Nicholas D. Lindley

Pyruvate Overflow and Carbon Flux Within the Central Metabolic Pathways of Corynebacterium-Glutamicum During Growth on Lactate

Abstract Growth of Corynebacterium glutamicum was characterized kinetically for chemostat cultures using lactate as the sole carbon substrate, and the concentration profile of various enzymes of central metabolism was established over a range of growth rates. Pyruvate overflow, together with incomplete lactate consumption, was observed under conditions for which the specific activity of pyruvate dehydrogenase no longer increased as a function of the growth rate. Biomass yields at such regimes were close to those obtained under exponential growth in batch cultures and were significantly higher than those associated with low growth rates. This shift in carbon conversion efficiency was correlated with increased malic enzyme and pyruvate carboxylase activities, suggesting the operation of a modified tricarboxylic acid cycle involving malate partitioning between malic enzyme (in conjunction with pyruvate carboxylation to replenish the C4 pool) and malate dehydrogenase. This metabolic deviation would provide the NADPH 2 necessary to sustain the observed biomass yields in the absence of NADH:NADP transhydrogenase activity. An analysis of the energetic potential of such a metabolic network indicates that NADH oxidase plays an important role in modulating the respiratory chain efficiency so as to avoid excessive production of adenosine triphosphate. Carbon flux through the central metabolic pathways of C . glutamicum is clearly a dynamic phenomenon whose regulatory complexity needs to be taken into account for future strain improvement strategies aimed at exploiting this organisms natural capacity to overproduce amino acids.

Modified carbon flux during oxygen limited growth of Corynebacterium glutamicum and the consequences for amino acid overproduction

SummaryThe amino acid producing bacterium Corynebacterium glutamicum accumulated lactate, succinate and acetate under oxygen-limited growth conditions. Significant restructuring of carbon flux through the central metabolic pathways occurred with a notable decrease in pentose pathway flux and the operation of the TCA cycle in a reductive mode. Simultaneous consumption of residual sugar and organic acids took place when oxygen sufficient conditions were restored though amino acids yields were significantly perturbed.

Carbon and energy flux constraints in continuous cultures of Alcaligenes eutrophus grown on phenol

Summary: The growth behaviour of Alcaligenes eutrophus on phenol was investigated in continuous cultures to identify the phenomena limiting both growth efficiency and substrate degradation rates. It was shown that the fixed stoichiometry of the meta pathway of phenol degradation, leading to equimolar quantities of pyruvate and acetate, and the structure of the central pathways, which do not allow gluconeogenesis of acetate during growth on phenol, provoke the accumulation of polyhydroxybutyrate (PHB) under certain growth conditions. Acetate is predominantly used as an energy source and PHB accumulates when the cells are carbon-limited rather than energy-limited. The maximum rates of phenol degradation can be attributed to the expression of the enzymes of the catabolic pathway. This is particularly true of phenol hydroxylase and 2-hydroxymuconate semialdehyde (2-hms) dehydrogenase, whose substrates accumulated to physiologically significant concentrations at high growth rates. Indeed the concentration of 2-hms that accumulated in the medium indicated that this enzyme was substrate-saturated at maximum growth rates. However, the specific activity profiles of other catabolic enzymes associated with phenol degradation were close to the estimated flux through the pathway. This suggests a complex control structure in which several enzymes contribute to the control of pathway flux, as would be expected in a catabolic pathway.

Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum

Abstract Growth of Corynebacterium glutamicum on mixtures of glucose and fructose leads to simultaneous consumption of both sugars in which the uptake of each sugar is directly related to the expression of the corresponding sugar uptake mechanism. The overall rate of sugar uptake was higher on sugar mixtures than on either glucose or fructose alone and was similar to that observed during sucrose metabolism. The results suggest that sugar uptake limits metabolic rates though, in the case of fructose, overflow metabolism of both lactate and dihydroxyacetone was observed. Such products could reflect a higher flux through glycolysis rather than the pentose pathway during catabolism of fructose.

Growth of Ralstonia eutropha on inhibitory concentrations of phenol: diminished growth can be attributed to hydrophobic perturbation of phenol hydroxylase activity

The effect of phenol concentration on growth and biodegradative capacity of Ralstonia eutropha regarding phenol was examined. Kinetic analysis indicated that phenol had a strong inhibitory effect on phenol metabolism and growth rate, although biomass yields remained constant, indicating that this phenomena was not caused by increased maintenance requirements. Measurements of specific enzyme activities involved specifically in the catabolic pathway of meta fission of phenol indicated that gene expression cannot explain the diminished metabolic rates at inhibitory phenol concentrations. This phenomenon is due to in vivo inhibition of enzyme activities and notably to phenol hydroxylase activity. Furthermore, other nonmetabolizable organic alcohols provoked a similar effect on both specific growth rate and phenol hydroxylase activity, indicating that inhibition was probably associated with modified membrane fluidity, partially offset by a change in the fatty acid composition of cellular lipids.

An improved temperature-triggered process for glutamate production with Corynebacterium glutamicum☆

An improved glutamate-producing fed-batch process, using a temperature-sensitive strain of Corynebacterium glutamicum, has been characterized. By a tight control of the culture temperature, it was possible to get industrially interesting performance as regards glutamate concentration, yield and productivity. A 24 h fermentation period enabled the production of 85 g/l of glutamate in the production phase induced after a temperature shift from 33°C to 39°C. The maximum specific production rate of glutamate was 0.63 g/g/h with a yield of 0.46 g of glutamate/gram of glucose. The two main co-products of the fermentation were lactate (11 g) and trehalose (12 g). Only trace amounts of other organic acids accumulated in the culture medium. This process offers an interesting alternative to currently employed fermentation strategies in which biotin limitation and/or surfactant addition is used to induce glutamate production. Simple control of fermentor cooling can be used to control the onset of the production phase, offering significant advantages from both an economic and a process robustness viewpoint.

Batch kinetics of Corynebacterium glutamicum during growth on various carbon substrates: use of substrate mixtures to localise metabolic bottlenecks

Growth of Corynebacterium glutamicum on various carbon substrates has been described kinetically under batch culture conditions on fully defined medium. The use of lactate (major fermentation end-product under O2 limitation) led to pyruvate overflow during exponential growth, although pyruvate was reconsumed late in the growth period when both specific rates and residual lactate concentration had diminished. During growth on glucose/lactate mixtures the metabolic architecture was such that simultaneous substrate consumption was observed and, in general, specific rates were correlated with mixture composition. Pyruvate overflow was, however, emphasised and correlated with the flux arriving at pyruvate, suggesting that pyruvate dehydrogenase activity may be rate-limiting under these conditions. The use of lactate/acetate mixtures, in which pyruvate overflow was not observed until all the acetate had been consumed, again suggests a metabolic bottleneck at the level of pyruvate dehydrogenase. The lack of detectable pyruvate overflow during growth on glucose, despite rapid rates of substrate consumption, can be attributed to carbon distribution through central metabolism and the tight control exerted over glucose uptake via the phosphotransferase system.

Influence of end‐products inhibition and nutrient limitations on the growth of Lactococcus lactis subsp. lactis

Lactococcus lactis was grown in a simple synthetic medium with glucose as substrate, enabling the precise quantification of each nutrients contribution to growth. As expected, for the growth of lactic acid bacteria, the growth rate decreased progressively during the cultivation after a short period of exponential growth. End‐products of fermentation, predominantly lactate and in minor amounts formate, acetate and ethanol, accumulated within the medium. Growth of the bacterium in fresh media supplemented with these end‐products showed that the concentrations attained in the fermentor had no significant influence on the growth rate. As regards nutrients, vitamins and magnesium were never limiting during the culture. On the other hand, amino acid concentrations decreased, some of them being totally consumed and exhausted from the medium before growth ceased. However, growth in reconstituted media constructed with the amino acid concentrations remaining at different times of cultivation showed that amino acid depletion could not account for the observed growth decrease. Batch culture supernatant fluid was used as cultivation medium. Growth rates observed in supernatant cultures supplemented with various nutrients, compared to non‐supplemented supernatant, showed that no addition improved growth. Finally, it was concluded that in the experimental conditions used in this study, growth inhibition was predominantly due to phenomena other than lactate inhibition and nutritional limitations, and hence associated with unidentified compounds produced in the fermentation.

Maintaining a controlled residual growth capacity increases the production of polyhydroxyalkanoate copolymers by Alcaligenes eutrophus

SummaryThe manner in which copolymer poly(HB-co-HV) production was influenced by different methods of limiting cell proliferation during the production phase was examined. Polymer production was significantly improved in fermentation strategies in which some growth was maintained, either by linear or exponential nitrogen source feeding as compared to cultures in which nitrogen supply was totally interrupted. Improved volumetric productivities were obtained in cultures fed with NH4OH and the proportion of 3HV incorporated was approximately twofold higher in these cultures. These performance improvements were due to higher specific rates of glucose and propionate consumption in cultures in which true growth capacity was maintained.

Flux limitations in the ortho pathway of benzoate degradation of Alcaligenes eutrophus: metabolite overflow and induction of the meta pathway at high substrate concentrations

The growth behaviour of Alcaligenes eutrophus using various concentrations of benzoate was investigated. In batch culture, growth was exponential and growth rate (mu) and yields (Y) were high [mu = 0.51 h-1 and Yx/benzoate = 0.56 mol carbon (mol carbon)-1] when low concentrations of benzoate (< 5 mM) were used. These kinetic parameters were close to the maxima determined in a benzoate-limited chemostat [mu(max) = 0.55 h-1 and YX/benzoatemax = 0.57 mol carbon (mol carbon)-1] and the part of the energy for maintenance was limited (mATP = 4.3 +/- 2.2 mmol ATP g-1 h-1). When higher concentrations of benzoate were used (up to 40 mM), several metabolic limitations appeared. The specific rate of benzoate consumption was not altered, whereas growth was inhibited [Ki(benzoate) approximately 27 mM]. Furthermore, high concentrations of catechol together with some 1,2-dihydro-1,2-dihydroxybenzoate (DHB) transiently accumulated in the medium. The accumulation of catechol was attributed to limiting flux through catechol 1,2-dioxygenase estimated to be 5.2 mmol g-1 h-1, whereas that of DHB was provoked by an imbalance in the NADH/NAD+ intracellular content. The direct consequence of DHB accumulation was the induction of the meta pathway for the degradation of catechol, and this pathway contributed up to 20% of the total flux of catechol to the central metabolism. Finally, when very high concentrations of benzoate were used (55 mM), both growth and the specific rate of benzoate degradation were diminished due to a strong decrease in benzoate 1,2-dioxygenase specific activity.