Effect of oxygenation and sulfate concentrations on pyruvate and lactate formation inKlebsiella oxytoca ZS growing in chemostat cultures

Klebsiella oxytoca ZS fermented glucose to ethanol and lactic, formic, and acetic acids, but, in contrast to many strains, accumulates pyruvic and acetic acids as the principal end products in aerobic growth conditions. This strain was grown in sulfate-limited chemostat at a fixed low dilution rate (D=0.033 h^–1) with glucose present in excess. When oxygen was supplied at a high level, pyruvate and acetate were produced, and the ratio NADH/NAD⁺ was low (0.04) while the internal pyruvate concentration increased to 100 mol (g dry wt)^–1. A shortage of oxygen supply was accompanied by lactate production, an increase of the ratio NADH/NAD⁺ (0.53), and an undetectable level in internal pyruvate concentration. The observed changes in LDH activity found in vitro in extracts of the cells are not strictly related to those found in vivo. In fact, the specific activity of LDH was essentially stable at 30% of dissolved oxygen tension (d.o.t.) and decreased slightly at 60% of d.o.t., whereas specific lactic acid production decreased rapidly. The in vitro LDH activity was strongly affected by the NADH/NAD⁺ ratio.     

Production of cyclodextrin glycosyltransferase by alkaliphilic <Emphasis Type="Italic">Bacillus circulans</Emphasis> in submerged and solid-state cultivation

Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an industrially important enzyme, which is used to produce cyclodextrins (CDs). In this research, we report the use of experimental factorial design to find the best conditions of pH and temperature for CGTase production by Bacillus circulans var. alkalophilus. The optimized calculated values for the tested variables were, respectively, pH 9.7 and temperature 36^oC, with a CGTase activity of 615 U mL⁻¹. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC) and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on SC was 1,155 U mL⁻¹ under aerobic conditions. Cell growth and CGTase synthesis in SSC using SIFR as substrate was excellent, with CGTase activity of 32,776 U g_(SIFR) ⁻¹. These results strongly support the use of SIFR for CGTase production since it is a non-expensive residue.     

Elimination of glycerol and replacement with alternative products in ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is a major by-product of ethanol fermentation by Saccharomyces cerevisiae and typically 2–3% of the sugar fermented is converted to glycerol. Replacing the NAD⁺-regenerating glycerol pathway in S. cerevisiae with alternative NADH reoxidation pathways may be useful to produce metabolites of biotechnological relevance. Under fermentative conditions yeast reoxidizes excess NADH through glycerol production which involves NADH-dependent glycerol-3-phosphate dehydrogenases (Gpd1p and Gpd2p). Deletion of these two genes limits fermentative activity under anaerobic conditions due to accumulation of NADH. We investigated the possibility of converting this excess NADH to NAD⁺ by transforming a double mutant (gpd1∆gpd2∆) with alternative oxidoreductase genes that might restore the redox balance and produce either sorbitol or propane-1,2-diol. All of the modifications improved fermentative ability and/or growth of the double mutant strain in a self-generated anaerobic high sugar medium. However, these strain properties were not restored to the level of the parental wild-type strain. The results indicate an apparent partial NAD⁺ regeneration ability and formation of significant amounts of the commodity chemicals like sorbitol or propane-1,2-diol. The ethanol yields were maintained between 46 and 48% of the sugar mixture. Other factors apart from the maintenance of the redox balance appeared to influence the growth and production of the alternative products by the genetically manipulated strains.     

Effects of overexpression of NAPRTase,NAMNAT, and NAD synthetase in the NAD(H) biosynthetic pathways on the NAD(H) pool,NADH/NAD+ ratio,and succinic acid production with different carbon sources by metabolically engineered Escherichia coli

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose anaerobically due to the inability to regenerate NAD⁺. To regulate NAD(H) pool size and NADH/NAD⁺ ratio, overexpression of the enzymes in the NAD(H) biosynthetic pathways in BA002 was investigated. The results clearly demonstrate that the increased NAD(H) pool size and the decreased NADH/NAD⁺ ratio improved the glucose consumption and cell growth, which improved succinic acid production. When the pncB and the nadD genes were co-overexpressed in CA102, the ratio of NADH/NAD⁺ was decreased from 0.60 to 0.12, and the concentration of NAD(H) was the highest among that of all the strains. Moreover, the dry cell weight (DCW), glucose consumption, and the concentration of succinic acid in CA102 were also the highest. Based on the sufficient NAD⁺ supply after gene modification in the NAD(H) biosynthetic pathways, reductive carbon sources with different amounts of NADH can further change the distribution of metabolites. When sorbitol was used as a carbon source in CA102, the byproducts were lower than those of glucose fermentation, and the yield of succinic acid was increased.     

Enhanced production of succinic acid by Actinobacillus succinogenes with reductive carbon source

Carbon sources with different oxidation states were used to investigate the possibility increasing the availability of NADH and the NADH/NAD⁺ ratio and to determine the effect of this manipulation on the distribution of metabolites in Actinobacillus succinogenes NJ113. The sugars glucose, sorbitol and gluconate were each used at an initial concentration of 40 g/L.The yield of succinic acid (0.75) and the ratio of succinic acid to acetic acid (5.06) were both higher for sorbitol than the values obtained with glucose (0.66 and 2.68, respectively). In contrast, with gluconate as the carbon source the yield of succinic acid was 0.54 and the ratio of succinic acid to acetic acid was only 1.70. This work showed that different levels of NADH availability and the NADH/NAD⁺ ratio can be achieved by using carbon sources that have different oxidation states.Highly reduced sorbitol was examined as a possible carbon substrate for maximizing the redox potential during the production of succinic acid.     

Carbohydrate metabolism in Thermoproteus tenax: in vivo utilization of the non-phosphorylative Entner-Doudoroff pathway and characterization of its first enzyme, glucose dehydrogenase

Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first enzyme of the ED pathway, glucose dehydrogenase, was characterized. The distribution of the ¹³C label in alanine synthesized by cells grown with [1-¹³C]glucose indicated that in vivo the EMP pathway and the modified ED pathway operate parallel, with glucose metabolization via the EMP pathway being prominent. To initiate studies on the regulatory mechanisms governing carbon flux via these pathways, the first enzyme of the ED pathway, glucose dehydrogenase, was purified to homogeneity and its phenotypic properties were characterized. The pyridine-nucleotide-dependent enzyme used both NAD⁺ and NADP⁺ as cosubstrates, showing a 100-fold higher affinity for NADP⁺. Besides glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the enzyme is the oxidation of glucose by NADP⁺. A striking feature was the influence of NADP⁺ and NAD⁺ on the quaternary structure and activity state of the enzyme. Without cosubstrate, the enzyme was highly aggregated (mol. mass > 600 kDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically active, homomeric dimers with a mol. mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarity to the respective partial sequences of alcohol dehydrogenases and sorbitol dehydrogenases, but no resemblance to the known pyridine-nucleotide-dependent archaeal and bacterial glucose dehydrogenases. Received: 25 October 1996 / Accepted: 15 April 1997     

The Increase in the Number of Accessible SH-Groups in the Enterococcal Membrane Vesicles by ATP and Nicotinamide Adenine Dinucleotides

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD⁺+NADH) to the vesicles caused a ∼4-fold or ∼1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD⁺+NADH both were added. The change was lacking in the presence of the F₀F₁-ATPase inhibitors N,N′-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F₀F₁-ATPase and, in addition, it was less in potassium ion–free medium. These results are correlated with data about K⁺-dependent F₀F₁-ATPase activity, suggesting a relationship between the F₀F₁-ATPase and K⁺ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.     

Effect of culture conditions on growth and esterase production by the moderate thermophile Bacillus circulans MAS2

Growth and esterase production (activity on p-nitrophenyl caprylate) by the newly isolated Bacillus circulans MAS2 bacterial strain were studied. The growth rate at 50°C was high (0.9 h^-1) on LB medium with glucose added. Esterase production followed growth with the majority of activity being intracellular during exponential growth phase. During stationary phase, the esterase activity was released in the culture medium. The strain was able to grow at 35– 55°C with maximum growth rate at 50°C, showing a pattern typical of a moderate thermophile. Growth occurred at pH 6–9 with a maximum at 8, with a similar pattern for the esterase production. Addition of glucose, fructose, sucrose or sodium acetate greatly promoted both growth and esterase production while starch, inulin, tributyrin or glycerol showed no effect. Complex nitrogen sources such as tryptone or yeast extract increased growth and esterase production while mineral sources (ammonium chloride or sulfate), glycine or glutamate showed no effect. An increase of tryptone plus yeast extract and glucose concentrations stimulated growth and esterase production which reached 160 U L⁻¹. Received 17 March 1999/ Accepted in revised form 25 June 1999     

Mitochondrial function in the yeast form of the pathogenic fungus <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I–V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH–ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD⁺-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast’s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.     

Extrusion of metabolites from baker’s yeast during glucose-induced acidification

Extrusion of metabolites (glycerol, lactic, malic, and succinic acid) during the medium acidification caused by resting baker’s yeast supplied with 200mm glucose was studied under aerobic and anaerobic conditions and in the absence and presence of 14mm KCl. The maximum levels of glycerol and of the sum of acids (about 13 and 8mm, respectively) were attained anaerobically; aerobiosis reduced the levels by 40–50 % and the presence of K⁺ ions by another 10–20 %. The time courses of glucose consumption and medium acidification were similar aerobically and anaerobically. The glucose consumption ourves exhibited a short plateau about 2 min after glucose addition, caused probably by a rapid osmotic equilibration of glucose across the cell mambrane. Metabolite extrusion indicates that at high glucose concentrations the alcohol dehydrogenase reaction is supplemented by other reactions aiding in the maintenance of a balanced NAD⁺/NADH ratio in the cells.     

Biochemical controls of citrate synthase in chickpea bacteroids

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The K _m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (K _is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K _is = 56 ± 3.8 μM and K _ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD⁺ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD⁺ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle. Received: 14 July 1999 / Accepted: 20 September 1999     

Production of nadh by microbial reduction of added nad

Summary Of thirteen bacterial strains and four strains of yeast-like organisms, permeabilized cells of two bacterial and one yeast strain effectively converted added NAD⁺ into NADH in the presence of glucose as substrate.Arthrobacter ureafaciens reduced more than 90% of 10 mM NAD⁺ into NADH during 1h.     

Glucose repression of anthracycline formation in Streptomyces peucetius var. caesius

The effect of glucose on growth and anthracycline production by Streptomyces peucetius var. caesius was examined in a chemically defined medium. Glucose concentrations above 100 mM inhibited anthracycline synthesis in the original strain without causing significant change in growth and final pH values. This effect was observed when the carbohydrate was added initially or after 24 h fermentation, but not when added during the stationary growth phase. When the microorganism was pregrown in 100 mM glucose and then transferred to a resting cell system with 444 mM glucose, no significant differences in antibiotic production were observed compared to the control without glucose. The negative effect of glucose on antibiotic synthesis was not observed in a mutant (2-dog^R–21) resistant to growth inhibition by 2-deoxyglucose. Glucose consumption by this mutant was approximately 30% of that utilized by the original strain. Compared to the original strain, the mutant 2-dog^R–21 exhibited a reduction of 50% in glucose transport and an 85% decrease in glucose kinase activity. The experimental evidence obtained suggests that glucose represses anthracycline formation in a transitory manner and that this effect is related to glucose transport and phosphorylation. Received: 15 January 1999 / Received revision: 7 April 1999 / Accepted: 1 May 1999     

Metabolism of acetaldehyde and Custers effect in the yeast

Brettanomyces abstinens growing on different initial glucose concentrations showed an anaerobic inhibition of fermentation. This Custers effect decreased as the initial glucose concentration in the medium increased. Two aldehyde dehydrogenases, one NAD⁺-linked and the other NADP⁺-linked were observed. The results suggest that the NAD⁺-linked enzyme is involved in the production of acetic acid and is repressed by glucose. The NADP⁺-linked enzyme seems to be a constitutive enzyme. Acetyl-CoA synthetase activity also was not greatly affected by the growth conditions.The results support the earlier hypothesis that the Custers effect in Brettanomyces is provoked by the reduction of NAD⁺ in the conversion of acetaldehyde to acetic acid.     

Regulation of glycerol metabolism in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> NBRC12010 under electrochemical conditions

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD⁺/NADH. At a NAD⁺/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD⁺/NADH. Exogenous NADH was oxidized to NAD⁺ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.     

The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In previous studies, we showed that cofactor manipulations can potentially be used as a tool in metabolic engineering. In this study, sugars similar to glucose, that can feed into glycolysis and pyruvate production, but with different oxidation states, were used as substrates. This provided a simple way of testing the effect of manipulating the NADH/NAD+ ratio or the availability of NADH on the metabolic patterns of Escherichia coli under anaerobic conditions and on the production of 1,2-propanediol (1,2-PD), which requires NADH for its synthesis. Production of 1,2-PD was achieved by overexpressing the two enzymes methylglyoxal synthase from Clostridium acetobutylicum and glycerol dehydrogenase from E. coli. In addition, the effect of eliminating a pathway competing for NADH by using a ldh ^– strain (without lactate dehydrogenase activity) on the production of 1,2-PD was investigated. The oxidation state of the carbon source significantly affected the yield of metabolites, such as ethanol, acetate and lactate. However, feeding a more reduced carbon source did not increase the yield of 1,2-PD. The production of 1,2-PD with glucose as the carbon source was improved by the incorporation of a ldh ^– mutation. The results of these experiments indicate that our current 1,2-PD production system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal. Electronic Publication     

Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae

Anaerobic fermentation was relatively difficult to optimize due to lack of monitoring parameters. In this paper, a new method was reported using extracellular oxidoreduction potential (ORP) to monitor 1,3-propanediol (1,3-PD) biosynthesis process by Klebsiella pneumoniae. In batch fermentation, cell growth, 1,3-propanediol production and by-products distribution were studied at four different ORP levels: 10, −140, −190 and −240 mV. From the results, the ORP level of −190 mV was preferable, which resulted in fast cell growth and high 1,3-propanediol concentration. The NAD⁺/NADH ratio was determined at different ORP levels, and a critical NAD⁺/NADH ratio of 4 was defined to divide fermentation environments into two categories: relatively oxidative environment (NAD⁺/NADH>4) and relatively reductive environment (NAD⁺/NADH<4). The former was correlative with high 1,3-propanediol productivity and high specific growth rate. The mechanism of ORP regulation was discussed. It is suggested that ORP regulation of fermentation might be due to its influence on the ratio of NAD⁺/NADH, which determined metabolic flux. Furthermore, a batch fermentation of modulating ORP following a profile in different levels corresponding to different fermentation stage was tested. The 1,3-PD concentration was 22.3% higher than that of constant ORP fermentation at −190 mV. Therefore, ORP is a valuable parameter to monitor and control anaerobic fermentation production.     

Effect of alternative NAD<Superscript>+</Superscript>-regenerating pathways on the formation of primary and secondary aroma compounds in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> glycerol-defective mutant

Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD⁺-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD⁺ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD⁺/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.