Utilization of Hydrocarbons and Vitamin B12 Production by Bacillus badius

The accumulation of vitamin B₁₂ by Bacillus badins grown on hydrocarbon was investigated. The bacterium could assimilate n-alkanes of C₁₁–C₁₈, ethanol, fumarate, α-ketoglutarate and malate. n-Alkanes of C₁₆–C₁₈, were the best for vitamin B₁₂ production. The bacterium utilized well all of the nitrogen sources tested. Above all, ammonium dihydrogen phosphate was the best for the bacteria] growth and vitamin B₁₂ production. Addition of organic nutrients such as malt extract and meat extract, and addition of metal ions such as ferrous and cobalt promoted the growth and vitamin B₁₂ production. Interestingly, vitamin B₁₂ was produced mostly in the supernatant. The cyanoform of the corrinoid predominantly formed in the supernatant would confirm the identity with cobalamin.     

Optimization of a Propionibacterium acidipropionici continuous culture utilizing sucrose

To produce propionic acid and vitamin B₁₂ from sucrose, the strain Propionibacterium acidipropionici NRRL B3569 was selected by screening a number of Propionibacterium strains. The nutrient composition and the fermentation conditions for this strain were optimized in continuous culture. The investigations show that within a concentration range of 30–170 g l^–1 of sucrose in the fermentation medium, no significant substrate inhibition occurred. For the production of propionic acid and vitamin B₁₂, concentrations of 1.5 mg FeSO₄·7H₂O g^–1 dry biomass, 0.75 mg cobalt ions g^–1 dry biomass, 0.3 mg 5,6-dimethylbenzimidazole g^–1 dry biomass, and 12 g yeast extract 1^–1 were necessary additions to the sources of nitrogen, phosphate, and magnesium ions. The extra addition of up to 2.8 g betaine g^–1 dry biomass significantly increases the production of vitamin B₁₂. In the optimization of the pH value, temperature, and aeration, it was established that the conditions for propionic acid production and vitamin B₁₂ production are different. Whereas the optimal production of propionic acid took place under completely anaerobic conditions with a pH value of 6.5 and a temperature of 37°C, optimal vitamin B₁₂ production required a temperature of 40°C and aerobic conditions (0.5 vvm aeration at 100 rpm) with a pH value of 6.5.     

High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels

Human lymphocyte kynureninase activity was assessed in homogenized cells by determination of 3-hydroxyanthranilic acid production as a function of time after addition of the substrate, 3-hydroxykynurenine. The product, 3-hydroxyanthranilic acid, was determined by isocratic high-performance liquid chromatography and fluorescence detection. Mean (± S.D.) lymphocyte kynureninase activity in a group (n = 12) of vitamin B₆-deficient men was 5.04 ± 0.81 pmol 3-hydroxyanthranilic acid formed per mg protein per min, which was significantly (p=0.005) lower than the 6.69 ± 1.70 pmol 3-hydroxyanthranilic acid formed per mg protein per min in men with a normal vitamin B₆ status. This indicates that lymphocyte kynureninase activity is depressed during a vitamin B₆ deficiency.     

Utilization of n-Paraffins and Vitamin B6 Production by Pichia guilliermondii Wickerham

The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK–2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀–C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal, The maximal vitamin B₆ production was approximately 25 mg per liter of the culture broth.     

Studies on the Biosynthesis of Vitamin B6

The production of vitamin B₆ (B₆) compounds with a cell-suspension of Achromobacter cycloclastes A.M.S. 6201 under various conditions were examined. An obvious accumulation of B₆ compounds in the incubation medium with a cell-suspension of the organism harvested at the middle to later part of exponential phase of growth was observed. γ-Aminobutyric acid or β-alanine was found to stimulate the B₆ production markedly, when they were added to the incubation mixture together with glycerol. Some discussion on the implication of these compounds as precursors of B₆ was presented.     

Production of vitamin B12 by immobilized cells of a propionic acid bacterium

Summary The ability of immobilized cells of propionic acid bacteria to form vitamin B₁₂ has been investigated. Propionibacterium arl AKU 1251 having a considerable activity to produce the vitamin was selected as a test organism among six strains of propionic acid bacteria tested. The whole cells were entrapped with urethane prepolymers, photo-crosslinkable resin prepolymers or several other materials such as -carrageenan, agar or sodium alginate, and their vitamin B₁₂ productivity was compared. Based on the criteria of the convenience of preparation and the stability of the cell-entrapping gels, a hydrophilic urethane prepolymer, PU-9, was employed as gel material. Satisfactory vitamin B₁₂ production was obtained when 5–10 g of wet cells precultured to the late exponential growth phase were entrapped with 1 g of the prepolymer. Addition of a suitable amount of cobaltous ion and of 5,6-dimethyl benzimidazole to the culture medium was effective for the production of the vitamin by the immobilized cells. The repeated use of the immobilized cells was successfully achieved when a suitable amount of cells were entrapped and allowed the proliferation of cells inside gel matrices.     

Isolation of a novel Pseudomonas species SP2 producing vitamin B12 under aerobic condition

Vitamin B₁₂ is a complex biomolecule that acts as a cofactor for a variety of enzymes in microbial metabolism. Pseudomonas denitrificans is exclusively used as an industrial strain for the production of vitamin B₁₂ under aerobic conditions. However, only a few strains of Pseudomonas have been reported to possess the capability of producing this vitamin and they are strongly patent-protected. To improve the applicability of the vitamin B₁₂-producing microorganisms, a new isolate was obtained from municipal waste samples and characterized for its biological properties. The new isolate, designated as SP2, was identified to be a Pseudomonas species based on the sequence homology of its 16S rDNA. Pseudomonas species SP2 had essential genes for vitamin B₁₂ synthesis such as cobB and cobQ and produced a similar amount of vitamin B₁₂ (10.6 ± 0.05 μg/mL) as P. denitrificans ATCC 13867 in 24 h flask culture. SP2 grew well under aerobic condition with the maximum specific growth rate (µ _max ) of 0.91 ± 0.03/h, but showed a poor growth under micro-aerobic conditions. SP2 was resistant to antibiotics like streptomycin, carbenicillin, ampicillin, cefpodoxime, colistin, nalidixic acid and sparfloxacin. The ability of SP2 to grow faster and produce vitamin B₁₂ under aerobic conditions makes it a promising host for the production of some biochemicals requiring a coenzyme B₁₂-dependent enzyme, such as glycerol dehydratase.     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

The influence of a specific microelemental environment in alginate gel beads on the course of propionic acid fermentation

In this work the exchange of calcium, cobalt, iron, magnesium, zinc and manganese ions between alginate gel beads and casein medium was investigated. The high release of calcium ions from alginate to the medium and the biosorption of some metal ions were observed. The pure alginate gel adsorbed all the metal ions examined, from a fermentative medium. Gel with immobilized cells of two strains of Propionibacterium freudenreichii subsp. shermanii showed an active ability to adsorb only cobalt, iron and zinc ions. In this way, a special microelemental environment was created in the alginate gel. This resulted in an increase of propionic acid production and a decrease of vitamin B₁₂ biosynthesis. Received: 30 April 1997 / Received revision: 2 July 1997 / Accepted: 4 July 1997     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium

Cobalamin (vitamin B₁₂) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B₁₂ biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG^AcbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B₁₂ riboswitch upstream of the cbiXJCDETLFGAcysG^AcbiYbtuR operon and the recombinant production of three different vitamin B₁₂ binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B₁₂-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B₁₂ concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.     

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek’s-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B₁ was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH₄)₂HPO₄ (1.55 gL^-1) and NaNO₂ (1.6 gL^-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B₁ production. Zn²⁺ and Co²⁺ supported significantly both fungal growth, as well as, aflatoxin B₁ production at the different tested concentrations. Zn²⁺ was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (a_w) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum a_w for the fungal growth was 0.8 on both coffee beans and rice grains, while a_w of 0.70 caused complete inhibition for the growth and aflatoxin B₁ production. H₂O₂ is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO₃ and C₆H₅COONa converted aflatoxin B₁ to water-soluble form which returned to aflatoxin B₁ by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B₁ production. Stearic acid supported the fungal growth and decreased the productivity of AFB₁ gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B₁ production. Vitamins C and D₂ were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B₁ production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B₁ synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B₁ production. All the tested enzymes as well as aflatoxin B₁ production were inhibited by either catechol or phenol.     

Nutritional requirements ofPrevotella sp. Isolated from the rumen of the goat

The nutritional requirements forPrevotella sp. 4PCCNB2 isolated from the rumen of a native goat in Korea and those of the ATCC 19189 strain isolated from the bovine rumen were investigated. The two strains grew well with ammonium sulfate as the sole added nitrogen source. However, neither a complex of amino acids nor casein hydrolysate effectively replaced ammonium sulfate. Biotin,p-aminobenzoic acid, and vitamin B₁₂ were essential to culture the ATCC 19189 strain. Unlike the ATCC 19189 strain, however, B₁₂ was only stimulatory for the growth of the 4PCCNB2 strain. The 4PCCNB2 strain grew well in the basal medium without an individual acid such as acetic acid or valeric acid. In contrast, either acetic or valeric acid was absolutely required for the growth of the ATCC 19189 strain.     

Biosynthesis of folic acid

Summary The influence of various vitamins on the biogenesis of folic acid has been studied in microorganisms requiring these as growth factors. In L. arabinosus, the folic acid synthesised was directly proportional to the availability of both riboflavin and pantothenic acid. The influence of cyanocobalamin on folic synthesis varied radically in different organisms. In case of the B₁₂/methionine auxotroph of E. coli there was an inverse relationship of vitamin B₁₂ to folic acid synthesis, while in Euglena the folic acid elaborated was in proportion to cyanocobalamin supplied. Synthesis of both folic acid and vitamin B₁₂ was depressed when thymine supply was adequate in the nutrition of E. coli 15 T ^-, a thymine auxotroph.     

Effect of chemical and microbial vitamin B<Subscript>12</Subscript> analogues on production of vitamin B<Subscript>12</Subscript>

Strain improvement by genetic manipulation or optimization of fermentation conditions for overproduction of vitamin B₁₂ has a drawback due to feed back inhibition. To resist the feed back inhibition by analogues of vitamin B₁₂ in Propionibacterium freudenrechii subsps. shermanii (OLP-5), we have tested with microbially separated B₁₂ analogues from three different strains. Microbial analogues were differentiated from commercially available vitamin B₁₂ by high pressure liquid chromatography and spectrophotometric method. An analogue isolated from NRRL-B-4327 was shown to increase vitamin B₁₂ concentration from 18.53 ± 0.15 to 31.67 ± 0.58 mg/l in OLP-5 strain. The presence of chemical analogue (ICH₂ Co(DH)₂ (H₂Py)₄) increased vitamin B₁₂ production from 16.13 ± 0.15 to 18.53 ± 0.15 mg/l in OLP-5. These findings revealed that addition of B₁₂ analogues in fermentation media have developed strain resistance to feed back inhibition by vitamin B₁₂.     

SECRETION OF VITAMINS AND AMINO ACIDS INTO THE ENVIRONMENT BY OCHROMONAS DANICA1,2

Ochromonas danica grown on a chemically defined medium under controlled conditions in the light synthesized the following vitamins: ascorbate, B₆, N⁵-methyltetrahydrofolate, tetrahydrofolate polyglutamates, oxidized folate monoglutamates, nicotinate, pantothenate, riboflavin, vitamin A, β-carotene, and vitamin E but no vitamin. B₁₂. The cells also secreted molecules into their growth medium including the vitamins ascorbate, B₆, the above folates, nicotinate, pantothenate, riboflavin, vitamin E, and the amino acids alanine, aspartic acid, leucine, and valine. The role of such secretions in nature is discussed.     

Effects of Vitamin B6 Deficiency on the Conversion Ratio of Tryptophan to Niacin

To investigate how vitamin B₆ (B₆) deficiency affects the whole metabolism of tryptophan-niacin, rats were fed for 19 days with each of the following four kinds of diets; a complete 20% casein diet (control diet), the control diet without B₆, the control diet without nicotinic acid, and the control diet without nicotinic acid and B₆, and the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone3- carboxamide each and the enzyme activities involved in tryptophan-niacin pathway were measured. The urinary excretion of kynurenic acid decreased while that of xanthurenic acid increased drastically in the two B₆-deficient groups, when compared with the B₆-containing groups. These results indicate that the rats fed with the B₆-free diets were in the vitamin-deficient state. The conversion ratio was calculated from the ratio of the urinary excretion of sum of nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5carboxamide, and N¹-methyl-4-pyridone-3-carboxamide, to the Trp intake. The ratio was statistically lower in the B₆-free diet than in the B₆-containing diet under the niacin-free conditions.     

Production of 3‐hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B12 naturally

3‐Hydroxypropionic acid (3‐HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant Escherichia coli stains can produce 3‐HP from glycerol in the presence of vitamin B₁₂ (coenzyme B₁₂), when overexpressed with a coenzyme B₁₂‐dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3‐HP in recombinant Klebsiella pneumoniae strains, which naturally synthesizes vitamin B₁₂ and does not require supplementation of the expensive vitamin. The NAD⁺‐dependent gamma‐glutamyl‐gamma‐aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B₁₂ addition, the recombinant K. pneumoniae ΔdhaTΔyqhD overexpressing PuuC could produce ～3.8 g/L 3‐HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3‐propanediol (1,3‐PDO) (instead of 3‐HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3‐HP and 1,3‐PDO were produced when aeration was too high. The production of a small amount of 3‐HP under improper aeration conditions was attributed to either slow NAD⁺ regeneration (under low aeration) or reduced vitamin B₁₂ synthesis (under high aeration). In a glycerol fed‐batch bioreactor experiment under a constant DO of 5%, the strain, K. pneumoniae ΔdhaTΔyqhD, overexpressing both PuuC and DhaB could produce >28 g/L 3‐HP in 48 h with a yield of >40% on glycerol. Only small amount of 3‐HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that K. pneumoniae is potentially useful for the production of 3‐HP in an economical culture medium that does not require vitamin B₁₂. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of 3‐HP while using this strain. Biotechnol. Bioeng. 2013; 110: 511–524. © 2012 Wiley Periodicals, Inc.     

Production of vitamin B12 by a fermentor with a hollow-fiber module

Summary To improve the microbial production of vitamin B₁₂, we applied a hollow-fiber module to cultivation of the vitamin producers. By the removal of growth inhibitors, very high concentrations of cells and vitamin B₁₂ were obtained comparing to the batch culture. We obtained 227 g dry cells/l and 52 mg vitamin B₁₂/l with Propionibacterium shermanii and 33.4 g dry cell/l and 92.5 mg vitamin B₁₂/l with Butyribacterium methylotrophicum by this cultivation.     

Life's utilization of B vitamins on early Earth

Coenzymes are essential across all domains of life. B vitamins (B₁‐thiamin, B₂‐riboflavin, B₃‐niacin, B₅‐pantothenate, B₆‐pyridoxine, B₇‐biotin, and B₁₂‐cobalamin) represent the largest class of coenzymes, which participate in a diverse set of reactions including C₁‐rearrangements, DNA repair, electron transfer, and fatty acid synthesis. B vitamin structures range from simple to complex heterocycles, yet, despite this complexity, multiple lines of evidence exist for their ancient origins including abiotic synthesis under putative early Earth conditions and/or meteorite transport. Thus, some of these critical coenzymes likely preceded life on Earth. Some modern organisms can synthesize their own B vitamins de novo while others must either scavenge them from the environment or establish a symbiotic relationship with a B vitamin producer. B vitamin requirements are widespread in some of the most ancient metabolisms including all six carbon fixation pathways, sulfate reduction, sulfur disproportionation, methanogenesis, acetogenesis, and photosynthesis. Understanding modern metabolic B vitamin requirements is critical for understanding the evolutionary conditions of ancient metabolisms as well as the biogeochemical cycling of critical elements such as S, C, and O.