Optimization of Ethanol Production in Saccharomyces cerevisiae by Metabolic Engineering of the Ammonium Assimilation

Nissen, Torben L.; Kielland‐Brandt, Morten C.; Nielsen, Jens; Villadsen, John

doi:10.1006/mben.1999.0140

Cited by 201 publications

(164 citation statements)

References 23 publications

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“…It therefore appears that turnover of the net excess NADH formed in biosynthesis and product formation is an important factor for determining the biomass yield. This is also supported by the observation that overexpression of the GDH2 gene, encoding the NADH-dependent glutamate dehydrogenase, in a gdh1 gene, lacking the NADPH-dependent enzyme, increases the biomass yield by more than 10% (Nissen et al, 2000). The activity of Gdh1p is about 70 times higher than that of Gdh2p in wild-type cells growing anaerobically on glucose with ammonium as the nitrogen source (Nissen et al, 1997(Nissen et al, , 2000.…”

Section: Nadh and Nadph Turnovermentioning

confidence: 66%

Metabolic flux analysis of RQ‐controlled microaerobic ethanol production by Saccharomyces cerevisiae

Franzén

2002

Yeast

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Microaerobic ethanol production by Saccharomyces cerevisiae CBS 8066 was investigated at different growth rates in respiratory quotient (RQ)-controlled continuous culture. The RQ was controlled by changing the inlet gas composition by a feedback controller while keeping other parameters constant. The ethanol yield increased slightly from the anaerobic values with decreasing RQ, reaching a broad maximum at RQ 20 to 12. There was little or no glycerol production at RQ values below 17 over a wide range of dilution rates. Metabolic flux analysis revealed that a decrease in the ethanol yield at RQ 6 coincided with the cyclic, oxidative operation of the TCA cycle reactions. The model indicated that respiratory dissimilation of glucose only occurs when the oxygen uptake rate is high enough to completely substitute for glycerol formation. The cytosolic and the mitochondrial NADH balances were important for determining the flux distributions. The smallest deviations between estimated and measured product yields were obtained when the unknown co-factor requirements of a limited number of biosynthetic reactions were chosen so that the amount of excess NADH formed in biosynthesis was minimized. The biomass yield was positively correlated with the net amount of NADH reoxidized in respiration and glycerol formation, indicating that the turnover of excess NADH from biosynthesis is an important factor influencing the biomass yield under oxygen-limiting conditions.

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Section: Nadh and Nadph Turnovermentioning

confidence: 66%

Metabolic flux analysis of RQ‐controlled microaerobic ethanol production by Saccharomyces cerevisiae

Franzén

2002

Yeast

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“…Attempts to engineer microbial metabolic fluxes through manipulations at the level of NADP-GDH (Marx et al, 1999;Nissen et al, 2000) clearly suggest the significance of this enzyme at the carbon-nitrogen interface of metabolism. It is interesting that, although NADP-GDH is central to aspergillus nitrogen metabolism (and ammonia assimilation), its activity is amenable to regulation by 2-oxoglutarate.…”

Section: Discussionmentioning

confidence: 99%

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Noor

Punekar

2005

Microbiology

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NADP-dependent glutamate dehydrogenase (NADP-GDH) mediates fungal ammonium assimilation through reductive synthesis of glutamate from 2-oxoglutarate. By virtue of its position at the interface of carbon and nitrogen metabolism, biosynthetic NADP-GDH is a potential candidate for metabolic control. In order to facilitate characterization, a new and effective dye-affinity method was devised to purify NADP-GDH from two aspergilli, Aspergillus niger and Aspergillus nidulans. The A. niger NADP-GDH was characterized at length and its kinetic interaction constants with glutamate (K m 34?7 mM) and ammonium (K m 1?05 mM; K i 0?4 mM) were consistent with an anabolic role. Isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate were significant inhibitors, with respective K i values of 6?9, 9?2 and 202?0 mM. The A. niger enzyme showed allosteric properties and a sigmoid response (n H =2?5) towards 2-oxoglutarate saturation. The co-operative behaviour was a feature common to NADP-GDH from Aspergillus awamori, A. nidulans and Aspergillus oryzae. NADP-GDH may therefore be a crucial determinant in adjusting 2-oxoglutarate flux between the tricarboxylic acid cycle and glutamate biosynthesis in aspergilli.

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“…It is likely that deletion of FPS1 in engineered yeast strains here hampered the glycerol production pathway, and the capability to deal with the redox imbalance problem in xylose fermentation under anaerobic conditions became less efficient. However, there was no significant difference in the NAD ϩ /NADH ratio between the two strains SR8-fps1⌬ and SR8, indicating that FPS1 deletion might not drastically influence the cofactor balance or that some alternative pathway other than glycerol formation could contribute to redox balancing (62). The reason for slightly higher NADP ϩ /NADPH in the SR8-fps1⌬ strain is unclear.…”

Section: Discussionmentioning

confidence: 58%

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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Optimization of Ethanol Production in Saccharomyces cerevisiae by Metabolic Engineering of the Ammonium Assimilation

Cited by 201 publications

References 23 publications

Metabolic flux analysis of RQ‐controlled microaerobic ethanol production by Saccharomyces cerevisiae

Metabolic flux analysis of RQ‐controlled microaerobic ethanol production by Saccharomyces cerevisiae

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

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

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