Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production

The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 × 10³-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. amminoagenes. The FAD-synthetase-gene-amplified C. amminoagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn²⁺ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyltransferase activity and resulted in the accumulation of FMN.     

Respiration in Organic Acid-Forming Molds

Cytochrome c, coenzyme Q and lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1, 1, 1, 27) in Rhizopus oryzae were studied in order to investigate the connection between the mechanism of lactate formation and terminal respiration.

Cytochrome c was extracted easily and in good yield by the addition of cetyltrimethyl ammonium bromide to mycelial suspensions. It was purified by calcium phosphate gel and Amberlite IRC-50 resin chromatography.

Coenzyme Q was extracted with ethanol, purified by chromatography on silicic acid, and, following crystallization from a mixture of ethanol and methanol, was identified as coenzyme Q₀.

Lactic dehydrogenase was partially purified and some of its properties were investigated.

Rhizopus oryzae at an early growth stage in shake culture produced almost no lactate. At this stage, the mycelia were rich in cytochrome c and FAD. On the contrary, those of later growth stages fermented a larger amount of the glucose to lactate and the contents of cytochrome c and FAD were lower than in the young mycelia.

Surface cultures produced lactate at a rate very nearly equivalent to the rate of glucose consumption. Addition of zinc to the medium resulted in decreased lactate production, but no increase was observed in the mycelial content of either cytochrome c or FAD in this case. On the other hand, increased quantities of FMN were found in mycelia from shake or surface cultures when zinc was added.     

One-step purification procedure for UDP-N-acetylmuramyl-peptide murein precursors from Bacillus cereus

A method is described for the rapid isolation of the activated murein precursors UDP-N-acetyl-muramyl-pentapeptide (UDP-MurNAc-pentapeptide) and UDP-MurNAc-tripeptide from Bacillus cereus. After accumulation of the precursors by inhibition of murein synthesis either in the presence of vancomycin (for the pentapeptide precursor) or D-cycloserine (for the tripeptide precursor) the cells were extracted with boiling water. Prior to high pressure liquid chromatography the material was freed from acid precipitable material. UDP-MurNAc-penta- and tripeptide were separated from other components by reversed-phase HPLC on Hypersil ODS using isocratic elution conditions with sodium phosphate buffer. The precursors were obtained with at least 98% purity and a yield of about 50 mumol from a 10-l culture of B. cereus.     

Metabolic and bioprocess engineering of the yeast Candida famata for FAD production

Flavins in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) play an important role in metabolism as cofactors for oxidoreductases and other enzymes. Flavin nucleotides have applications in the food industry and medicine; FAD supplements have been efficiently used for treatment of some inheritable diseases. FAD is produced biotechnologically; however, this compound is much more expensive than riboflavin. Flavinogenic yeast Candida famata synthesizes FAD from FMN and ATP in the reaction catalyzed by FAD synthetase, a product of the FAD1 gene. Expression of FAD1 from the strong constitutive promoter TEF1 resulted in 7- to 15-fold increase in FAD synthetase activity, FAD overproduction, and secretion to the culture medium. The effectiveness of FAD production under different growth conditions by one of these recombinant strains, C. famata T-FD-FM 27, was evaluated. First, the two-level Plackett–Burman design was performed to screen medium components that significantly influence FAD production. Second, central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum FAD yield. FAD production varied most significantly in response to concentrations of adenine, KH₂PO₄, glycine, and (NH₄)₂SO₄. Implementation of these optimization strategies resulted in 65-fold increase in FAD production when compared to the non-optimized control conditions. Recombinant strain that has been cultivated for 40 h under optimized conditions achieved a FAD accumulation of 451 mg/l. So, for the first time yeast strains overproducing FAD were obtained, and the growth media composition for maximum production of this nucleotide was designed.     

Purification of bovine protoporphyrinogen oxidase: immunological cross-reactivity and structural relationship to ferrochelatase

Protoporphyrinogen oxidase, the penultimate enzyme in the haem biosynthetic pathway has been purified to apparent homogeneity from bovine liver mitochondria, by a published method (Dailey, H.A. and Fleming, J.E., (1983)), with an additional ion-exchange chromatography step, using a Mono Q column on an FPLC-system. This gave a product with a 68% yield and 870-fold purification. Protoporphyrinogen oxidase (EC 1.3.3.4) has an apparent Mr of 57,000 and the Km for protoporphyrinogen IX was 16.6 microM. Activity of the isolated enzyme was increased by 66% in the presence of oleic acid, and evidence was obtained for a FAD prosthetic group. Ferrochelatase (EC 4.99.1.1) was purified and antibodies were raised in rabbits against ferrochelatase and protoporphyrinogen oxidase, respectively. Anti-protoporphyrinogen oxidase IgG showed marked cross-reactivity with ferrochelatase and anti-ferrochelatase IgG cross-reacted with protoporphyrinogen oxidase. In addition, radiolabelled peptides of both enzymes, generated by chymotrypsin, demonstrated common peptides when analysed by two-dimensional chromatography.     

The photodynamic action of riboflavine on erythrocytes

The photodynamic action of riboflavine and riboflavine phosphate on erythrocytes was studied. It was found that irradiation of rat erythrocyte suspension in the presence of riboflavine phosphate resulted in an enhancement in erythrocyte potassium losses. Also, in the presence of riboflavine and light the release of lactose isonicotinoyl-hydrazone entrapped in released mouse erythrocytes, was markedly increased. The delayed photosensitizing effect, after keeping the irradiated samples for 20 hours in the dark, was more pronounced. No significant photohemolysis of erythrocytes could be demonstrated. Products of lipid peroxidation were formed, and was assumed to be responsible for the alteration in membrane permeability.     

Enhancement of Sf-9 cell growth and longevity through supplementation of culture medium with hemolymph

The benefits of insect cell culture medium supplementation with hemolymph of Lonomia obliqua were investigated. The addition of hemolymph to the medium induced high levels of cell growth, and the viability was maintained for longer periods. The maximum cell yield increased almost 3-fold after hemolymph supplementation. Cultures in their stationary phase were rescued through hemolymph supplementation, also reaching high cell concentrations. These actions were much dependent on the concentration of hemolymph; low hemolymph concentration had a positive effect in cell growth, whereas high hemolymph concentration showed a deleterious effect. Fractionation of hemolymph by gel filtration chromatography showed the presence of three factors with different activity in insect cell culture: an potential anti-apoptotic factor, a growth-promoting factor, and an enzyme that hydrolyzes sucrose. Addition of hemolymph to the medium induced high levels of glucose production. The sucrose to glucose conversion was also linearly dependent upon the hemolymph concentration. Therefore, we conclude that cell growth and longevity can be increased by supplementation of the culture medium with hemolymph.     

A Point Mutation in cycA Partially Contributes to the D-cycloserine Resistance Trait of Mycobacterium bovis BCG Vaccine Strains

In mycobacteria, CycA a D-serine, L- and D-alanine, and glycine transporter also functions in the uptake of D-cycloserine, an important second-line anti-tubercular drug. A single nucleotide polymorphism identified in the cycA gene of BCG was hypothesized to contribute to the increased resistance of Mycobacterium bovis bacillus Calmette-Guérin (BCG) to D-cycloserine compared to wild-type Mycobacterium tuberculosis or Mycobacterium bovis. Working along these lines, a merodiploid strain of BCG expressing Mycobacterium tuberculosis CycA was generated and found to exhibit increased susceptibility to D-cycloserine albeit not to the same extent as wild-type Mycobacterium tuberculosis or Mycobacterium bovis. In addition, recombinant Mycobacterium smegmatis strains expressing either Mycobacterium tuberculosis or Mycobacterium bovis CycA but not BCG CycA were rendered more susceptible to D-cycloserine. These findings support the notion that CycA-mediated uptake in BCG is impaired as a result of a single nucleotide polymorphism; however, the partial contribution of this impairment to D-cycloserine resistance suggests the involvement of additional genetic lesions in this phenotype.     

Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.     

Selection of Microorganisms Producing Flavin-Adenine Dinucleotide from FMN and Adenine (AMP) and Production of Flavin-Adenine Dinucleotide by Sarcina lutea

For the purpose of the fermentative production of flavin-adenine dinucleotide (FAD), the authors attempted to select the microorganisms which could produce FAD in culture medium from flavin mononucleotide and adenylic acid for adenine, and discovered that bacteria and actinomycetales could accumulate FAD more or less, but yeasts and moulds could not. Among the microorganisms used in the experiments, the strains belonging to the genera Sarcina and Brevibacterium accumulated relatively large amounts of FAD. Especially, Sarcina lutea and Sarcina aurantiaca seemed to be the most favorable microorganisms for the fermentative production of FAD. By using Sarcina lutea the investigation of the culture conditions for the production of FAD was carried out.     

High pressure refolding, purification, and crystallization of flavin reductase from Sulfolobus tokodaii strain 7

Flavin reductase HpaC(St) catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaC(St), which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaC(St) was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS-PAGE. The purified refolded HpaC(St) did not contain FMN, unlike the same enzyme expressed as a soluble protein. After the addition of FMN to the protein solution, the refolded enzyme showed a higher activity than the enzyme expressed as the soluble protein. Crystals of the refolded enzyme were obtained by adding FMN, FAD, or riboflavin to the protein solution and without the addition of flavin compound.     

Bioconversion of poultry wastes. I--Factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus, A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, although no significantly increased uricase production was found.     

Bioconversion of poultry wastes I-factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus. A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for both A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, but did not significantly increase uricase production.     

Enhanced taxol production and release in Taxus chinensis cell suspension cultures with selected organic solvents and sucrose feeding

Suspension culture of Taxus chinensis cells was carried out in aqueous-organic two-phase systems for the production and in situ solvent extraction of taxol (paclitaxel). Three organic solvents, hexadecane, decanol, and dibutylphthalate, were tested at 5-20% (v/v) in the culture liquid. All of these solvents stimulated taxol release and the yield per cell, though decanol and higher concentrations of the other two solvents depressed biomass growth significantly. Ten percent dibutylphthalate was the optimal solvent for improving taxol production and release with minimal cell growth inhibition. The time of solvent addition to the culture also affected taxol production, with the addition during the late-log growth phase being most favorable. By feeding sucrose to the culture near the stationary growth phase, the cell growth and taxol production period was extended from 27 to 42 days. The combining of the two-phase culture and sucrose feeding increased the taxol yield by about 6-fold compared with the single-phase batch culture, to 36.0 +/- 3.5 mg/L, with up to 63% taxol released. This study shows that in situ solvent extraction combined with nutrient feeding is an effective process strategy for production and recovery of secondary metabolites in plant cell suspension culture.     

Effect of IPTG amount on apo- and holo- forms of glycerophosphate oxidase expressed in Escherichia coli

Escherichia coli has proved to be a successful host for the expression of many heterologous proteins, and much efforts have been made toward improving recombinant protein expression including the usage of strong promoters and co-expression with chaperones. But little attention was paid on the relation between expression level and function of the target protein. Glycerophosphate oxidase (GPO) is a protein with FAD cofactor (without free cysteine and disulfide bonds).It was observed that the specific activity of GPO dramatically decreased with the increase of inducer IPTG. In addition, the stability of it decreased correspondingly. The structural difference of samples expressed under varying IPTG was investigated using size-exclusion and reverse-phase high performance liquid chromatography, together with CD spectrum. It was found that the conformation of peptide and organization of subunits were not affected. The loss of specific activity and stability were correlated to incomplete attachment of FAD onto GPO. These results revealed that synthesis speed should be controlled either by reduction of IPTG amount or using weak promoters in the production of GPO.     

High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris

Dipetarudin is a potent direct thrombin inhibitor that was genetically engineered as a chimera between dipetalogastin II and hirudin. Dipetarudin was initially cloned and purified from Escherichia coli, but with a very low yield of about 0.3 mg/l of culture medium. In this study, we report the production of dipetarudin in the methylotrophic yeast Pichia pastoris using pPIC9 vector. The His+ transformants were screened for the best expression performances by prolongation of the ecarin clotting time. An optimal dipetarudin's expression was reached by addition of methanol in culture medium to a final concentration of 0.5%, every 8h during 4 days. Secreted dipetarudin was purified essentially using a two-step purification scheme: anion exchange chromatography in a Resource Q column, followed by C18-reversed phase HPLC. About 150 mg purified dipetarudin was obtained from 1l culture supernatant. This yield is 500-fold higher than the yield obtained with the E. coli system. The molecular mass of dipetarudin calculated by MALDI-TOF (7450 Da) was in agreement with the mass calculated by the amino acid composition (7454 Da), indicating correct processing of the signal sequence. The Ki value of dipetarudin was 399+/-83 fM, which is in agreement with that calculated for the inhibitor isolated from E. coli. This efficient and cost-effective expression system facilitates large-scale production and purification of dipetarudin for further structural, functional and pharmacological investigations.     

The thermochemical characterization of sodium dithionite, flavin mononucleotide, flavin-adenine dinucleotide and methyl and benzyl viologens as low-potential reductants for biological systems.

The heat of reaction (deltaH) of Fe(CN)63-, Methyl Viologen, FMN and FAD with S2O42- in aqueous buffer solutions was measured calorimetrically. In addition deltaH values for reduction of Fe(CN)63-, FMN and FAD by reduced Methyl Viologen were determined. The resulting calorimetric data and corresponding E0 values were combined to yield thermodynamic data for these simple reducing agents in a form useful for applications to biological reactions. Thermodynamic data for the reduction of spinach ferredoxin are also presented.     

Marinobacterium sp. strain DMS-S1 uses dimethyl sulphide as a sulphur source after light-dependent transformation by excreted flavins

Marinobacterium sp. strain DMS-S1 is a unique marine bacterium that can use dimethyl sulphide (DMS) as a sulphur source only in the presence of light. High-performance liquid chromatography (HPLC) analyses of the culture supernatant revealed that excreted factors, which could transform DMS to dimethyl sulphoxide (DMSO) under light, are FAD and riboflavin. In addition, FAD appeared to catalyse the photolysis of DMS to not only DMSO but also methanesulphonate (MSA), formate, formaldehyde and sulphate. As strain DMS-S1 can use sulphate and MSA as a sole sulphur source independently of light, the excretion of flavins appeared to support the growth on DMS under light. Furthermore, three out of 12 marine bacteria from IAM culture collection were found to be able to grow on DMS with the aid of photolysis by the flavins excreted. This is the first report that bacteria can use light to assimilate oceanic organic sulphur compounds outside the cells by excreting flavins as photosensitizers.     

Collagenase synthesis in clonal monkey kidney epithelial cells (JTC-12 cells)

Cells of clone JTC-12 derived from monkey kidney epithelial cells were found to produce latent collagenase in serum-containing cultures. Collagenase production by JTC-12 cells was stimulated more than ten-fold with the addition of 12-0-tetradecanoyl-phorbol-13-acetate. The JTC-12 cell collagenase was purified 2,370-fold over the conditioned medium to a specific activity of 5,940 units/mg with a yield of 79% by heparin-Sepharose, ion-exchange and immunoadsorption chromatography. Thus the JTC-12 cell culture appears to be a useful model for studying the initial degradation of surrounding connective tissues by epithelial tissue in carcinogenesis.