Resonant Raman quantification of zeaxanthin production from Flavobacterium multivorum

Resonant Raman scattering was used as a novel, rapid, non-destructive optical technique to measure zeaxanthin levels in Flavobacterium multivorum ATCC 55238. Culture broth, after bacterial growth for 40 h, exhibited characteristic resonance Raman vibrational modes at 1159 cm^–1 (C-C stretch) and 1525 cm^–1 (C=C stretch) upon excitation at 488 nm. A striking correlation was observed between the carotenoid level as estimated by HPLC and by resonance Raman spectroscopy.     

Utilization of the tricarboxylic acid cycle, a reactor design criterion for the microaerobic production of 2,3-butanediol

A new parameter, the relative utilization of tricarboxylic acid (TCA) cycle beta, is introduced to quantitatively account for the involvement of fermentation pathways and TCA cycle in the utilization of oxygen under oxygen-limiting (microaerobic) conditions. With the facultative anaerobe Enterobacter aerogenes, which produces 2,3-butanediol, a method is proposed to calculate beta from measurement of metabolites and exhaust gas. In continuous culture beta was found to be small under oxygen limitation, indicating that the fermentation pathways were preferred over the TCA cycle and oxygen was almost entirely consumed through oxidation of reduced nicotinamide adenine dinucleotide (NADH(2)) released by fermentation under these conditions. The increase of beta at high oxygen supply revealed a saturation of oxygen utilization through fermentation pathways. It could be concluded that, for the optimal performance of a microaerobic culture, oxygen uptake rate must be kept at such a level that as much NADH(2) as possible from fermentation pathways is oxidized by oxygen, and at the same time the utilization of TCA cycle is kept at a minimum. As the dynamics of the microaerobic culture can be fast, a significant effect of reactor hydrodynamics, i.e., mixing, on the overall performance can be expected. This was confirmed experimentally, and the parameter beta proved to be a useful reactor design criterium for the microaerobic cultivation. (c) 1992 John Wiley & Sons, Inc.     

Utilization of the tricarboxylic acid cycle intermediates and symbiotic effectiveness inRhizobium meliloti

Summary The utilization of the tricarboxylic acid cycle intermediates and related compounds was studied in strains ofRhizobium meliloti having different symbiotic effectiveness. In general, the very effective (VE) strains used these compounds as sole carbon source better than the ineffective (I) strains. However, a significant different was observed between VE and I strains in their ability to use acetate or oxaloacetate for growth. In fact, at a concentration of 2 mM, 80% of the VE strains used acetate or oxaloacetate white 50% of the I strains used acetate and none was able to grow on oxaloacetate. No correlation was found between the symbiotic effectiveness of the strains and their ATP content, when grown on mannitol. The highest ATP content (9.21 nM×g protein^–1) was found in the I strain S₂₀ and the lowest (0.69 nM×g protein^–1 was found in the effective strain S₈. Numerical analysis of the patterns of utilization of the TCA cycle intermediates and related compounds indicated that the 49 strains tested formed 11 distinct groups at 86% similarity, according to Jaccard's coefficient. Several strains showed unique patterns of utilization and can be clearly identified under laboratory conditions.Contribution no.225 Station de Recherches, Agriculture Canada.     

An optimization study of carotenoid production by Rhodotorula glutinis DBVPG 3853 from substrates containing concentrated rectified grape must as the sole carbohydrate source

A multivariate statistical approach was employed for the optimization of conditions for carotenoid production by Rhodotorula glutinis DBVPG 3853 from a substrate containing concentrated rectified grape must as the sole carbohydrate source. Several experimental parameters (carbohydrate, yeast autolysate and salt concentrations, and pH) were tested at two levels by following a fractional factorial design. Carotenogenesis was most sensitive to both initial pH and yeast autolysate concentration. A Central Composite Design experiment was then performed by obtaining both second-order polynomial models and isoresponse diagrams where initial pH and yeast autolysate concentration were considered as variables. In this way it was possible to determine the conditions (pH = 5.78, yeast autolysate = 4.67 g L⁻¹) which maximize both the concentration of total carotenoids and that of β-carotene (6.9 mg L⁻¹ and 1100 μg L⁻¹ of culture fluid, respectively, after 120 h of fermentation). Journal of Industrial Microbiology & Biotechnology (2000) 24, 41–45. Received 23 February 1999/ Accepted in revised form 14 September 1999     

Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.     

Systematic engineering of TCA cycle for optimal production of a four-carbon platform chemical 4-hydroxybutyric acid in Escherichia coli

To address climate change and environmental problems, it is becoming increasingly important to establish biorefineries for the production of chemicals from renewable non-food biomass. Here we report the development of Escherichia coli strains capable of overproducing a four-carbon platform chemical 4-hybroxybutyric acid (4-HB). Because 4-HB production is significantly affected by aeration level, genome-scale metabolic model-based engineering strategies were designed under aerobic and microaerobic conditions with emphasis on oxidative/reductive TCA branches and glyoxylate shunt. Several different metabolic engineering strategies were employed to develop strains suitable for fermentation both under aerobic and microaerobic conditions. It was found that microaerobic condition was more efficient than aerobic condition in achieving higher titer and productivity of 4-HB. The final engineered strain produced 103.4 g/L of 4-HB by microaerobic fed-batch fermentation using glycerol. The aeration-dependent optimization strategy of TCA cycle will be useful for developing microbial strains producing other reduced derivative chemicals of TCA cycle intermediates.     

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production

trans-4-Hydroxy- l -proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l -proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L ⁻¹·hr ⁻¹ in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.     

Factorial design for the optimization of enzymatic detection of cadmium in aqueous solution using immobilized urease from vegetable waste

Free as well as alginate immobilized urease was utilized for detection and quantitation of cadmium (Cd2+) in aqueous samples. Urease from the seeds of pumpkin (Cucumis melo), being a vegetable waste, was extracted and purified to apparent homogeneity (Sp. Activity 353 U/mg protein; A280/A260=1.12) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization and no leaching of the enzyme was found over a period of 15 days at 4 degrees C. Urease catalyzed urea hydrolysis by both soluble and immobilized enzyme revealed a clear dependence on the concentration of Cd2+. The inhibition caused by Cd2+ was non-competitive (Ki=1.41 x 10(-5) M). The time dependent inhibition both in the presence and in absence of Cd2+ ion revealed a biphasic inhibition in the activity. A Response Surface Methodology (RSM) for the parametric optimization of this process was performed using two-level-two-full factorial (2(2)), central composite design (CCD). The regression coefficient, regression equation and analysis of variance (ANOVA) was obtained using MINITAB 15 software. The predicted values thus obtained were closed to the experimental value indicating suitability of the model. In addition to this 3D response surface plot and isoresponse contour plot were helpful to predict the results by performing only limited set of experiments.     

Characterization of water-soluble products of palmitic acid beta-oxidation by a rat brain preparation

Abstract: (1) [1-¹⁴C]Palmitic acid was oxidized to CO₂ and a water-soluble material by a rat brain preparation. The radioactive CO₂ and water-soluble material were produced in a ratio of 1.0:1.3 when the mitochondrial fraction was used, and 1.0:10 or more with the postnuclear fraction. There was a lag period of 10 min for CO₂ production. These conversions were stimulated by carnitine and inhibited by cyanide. (2) Of the total radioactivity in the water-soluble material obtained with the mitochondrial fraction, 65% after 10 min of incubation and 80% thereafter were associated with amino acids, mostly with aspartate and glutamate. The remaining radioactivity, 35 and 20%, respectively, was associated with organic acids, 60–65% in citrate. The water-soluble material obtained with the postnuclear fraction contained an equal amount of radioactivity in organic and amino acids during the course of the experiment. In the organic acids, succinate was the highest labeled product during 10–40 min of incubation, whereas citrate was the highest labeled at the end of 60 min of incubation. After 60 min, the radioactivity in the amino acids was markedly associated with glutamate, and its radioactivity was 10 times greater with the postnuclear fraction than with the mitochondrial one. (3) An experiment with rat liver preparations was also camed out. The liver mitochondrial fraction showed an accumulation of radioactive organic acids within 10 min of incubation, which was followed by a linear production of ¹⁴CO₂. With the liver postnuclear fraction, the radioactivity was found mostly in the organic acids during the course of the experiment. In the liver system, the radioactive amino acids accounted for only 25% or less of the total radioactivity in the water-soluble material.     

Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii displays some unusual localisations of carbohydrate converting enzymes, which is due to the presence of a vestigial, non-photosynthetic plastid, referred to as the apicoplast. It was recently demonstrated that the single pyruvate dehydrogenase complex (PDH) in T. gondii is exclusively localised inside the apicoplast but absent in the mitochondrion. This raises the question about expression, localisation and function of enzymes for the tricarboxylic acid (TCA)-cycle, which normally depends on PDH generated acetyl-CoA. Based on the expression and localisation of epitope-tagged fusion proteins, we show that all analysed TCA cycle enzymes are localised in the mitochondrion, including both isoforms of malate dehydrogenase. The absence of a cytosolic malate dehydrogenase suggests that a typical malate-aspartate shuttle for transfer of reduction equivalents is missing in T. gondii. We also localised various enzymes which catalyse the irreversible steps in gluconeogenesis to a cellular compartment and examined mRNA expression levels for gluconeogenesis and TCA cycle genes between tachyzoites and in vitro bradyzoites. In order to get functional information on the TCA cycle for the parasite energy metabolism, we created a conditional knock-out mutant for the succinyl-CoA synthetase. Disruption of the sixth step in the TCA cycle should leave the biosynthetic parts of the cycle intact, but prevent FADH2 production. The succinyl-CoA synthetase depletion mutant displayed a 30% reduction in growth rate, which could be restored by supplementation with 2 microM succinate in the tissue culture medium. The mitochondrial membrane potential in these parasites was found to be unaltered. The lack of a more severe phenotype suggests that a functional TCA cycle is not essential for T. gondii replication and for maintenance of the mitochondrial membrane potential.     

Use of a structured kinetic model of antibody synthesis and secretion for optimization of antibody production systems: I. Steady-state analysis

Steady-state simulations using our previously developed structured kinetic model of antibody synthesis and secretion by hybridoma cells are used here in conjunction with factorial design analysis to identify intracellular parameters important in determining the specific antibody secretion rate and predict the dependence of this rate on cell specific growth rate. Simulation results suggest that the specific growth rate, the assembly rate of the heavy and light chains and the heavy- and -chain gene dosage can significantly affect the rate of antibody secretion. Based on these results, environmental and/or genetic manipulation approaches are proposed for maximizing the specific antibody secretion rate and the antibody volumetric productivity in large-scale antibody production systems.     

Enzymatic esterification of an acid with an epoxide using an immobilized lipase from Mucor miehei as catalyst: optimization of the yield and isomeric excess of ester by statistical analysis

We have developed the methodology for the esterification of an acid with an epoxide using 2-chlorobutyric acid and 1,2-epoxy-5-hexene catalysed by a Mucor miehei-immobilized lipase. Thus, this methodology could be applied to obtain 2-chloroesters. A factorial design of experiments and a central composite design have been used to optimise the synthesis of these esters. The variables chosen were temperature and initial catalyst concentration, while the responses were yield and isomeric excess of the ester. According to this study, temperature was the most important factor, having a positive influence on the yield and a small negative influence on the isomeric excess of the ester. The yield and isomeric excess of the ester show a greater dependence on temperature compared to the catalyst concentration. Although the effect of the catalyst concentration on both responses is smaller than the temperature effect, the higher selectivity presented by the biocatalyst towards the studied ester considerably decreased the final product distribution. Journal of Industrial Microbiology & Biotechnology (2002) 28, 173–179 DOI: 10.1038/sj/jim/7000218 Received 27 June 2001/ Accepted in revised form 25 October 2001     

Application of statistically-based experimental designs for the optimization of nisin production from whey

Statistically-based experimental designs were applied for the optimization of nisin production by Lactococcus lactis in a whey-based medium. Yeast extract, KH₂PO₄, and MgSO₄ were identified to have significant effects on nisin biosynthesis by a Plackett–Burman design. These three significant factors were subsequently optimized using central composite design, and the optimal conditions were determined to be 12.067 g l^–1 for yeast extract, 0.569 g l^–1 for KH₂PO₄, and 0.572 g l^–1 for MgSO₄. The validity of the optimal conditions was verified by a separate experiment.     

Evidence for functional and regulatory cross-talk between the tricarboxylic acid cycle 2-oxoglutarate dehydrogenase complex and 2-oxoadipate dehydrogenase on the l-lysine,l-hydroxylysine and l-tryptophan degradation pathways from studies in vitro

Herein are reported findings in vitro suggesting both functional and regulatory cross-talk between the human 2-oxoglutarate dehydrogenase complex (hOGDHc), a key regulatory enzyme within the tricarboxylic acid cycle (TCA cycle), and a novel 2-oxoadipate dehydrogenase complex (hOADHc) from the final degradation pathway of l-lysine, l-hydroxylysine and l-tryptophan. The following could be concluded from our studies by using hOGDHc and hOADHc assembled from their individually expressed components in vitro: (i) Different substrate preferences (k_cat/K_m) were displayed by the two complexes even though they share the same dihydrolipoyl succinyltransferase (hE2o) and dihydrolipoyl dehydrogenase (hE3) components; (ii) Different binding modes were in evidence for the binary hE1o-hE2o and hE1a-hE2o subcomplexes according to fluorescence titrations using site-specifically labeled hE2o-derived proteins; (iii) Similarly to hE1o, the hE1a also forms the ThDP-enamine radical from 2-oxoadipate (electron paramagnetic resonance detection) in the oxidative half reaction; (iv) Both complexes produced superoxide/H₂O₂ from O₂ in the reductive half reaction suggesting that hE1o, and hE1a (within their complexes) could both be sources of reactive oxygen species generation in mitochondria from 2-oxoglutarate and 2-oxoadipate, respectively; (v) Based on our findings, we speculate that hE2o can serve as a trans-glutarylase, in addition to being a trans-succinylase, a role suggested by others; (vi) The glutaryl-CoA produced by hOADHc inhibits hE1o, as does succinyl-CoA, suggesting a regulatory cross-talk between the two complexes on the different metabolic pathways.     

Metabolic engineering of Mannheimia succiniciproducens for succinic acid production based on elementary mode analysis with clustering

Mannheimia succiniciproducens, a capnophilic gram‐negative rumen bacterium, has been employed for the efficient production of succinic acid. Although M. succiniciproducens metabolism was previously studied using a genome‐scale metabolic model, more metabolic characteristics are to be understood. To this end, elementary mode analysis accompanied with clustering (‘EMC’ analysis) is used to gain further insights on metabolic characteristics of M. succiniciproducens allowing efficient succinic acid production. Elementary modes (EMs) generated from the central carbon metabolic network of M. succiniciproducens are clustered to systematically analyze succinic acid production routes. Based on the results of EMC analysis, zwf gene is identified as a novel overexpression target for the improved succinic acid production. This gene is overexpressed in a previously constructed succinic acid‐overproducing M. succiniciproducens LPK7 strain. Heterologous NADPH‐dependent mdh is later intuitively selected for overexpression to synergistically improve succinic acid production by utilizing abundant NADPH pool mediated by the overexpressed zwf. The LPK7 strains co‐expressing mdh alone and both zwf and mdh genes are subjected to fed‐batch fermentation to better examine their succinic acid production performances. Strategies of EMC analysis will be useful for further metabolic engineering of M. succiniciproducens and other microorganisms to improve production of succinic acid and other chemicals of interest.     

Cloning of two carotenoid ketolase genes from Nostoc punctiforme for the heterologous production of canthaxanthin and astaxanthin

For the heterologous synthesis of keto-carotenoids such as astaxanthin, two carotenoid ketolase genes crtW38 and crtW148, were cloned from the cyanobacterium, Nostoc punctiforme PCC 73102 and functionally characterized. Upon expression in Escherichia coli, both genes mediated the conversion of beta-carotene to canthaxanthin. However in a zeaxanthin-producing E. coli, only the gene product of crtW148 introduced 4-keto groups into the 3,3'-dihydroxy carotenoid zeaxanthin yielding astaxanthin. The gene product of crtW38 was unable to catalyze this reaction. Both ketolases differ in their interaction with a hydroxylase in the biosynthetic pathway from beta-carotene to astaxanthin.     

Molecular analysis of the nitrogen cycle in deep-sea microorganisms from the Nankai Trough: genes for nitrification and denitrification from deep-sea environmental DNA

Nitrification and denitrification are bacterial functions, which are important for the global nitrogen cycle. Thus, it is important to study the diversity and distribution of bacteria in the environment, which are involved in the nitrogen cycle on the earth. Ammonia monooxygenase encoded by the amoA gene and nitrite reductase encoded by nirK or nirS are essential enzymes for nitrificaton and denitrification, respectively. These genes can be used as markers for the identification of organisms in the nitrogen cycle. In this study, we identified amoA (42 clones) and nirS (98 clones) genes in parallel from samples recovered from the deep-sea of the Nankai Trough. Genes for nirK could not be amplified from these samples. The obtained amoA sequences were not so closely related to those of amoA genes from previously isolated environmental organisms and those of genes from environmental DNAs. On the other hand, the nirS genes sequenced showed some relationship to some extent with the latter genes. However, some of the newly sequenced genes formed clusters, which contained no previously identified genes on a phylogenetic tree. These are likely present in specific denitrifiers from the deep-sea. The results of this study further suggest that nitrifiers and denitrifiers live in the same area of the Nankai Trough and the nitrogen cycle exists even in the deep-sea.     

Application of statistically-based experimental designs in medium optimization for spore production of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> from distillery effluent

Spore production of Bacillus subtilis from distillery effluent was optimized using statistically-based experimental designs. The two-level Plackett–Burman design was applied to choose the nutrient supplements significantly influencing spore production. Among the seven variables we tested, the most significant variables influencing spore production were statistically elucidated for optimization, and included (NH₄)₂SO₄, corn flour and MgSO₄. The optimum concentration of each significant variable was then predicted using Box–Behnken design. A second-order polynomial was determined by the multiple regression analysis of this experimental data. The optimum values for the critical nutrient supplements for the maximum were obtained as followed: (NH₄)₂SO₄, 4.54%; corn flour, 1.2%; MgSO₄, 0.56% with the corresponding value of maximum spore production of 7.24 × 10⁸ spores/ml. A verification experiment performed under the optimum conditions resulted in 6.95 × 10⁸ spores/ml. The determination coefficient (R ²) was 0.98, which ensure an adequate credibility of the model.     

Mixture design as a first step for optimization of fermentation medium for cutinase production from <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

Cutinase enzymes from fungi have found diverse applications in industry. However, most of the available literature on cutinase production is related to the cultivation of genetically engineered bacteria or yeast cells. In the present study, we use mixture design experiments to evaluate the influence of six nutrient elements on production of cutinase from the fungus Colletotrichum lindemuthianum. The nutritional elements were starch, glucose, ammonium sulfate, yeast extract, magnesium sulfate, and potassium phosphate. In the experimental design, we imposed the constraints that exactly one factor must be omitted in each set of experiments and no factor can account for more than one third of the mixture. Thirty different sets of experiments were designed. Results obtained showed that while starch is found to have negative influence on the production of the enzyme, yeast extract and potassium phosphate have a strong positive influence. Magnesium sulfate, ammonium sulfate, and glucose have low positive influence on the enzyme production. Contour plots have also been created to obtain information concerning the interaction effects of the media components on enzyme production.