Flux Redistribution Research Articles

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1mol of CO2 was fixated in 1mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD+ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD+ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72h, 14.5gL−1 of glucose was consumed to generate 12.08gL−1 of succinic acid. Furthermore, under optimized condition of CO2 supply, the succinic acid productivity and the CO2 fixation rate reached 223.88mgL−1h−1 and 83.48mgL−1h−1, respectively.

Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived 13C-labelling data from proteinogenic amino acids

The yeast Pichia pastoris has emerged as one of the most promising yeast cell factories for the production of heterologous proteins. The readily available genetic tools and the ease of high-cell density cultivations using methanol or glycerol/methanol mixtures are among the key factors for this development. Previous studies have shown that the use of mixed feeds of glycerol and methanol seem to alleviate the metabolic burden derived from protein production, allowing for higher specific and volumetric process productivities. However, initial studies of glycerol/methanol co-metabolism in P. pastoris by classical metabolic flux analyses using (13)C-derived Metabolic Flux Ratio (METAFoR) constraints were hampered by the reduced labelling information obtained when using C3:C1 substrate mixtures in relation to the conventional C6 substrate, that is, glucose. In this study, carbon flux distributions through the central metabolic pathways in glycerol/methanol co-assimilation conditions have been further characterised using biosynthetically directed fractional (13)C labelling. In particular, metabolic flux distributions were obtained under 3 different glycerol/methanol ratios and growth rates by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids using the software tool (13)CFlux2. Specifically, cells were grown aerobically in chemostat cultures fed with 80:20, 60:40 and 40:60 (w:w) glycerol/methanol mixtures at two dilutions rates (0.05 hour(-1) and 0.16 hour(-1)), allowing to obtain additional data (biomass composition and extracellular fluxes) to complement pre-existing datasets. The performed (13)C-MFA reveals a significant redistribution of carbon fluxes in the central carbon metabolism as a result of the shift in the dilution rate, while the ratio of carbon sources has a lower impact on carbon flux distribution in cells growing at the same dilution rate. At low growth rate, the percentage of methanol directly dissimilated to CO2 ranges between 50% and 70%. At high growth rate the methanol is completely dissimilated to CO2 by the direct pathway, in the two conditions of highest methanol content.

Enhancing l-Isoleucine Production by thrABC Overexpression Combined with alaT Deletion in Corynebacterium glutamicum

L-isoleucine is synthesized from 2-ketobutyrate and pyruvate in Corynebacterium glutamicum, and the supplies of these two precursors are important for L-isoleucine synthesis. C. glutamicum YILWΔalaT with alaT gene deletion (encoding alanine aminotransferase, a principal enzyme for L-alanine synthesis) was constructed to increase intracellular pyruvate availability, and the thrABC genes from Escherichia coli (encoding bifunctional aspartate kinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase) were overexpressed in C. glutamicum YILW and YILWΔalaT to increase the supply of intracellular 2-ketobutyrate. In the fed-batch fermentation, YILWpXMJ19thrABC, YILWΔalaT, and YILWΔalaTpXMJ19thrABC exhibited 5.3, 17.6, and 8.4 % higher L-isoleucine production than the original strain, respectively. Both YILWpXMJ19thrABC and YILWΔalaT excreted lower concentrations of L-lysine, L-alanine, and L-valine. YILWΔalaTpXMJ19thrABC exhibited a cumulative reduction of these by-products excretion, which indicated that thrABC overexpression combined with alaT deletion resulted in the metabolic flux redistribution from 2-ketobutyrate and pyruvate to L-isoleucine synthesis, and decreased the fluxes to by-products synthesis accordingly.

‘Super-Chandrasekhar’ Type Ia Supernovae at nebular epochs★

We present a first systematic comparison of superluminous Type Ia supernovae (SNe Ia) at late epochs, including previously unpublished photometric and spectroscopic observations of SN 2007if, SN 2009dc and SNF20080723-012. Photometrically, the objects of our sample show a diverse late-time behaviour, some of them fading quite rapidly after a light-curve break at ~150-200d. The latter is likely the result of flux redistribution into the infrared, possibly caused by dust formation, rather than a true bolometric effect. Nebular spectra of superluminous SNe Ia are characterised by weak or absent [Fe III] emission, pointing at a low ejecta ionisation state as a result of high densities. To constrain the ejecta and 56Ni masses of superluminous SNe Ia, we compare the observed bolometric light curve of SN 2009dc with synthetic model light curves, focusing on the radioactive tail after ~60d. Models with enough 56Ni to explain the light-curve peak by radioactive decay, and at the same time sufficient mass to keep the ejecta velocities low, fail to reproduce the observed light-curve tail of SN 2009dc because of too much gamma-ray trapping. We instead propose a model with ~1 solar mass of 56Ni and ~2 solar masses of ejecta, which may be interpreted as the explosion of a Chandrasekhar-mass white dwarf (WD) enshrouded by 0.6-0.7 solar masses of C/O-rich material, as it could result from a merger of two massive C/O WDs. This model reproduces the late light curve of SN 2009dc well. A flux deficit at peak may be compensated by light from the interaction of the ejecta with the surrounding material.

Coupling IR Thermography and BIA to analyse body reaction after one acupuncture session

Coupling both thermography and bio-Impedance, some biophysical acupuncture mechanisms are statically studied on a small population of 18 subjects. Results show that a possible way of understanding acupuncture, in an electrical way, should be to consider ionic flux redistribution between vascular and extra cell compartments. This is a two steps mechanism. The first one is starting with needles insertion and the second one is lasting with more intensity after removing them from skin.

Improvement of welan gum production and redistribution of metabolic flux under pH control process in Alcaligenes sp. CGMCC2428

Batch fermentations of welan gum from Alcaligenes sp. CGMCC2428 at pH values of 5.5 ∼ 7.0 were studied. Based on the kinetic analysis, a pH control process for improving welan production was developed. By maintaining the culture pH at 7.0, the process significantly improved the maximal welan concentration and productivity to reach 25.1 ± 0.65 g/L and 0.42 ± 0.003 g/L/h, respectively, compared with those in native pH processes where pH value would decrease from 7.0 to 5.1 (18.5 ± 0.72 g/L and 0.28 ± 0.002 g/L/h). This improvement may be due to the increased metabolic flux of glucose-1-phosphate to welan induced by pH control process. Furthermore, scale-up fermentation under controlled pH was implemented using 300-L fermentor. The highest welan concentration of 27.5 ± 0.97 g/L was obtained. This simplified process proved effective in industry fermentation for high welan production.

Metabolic responses of Saccharomyces cerevisiae to valine and ammonium pulses during four-stage continuous wine fermentations.

Nitrogen supplementation, which is widely used in winemaking to improve fermentation kinetics, also affects the products of fermentation, including volatile compounds. However, the mechanisms underlying the metabolic response of yeast to nitrogen additions remain unclear. We studied the consequences for Saccharomyces cerevisiae metabolism of valine and ammonium pulses during the stationary phase of four-stage continuous fermentation (FSCF). This culture technique provides cells at steady state similar to that of the stationary phase of batch wine fermentation. Thus, the FSCF device is an appropriate and reliable tool for individual analysis of the metabolic rerouting associated with nutrient additions, in isolation from the continuous evolution of the environment in batch processes. Nitrogen additions, irrespective of the nitrogen-containing compound added, substantially modified the formation of fermentation metabolites, including glycerol, succinate, isoamyl alcohol, propanol, and ethyl esters. This flux redistribution, fulfilling the requirements for precursors of amino acids, was consistent with increased protein synthesis resulting from increased nitrogen availability. Valine pulses, less efficient than ammonium addition in increasing the fermentation rate, were followed by a massive conversion of this amino acid in isobutanol and isobutyl acetate through the Ehrlich pathway. However, additional routes were involved in valine assimilation when added in stationary phase. Overall, we found that particular metabolic changes may be triggered according to the nature of the amino acid supplied, in addition to the common response. Both these shared and specific modifications should be considered when designing strategies to modulate the production of volatile compounds, a current challenge for winemakers.

Numerical study of conjugate heat transfer of cryogenic methane in rectangular engine cooling channels at supercritical pressures

Three-dimensional conjugate heat transfer of cryogenic methane in rectangular engine cooling channels at supercritical pressures with asymmetric heating imposed on the top channel surface is numerically investigated, focusing mainly on effects of the thermal conductivity of the solid channel material and the geometric aspect ratio of the channel on fluid flow and heat transfer. Results indicate that variations of the thermal conductivity in the solid fin produce significant impact on conjugate heat transfer owing to heat flux redistribution in solid region and convective heat transfer variation in fluid phase. The latter factor is dictated by strong variations of fluid thermophysical properties at a supercritical pressure. At a constant inlet mass flow rate, variations of the geometric aspect ratio of the channel exert strong influence on both heat transfer and pressure loss due mainly to fluid velocity variations. Results indicate that convective heat transfer plays a more dominating role than thermal conduction in the solid fin. A thermal performance factor can be used for the combined evaluation of heat transfer and pressure loss. The modified Jackson & Hall empirical heat transfer expression is tested to be applicable for heat transfer prediction of cryogenic methane at supercritical pressures.

Establishment, in silico analysis, and experimental verification of a large-scale metabolic network of the xanthan producing Xanthomonas campestris pv. campestris strain B100

The γ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) B100 synthesizes the polysaccharide xanthan, a commercially important viscosifier. Since the complete genome of Xcc B100 is available, systems biology tools were applied to obtain a deeper understanding of the metabolism involved in xanthan biosynthesis. A large-scale metabolic network was reconstructed and manually curated. The reconstructed network included 352 genes, 437 biochemical reactions, 10 transport reactions, and 338 internal metabolites. To use this network for flux balance analysis, the biomass composition of Xcc B100 was determined. The comprehensive model obtained was applied for in silico analyses to predict biomass generation and gene essentiality. Predictions were extensively validated by analyzing batch culture performance and by carbon balancing including xanthan production. Single gene deletion mutants causing deficiencies in the central carbohydrate metabolism were constructed to enforce major flux redistributions. The impact of xanthan production was studied in vivo and in silico, comparing the physiology of a gumD mutant, negative in xanthan production, with the original strain. The results indicate a redistribution of resources from xanthan to biomass, rather than a reduction in carbon uptake. With this high quality metabolic model, both systems biology analyses and synthetic biology reengineering of Xcc gained an important tool.

Enhanced production of l-tryptophan with glucose feeding and surfactant addition and related metabolic flux redistribution in the recombinant Escherichia coli

The production of l-tryptophan was investigated in a recombinant strain Escherichia coli W3110-ZDrr. It was observed that phosphate and feeding strategies are key factors to ensure the good cell growth and high production of l-tryptophan. The simple exponential feeding strategy could only produce 10.6 g/L l-tryptophan due to the improper feeding rate; while the manual glucose-feedback feeding approach could effectively control the substrate and inhibit the formation of acetate, and thus improvemed the l-tryptophan production to 25.5 g/L. The modified exponential feeding approach avoided overfeeding or underfeeding and achieved high production of l-tryptophan. Moreover, the addition of Tween 60 or PL61 could enhance the cell growth and the production of l-tryptophan in the fed-batch mode. Subsequent metabolic flux analysis showed that more carbon flux was distributed into the biosynthesis of l-tryptophan when Tween 60 or PL61 was supplied. The present work presents one base for further large-scale production of this important amino acid.

Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae

BackgroundAcetoin is an important bio-based platform chemical. However, it is usually existed as a minor byproduct of 2,3-butanediol fermentation in bacteria.ResultsThe present study reports introducing an exogenous NAD+ regeneration sysytem into a 2,3-butanediol producing strain Klebsiella pneumoniae to increse the accumulation of acetoin. Batch fermentation suggested that heterologous expression of the NADH oxidase in K. pneumoniae resulted in large decreases in the intracellular NADH concentration (1.4 fold) and NADH/NAD+ ratio (2.0 fold). Metabolic flux analysis revealed that fluxes to acetoin and acetic acid were enhanced, whereas, production of lactic acid and ethanol were decreased, with the accumualation of 2,3-butanediol nearly unaltered. By fed-batch culture of the recombinant, the highest reported acetoin production level (25.9 g/L) by Klebsiella species was obtained.ConclusionsThe present study indicates that microbial production of acetoin could be improved by decreasing the intracellular NADH/NAD+ ratio in K. pneumoniae. It demonstrated that the cofactor engineering method, which is by manipulating the level of intracellular cofactors to redirect cellular metabolism, could be employed to achieve a high efficiency of producing the NAD+-dependent microbial metabolite.

Equirate magnetron sputtering of mosaic copper-graphite targets

Based on experimental data on sputtering of copper-graphite cathodes, we propose a mechanism of equirate sputtering of elements of mosaic targets prepared from materials the sputtering yields of which differ by almost an order of magnitude. The mechanism takes into account the occurrence of a height discontinuity between the mosaic elements at the initial stage of sputtering, the redistribution of ion fluxes between the elements, and the effects related to the mass transfer in plasma and the implantation of sputtered atoms into the mosaic elements of the different variety.

Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress

The ability of a microorganism to adapt to changes in the environment, such as in nutrient or oxygen availability, is essential for its competitive fitness and survival. The cellular objective and the strategy of the metabolic response to an extreme environment are therefore of tremendous interest and, thus, have been increasingly explored. However, the cellular objective of the complex regulatory structure of the metabolic changes has not yet been fully elucidated and more details regarding the quantitative behaviour of the metabolic flux redistribution are required to understand the systems-wide biological significance of this response. In this study, the intracellular metabolic flux ratios involved in the central carbon metabolism were determined by fractional (13)C-labeling and metabolic flux ratio analysis (MetaFoR) of the wild-type E. coli strain JM101 at an oxidative environment in a chemostat. We observed a significant increase in the flux through phosphoenolpyruvate carboxykinase (PEPCK), phosphoenolpyruvate carboxylase (PEPC), malic enzyme (MEZ) and serine hydroxymethyltransferase (SHMT). We applied an ε-constraint based multi-objective optimization to investigate the trade-off relationships between the biomass yield and the generation of reductive power using the in silico iJR904 genome-scale model of E. coli K-12. The theoretical metabolic redistribution supports that the trans-hydrogenase pathway should not play a direct role in the defence mounted by E. coli against oxidative stress. The agreement between the measured ratio and the theoretical redistribution established the significance of NADPH synthesis as the goal of the metabolic reprogramming that occurs in response to oxidative stress. Our work presents a framework that combines metabolic flux ratio analysis and multi-objective optimization to investigate the metabolic trade-offs that occur under varied environmental conditions. Our results led to the proposal that the metabolic response of E. coli to paraquat-induced oxidative stress is globally conserved and coordinated.

The main byproducts and metabolic flux profiling of γ-PGA-producing strain B. subtilis ZJU-7 under different pH values

Poly-γ-glutamic acid (γ-PGA) is an extracellular anionic polymer with many potential applications. Although microbial fermentation is the common approach to produce γ-PGA, the broth at the latter stage usually becomes very viscous and severely influences the metabolites producing pattern of target microbe. In this study, acetoin and 2,3-butanediol were confirmed to be the main byproducts of γ-PGA-producing strain B. subtilis ZJU-7 (B. subtilis CGMCC1250), and their effects on the cell growth and γ-PGA biosynthesis were further investigated in shake flasks. The outcome indicated that both acetoin and 2,3-butanediol showed clear impairment on γ-PGA production of B. subtilis ZJU-7. Moreover, the extracellular metabolites profiles of fermentation under three different pH values were acquired and the metabolic flux redistribution of pathways related to γ-PGA biosynthesis was calculated based on the collected data. As a result, the metabolic flux favored to distribute toward glycolytic pathway at pH 6.5, in which the ingestion rate of extracellular glutamic acid was higher and the subsequent γ-PGA biosynthesis was enhanced. The present work provided us a deep insight into the metabolic flux control of γ-PGA biosynthesis, which will stimulate some novel metabolic engineering strategies to improve the productivity of γ-PGA in future.

Comparison of metabolic pathway for hydrogen production in wild-type and mutant Clostridium tyrobutyricum strain based on metabolic flux analysis

There has been a great interest in fermentative hydrogen production during recent decades. However, the low H2 yield associated with fermentative hydrogen production process continues to hinder its industrial application. It is delectable that a maximum 3.9 mol H2 per mol glucose was obtained in fed-batch fermentation mode with a butyric acid over-producing Clostridium tyrobutyricum mutant, which to our knowledge is the highest H2 yield ever got in the fermentation process with Clostridium sp. This study aimed to better understand the change of flux profile within the whole metabolic network and to conduct the metabolic flux analysis of fermentative hydrogen production. For the first time, we constructed a metabolic flux model for the anaerobic glucose metabolism of C. tyrobutyricum ATCC 25755, and revealed the internal mechanism responsible for the redistribution of the carbon flux in the mutant strain in comparison with the wide-type. The MFA methodology was used to study the fractional flux response to variations in operational pH, and revealed that pH was a significant operational parameter effecting on the fermentative hydrogen production process. Furthermore, the presence of NADH-ferredoxin oxidoreductase activity in this anaerobe was demonstrated. By measuring the activities of related enzymes in the biosynthesis pathway of hydrogen, we thus concluded that the increased specific activities of both NFOR and hydrogen-catalyzing enzyme (hydrogenase) would be attributed to the hydrogen over-producing.

Copper-Capped Carbon Nanocones on Silicon: Plasma-Enabled Growth Control

Controlled self-organized growth of vertically aligned carbon nanocone arrays in a radio frequency inductively coupled plasma-based process is studied. The experiments have demonstrated that the gaps between the nanocones, density of the nanocone array, and the shape of the nanocones can be effectively controlled by the process parameters such as gas composition (hydrogen content) and electrical bias applied to the substrate. Optical measurements have demonstrated lower reflectance of the nanocone array as compared with a bare Si wafer, thus evidencing their potential for the use in optical devices. The nanocone formation mechanism is explained in terms of redistribution of surface and volumetric fluxes of plasma-generated species in a developing nanocone array and passivation of carbon in narrow gaps where the access of plasma ions is hindered. Extensive numerical simulations were used to support the proposed growth mechanism.

Ultraviolet variability of the helium-peculiar staraCentauri

The spectrophotometric variability of the classical magnetic chemically peculiar star a Cen in the ultraviolet spectral region from 1150 to 3100 A is investigated. This study is based on archival International Ultraviolet Explorer data obtained at various phases of the rotational cycle. The light variations in wavelengths shorter than λ1616 A are mainly in antiphase to the light variations in the longer-wavelength region. The brightness of the star in the spectral region λλ1616–1640 A is constant over the period of rotation, which means that a so-called ‘null wavelength region’ exists over these wavelengths. There is also a second ‘null wavelength region’ in the spectrum of a Cen. According to our investigation, the semi-amplitude of the light variations is practically zero over the period of rotation in the spectral region λλ1244–1343 A. The exception is the light variation in the depressions at λλ1265 and 1300 A, which are formed by resonance lines of silicon. However, the fluxes are constant at the cores of some features and in small depressions. An explanation for the light variations of a Cen is flux redistribution through bound–free and bound–bound silicon transitions combined with an inhomogeneous surface distribution of silicon on the stellar surface. The influence of other elements on the flux redistribution is discussed.

Flux Analysis of the Trypanosoma brucei Glycolysis Based on a Multiobjective-Criteria Bioinformatic Approach.

Trypanosoma brucei is a protozoan parasite of major of interest in discovering new genes for drug targets. This parasite alternates its life cycle between the mammal host(s) (bloodstream form) and the insect vector (procyclic form), with two divergent glucose metabolism amenable to in vitro culture. While the metabolic network of the bloodstream forms has been well characterized, the flux distribution between the different branches of the glucose metabolic network in the procyclic form has not been addressed so far. We present a computational analysis (called Metaboflux) that exploits the metabolic topology of the procyclic form, and allows the incorporation of multipurpose experimental data to increase the biological relevance of the model. The alternatives resulting from the structural complexity of networks are formulated as an optimization problem solved by a metaheuristic where experimental data are modeled in a multiobjective function. Our results show that the current metabolic model is in agreement with experimental data and confirms the observed high metabolic flexibility of glucose metabolism. In addition, Metaboflux offers a rational explanation for the high flexibility in the ratio between final products from glucose metabolism, thsat is, flux redistribution through the malic enzyme steps.

Dietary Pulse Consumption and Arterial Damage in Type 2 Diabetes

s / Can J Diabetes 36 (2012) S2eS22 S10 tissues insulin resistance and inflammation. In the present study, we investigated the effect of 4-phenylbutyrate (4-PBA), a chemical chaperon that prevents ER stress, on the regulation of tissues’ nonesterified fatty acids metabolism and diet-induced insulin resistance. Research Design and Methods: Control (chow fed) vs. diabetic (high-fat/high-fructose fed + small dose streptozotocin-treated e HFHFS) male Wistar rats were treated with 4-PBA (1g/kg/day) by oral gavage for 6 weeks. At the end of the treatment period, [8, 9-3H]-palmitate was infused during an euglycemic ehyperinsulinemic clamp followed by an i.v. bolus of [1-14C]-2bromopalmitate at steady-state to determine oxidative and non-oxidative nonesterified fatty acid (NEFA) metabolism in tissues. Results: HFHFS-fed rats displayed insulin resistance with high levels of fasting glucose and insulin. Increased NEFA fractional uptake by the gastrocnemius and reduced myocardial NEFA nonoxidative disposal was observed. Animals treated with 4-PBA displayed greater plasmatic NEFA rate of appearance and clearance. 4-PBA normalized gastrocnemius fractional uptake and increased NEFA oxidation and fractional uptake by the liver. SCAT of treated rats are characterized larger SC adipocytes with greater fractional and net NEFA uptake. In vitro, 4-PBA inhibits differentiation of adipocytes precursors but stimulates fatty acids uptake by mature adipocytes. Conclusions: Redistribution of NEFA fluxes by 4-PBA treatment does not restore insulin sensitivity in a nutritional model of T2D but reduced fasting glucose.

Impairment of NADH dehydrogenase for increased hydrogen production and its effect on metabolic flux redistribution in wild strain and mutants of Enterobacter aerogenes

An NADH dehydrogenase encoded by the nuo cluster was isolated and impaired by knocking out the nuoB gene in Enterobacter aerogenes to examine its effect on hydrogen production. Three nuoB-deleted mutant strains were constructed from the wild-type strain E. aerogenes IAM1183 and two recombinant strains, IAM1183-A (ΔhycA) and IAM1183-O (ΔhybO), from which the hycA and hybO genes had already been deleted previously, respectively. Compared with the performance of the wild-type strain, the overall hydrogen production of the mutants IAM1183-B (ΔnuoB), IAM1183-AB (ΔhycA/ΔnuoB) and IAM1183-BO (ΔhybO/ΔnuoB) was increased by 49.2%, 54.0%, and 52.4% in batch culture, respectively. The hydrogen yields from glucose by the three mutants IAM1183-B, IAM1183-AB, IAM1183-BO were 1.38, 1.49, and 1.39 mol H2/mol glucose, respectively, while it was 1.16 mol H2/mol glucose in the wild-type strain. Metabolic flux analysis indicated that all three mutants exhibited reduced fluxes to lactate production, and enhanced fluxes toward the generation of hydrogen, acetate, ethanol, succinate and 2,3-butanediol. Both the formate pathway and the NADH pathway contributed to increased hydrogen production in the mutant strains. The assay of 4 NADH-mediated enzyme activities (H2ase, LDH, ADH and BDDH) was in accordance with the redistributions of the metabolic fluxes in the mutant strains.