of 2-DG are the consequence of a block in carbohydrate catalysis, implying that 2-DG treated cells, unable to metabolize glucose, stop growing as a result of a lack of energy and metabolic intermediates. However, several observations bring this assumption into question. First, only a moderate decline in ATP has been observed in 2-DG-treated eukaryotes (7,12). Second, a current study reveals that tumor cells react differently to 2-DG than to its close analogue 2-fluorod...

show abstract

Section: Resultsmentioning

confidence: 99%

“…When growing on pentoses, K. lactis avoids carbon exhaust by inducing the expression of this enzyme; GDP1 interferes with the interplay of glycolysis and the PPP by switching the glycolytic redox-cofactor from NAD(H) to NADP(H) (17,18). 2-DG tolerance was examined in the presence of GDP1.…”

Section: Resultsmentioning

confidence: 99%

A catabolic block does not sufficiently explain how 2-deoxy-d-glucose inhibits cell growth

Ralser

Wamelink

Struys

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

149

160

View full text Add to dashboard Cite

show abstract

“…Metabolic engineering of xylose metabolism would benefit on finding a mechanism for production of NADPH upstream in metabolism through a route, which would not result in loss of carbon. For example simultaneous deletion of glucose-6-phospate dehydrogenase encoding gene (Jeppsson et al, 2002) and expression of a partly NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene could direct the flow of carbon directly towards pyruvate and ethanol without the loss of carbon (Verho et al, 2002;Verho et al, manuscript in preparation). By using solely metabolic flux analysis it was not possible to study the effects of xylose uptake or pentose phosphate reactions, since their role as a limiting step originates from their kinetic properties such as K m values for uptake and Gibbs free energies for pentose phosphate pathway and thus cannot be studied in the present steady-state model.…”

Section: Discussionmentioning

confidence: 99%

Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture

Pitkänen

Aristidou

Salusjärvi

et al. 2003

Metabolic Engineering

Self Cite

108

View full text Add to dashboard Cite

This study focused on elucidating metabolism of xylose in a Saccharomyces cerevisiae strain that overexpresses xylose reductase and xylitol dehydrogenase from Pichia stipitis, as well as the endogenous xylulokinase. The influence of xylose on overall metabolism was examined supplemented with low glucose levels with emphasis on two potential bottlenecks; cofactor requirements and xylose uptake. Results of metabolic flux analysis in continuous cultivations show changes in central metabolism due to the cofactor imbalance imposed by the two-step oxidoreductase reaction of xylose to xylulose. A comparison between cultivations on 27:3 g/L xylose-glucose mixture and 10 g/L glucose revealed that the NADPH-generating flux from glucose-6-phosphate to ribulose-5-phosphate was almost tenfold higher on xylose-glucose mixture and due to the loss of carbon in that pathway the total flux to pyruvate was only around 60% of that on glucose. As a consequence also the fluxes in the citric acid cycle were reduced to around 60%. As the glucose level was decreased to 0.1 g/L the fluxes to pyruvate and in the citric acid cycle were further reduced to 30% and 20%, respectively. The results from in vitro and in vivo xylose uptake measurements showed that the specific xylose uptake rate was highest at the lowest glucose level, 0.1 g/L.

show abstract

“…From our proteomics results, we found that of the three GapDH isoenzymes in yeast, Tdh3p was the most highly expressed. We thus replaced TDH3 with either an NADPH-producing GapDH from Kluyveromyces lactis (KlGapDH) (Verho et al, 2002), or a non-phosphorylating GapDH from Bacillus cereus (BcGapN) in the baseline yL405 strain. Neither resulting strain produced more fatty alcohols than the parent (Fig.…”

Section: Optimizing Nadph/nadp+ and Nadh/nad+ Cofactor Usagementioning

confidence: 99%

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

d’Espaux

Ghosh

Runguphan

et al. 2017

Metabolic Engineering

102

View full text Add to dashboard Cite

A B S T R A C TFatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to~20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acidderived products.

show abstract

Identification of the First Fungal NADP-GAPDH from Kluyveromyces lactis

Cited by 60 publications

References 22 publications

A catabolic block does not sufficiently explain how 2-deoxy-d-glucose inhibits cell growth

A catabolic block does not sufficiently explain how 2-deoxy-d-glucose inhibits cell growth

Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Contact Info

Product

Resources

About