Formate gradients as a means for detecting the maximum carbon conversion efficiency of heterotrophic substrates: Correlation between formate utilization and biomass increase

Müller, Roland; Markuske, Klaus D.; Babel, Wolfgang

doi:10.1002/bit.260271114

Cited by 25 publications

(3 citation statements)

References 22 publications

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“…These results are likely to be a combination of effects from alleviating the feedback inhibition of the TCA cycle by mitochondrial NADH and increasing respiratory capacity due to improved efficiency of oxidative phosphorylation, as quantified by the P/O ratio (15). There are no reports that describe the effect of increasing NADH in S. cerevisiae, although formate has been used previously as a source of additional reducing power in S. cerevisiae (2,4,11,23,24,27). Formate (HCOO Ϫ ) is efficiently oxidized to CO 2 by NAD ϩ -dependent formate dehydrogenase (27) and, therefore, cannot be used as a carbon source for biomass synthesis.…”

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confidence: 99%

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Hou

Scalcinati

Oldiges

et al. 2010

Appl Environ Microbiol

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Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD ؉ -dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.The traditional use of baker's yeast, Saccharomyces cerevisiae, for ethanol production has resulted in the accumulation of substantial information about its genetics, metabolism, and process development. Consequently, the collection of compounds that are produced using S. cerevisiae has expanded to include organic acids and even secondary metabolites (1,25,28). Unlike ethanol, many of these products are not redox neutral relative to commonly used substrates such as glucose. Therefore, in addition to stoichiometry, redox constraints play an important role in the formation of the products. Additional reducing power has to be supplied to produce compounds whose degree of reduction is higher than that of the substrate. On the other hand, producing compounds with a degree of reduction lower than that of the substrate will force the synthesis of other compounds with higher degrees of reduction to compensate for excess reducing power generated from substrate oxidation. These constraints may decrease the product yield substantially.The catabolic currency that balances the degree of reduction between the substrate and the products is usually NADH. In S. cerevisiae, NADH is produced in the cytosol by mainly glyceraldehyde-3-phosphate dehydrogenase and other assimilatory reaction enzymes (35). In the mitochondria, NADH is formed in the tricarboxylic acid (TCA) cycle and the reaction of the pyruvate dehydrogenase complex. Cytosolic NADH is oxidized by the glycerol-3-phosphate shuttle or the external cytosolic NADH dehydrogenases, which are part of the electron transp...

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confidence: 99%

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Hou

Scalcinati

Oldiges

et al. 2010

Appl Environ Microbiol

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“…After reaching steady state at the respective dilution rate, 2,4-D was fed via a linear gradient (cf. Mu¨ller et al 1985) in addition to (RS)-2,4-DP. The 2,4-D concentration extended from 0 to 6 mM in two reservoir flasks which had a total volume of 6.8 l. The experiments were performed at dilution rates in a range of 0.06 -0.17 h )1 .…”

Section: Cultivationmentioning

confidence: 97%

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Hoffmann

Müller

2006

Biodegradation

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Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain's degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (mu max) of 0.158 h(-1). The half-maximum rate-associated substrate concentration (Ks) was 45 microM. At pH 8.5 mu max was only 0.05 h(-1) and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that mu max with dichlorprop was around 0.2 h(-1) at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with mu max of 0.147 h(-1) and Ks of 267 microM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 microM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)-2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h(-1) at pH 6.8 and up to D = 0.2 h(-1) at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.

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“…Hamenula polymorph (Tor~lopsis sp.) MH 26 is a methylotrophic yeast which assimilates glucose mainly via the HMP pathway [13,14]. This means that pentose phosphates are obligatorily included in the primary assimilatory pathway offering themselves as the C,-acceptor in the case of the simultaneous utilization of methanol.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous utilization of methanol and glucose by Hansenula polymorpha (Torulopsis sp.) MH 26, a chemostatic investigation on the distribution of ¹⁴C‐methanol

Müler

Babel

Uhlenhut³

1988

Acta Biotechnologica

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Simultaneous utilization of methanol and glucose by Hansenvla polymorpha (Toru1opsi.s sp.)M H 26 results in a n increase in growth yield of up to 18% in dependence of the mixing proportion.The distribution of carbon from 14C-methanol into biomass and carbon dioxide was investigated. The distribution pattern was strongly influenced by the mixing proportion showing that methanol plays an increasing role as an energy donor as the glucose content in the mixture increased. Due to these data and verified by theoretical considerations the effect on growth yield was discussed to be caused by a n interconnection in the conversion of the individual substrates. This is attributed to glucose delivering the acceptor for C,-assimilation and resulting in n more efficient utilization of glucose carbon on the one as well as the energy content of methanol on the other hand.

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Formate gradients as a means for detecting the maximum carbon conversion efficiency of heterotrophic substrates: Correlation between formate utilization and biomass increase

Cited by 25 publications

References 22 publications

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Simultaneous utilization of methanol and glucose by Hansenula polymorpha (Torulopsis sp.) MH 26, a chemostatic investigation on the distribution of ¹⁴C‐methanol

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Formate gradients as a means for detecting the maximum carbon conversion efficiency of heterotrophic substrates: Correlation between formate utilization and biomass increase

Cited by 25 publications

References 22 publications

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Simultaneous utilization of methanol and glucose by Hansenula polymorpha (Torulopsis sp.) MH 26, a chemostatic investigation on the distribution of 14C‐methanol

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Simultaneous utilization of methanol and glucose by Hansenula polymorpha (Torulopsis sp.) MH 26, a chemostatic investigation on the distribution of ¹⁴C‐methanol