Biosynthesis of Cobalamine,Erythromycin and Chlortetracycline by Streptomyces Species

Attempts were devoted to use Streptomyces aureofaciens and Streptomyces erythreus, the antibiotics producers as sources for the biosynthesis of cobalamine. The constituents of the fermentation medium and the strain play an important role in the biosynthesis of vitamin B₁₂. The same strain produced different amounts of antibiotic and vitamin on the two different constitutive media. The increase of the phosphorus concentration in the fermentation medium—within limits—increased the vitamin B₁₂ biosynthesis. The optimal concentration of phosphorus favourable for the synthesis of cobalamine was inhibitive for the antibiotic production. The phosphorus level in the fermentation medium plays an important role in the metabolism of carbohydrate and consequently on the biosynthesis of antibiotics. Low concentration of 5,6-dimethylbenzimidazole (cobalamine precursor) in the presence of suitable phosphorus induced the microorganism to increase its biosynthetic potentiality for the vitamin B₁₂ production.     

Simulation of fermentation conditions for vitamin B12 biosynthesis from whey

Vitamin B₁₂ production in fermentation of Propionibacterium shermanii and Propionibacterium arl AKU 1251 in whey permeate medium has been studied. The observed results and simulated expected values obtained by fitting statistical equations to the recorded data showed that 24 h old inoculum, 5 mg iron l^?1 and 4% whey lactose were optimal for vitamin B₁₂ biosynthesis in both strains when fermentation was carried out under anerobic (84 h) and aerobic (84 h) conditions at 30°C. The supplementation of whey medium with 0.5% (NH₄)₂HPO₄ enhanced further the metabolite yield; however, the preference for a mixed carbon source (lactose + d-glucose or lactose + d-fructose) at different levels varied in the strains under study. P. shermanii, under optimal cultural conditions, was found to be a better strain than Propionibacterium arl AKU, 1251 in fermenting whey lactose for product (vitamin B₁₂) formation.     

Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

脱氮假单胞菌生产维生素B12过程中粪卟啉Ⅲ的测定及发酵调控

脱氮假单胞菌发酵生产维生素B₁₂过程中,副产物粪卟啉Ⅲ的积累对产物的代谢合成和分离提取有很大的影响。建立了发酵液中粪卟啉Ⅲ的HPLC快速测定方法,发酵液处理后直接进样测定,检测线性范围为12~275 μg/ml,重复性好。研究了不同供氧水平、二氧化碳浓度和pH值对发酵过程中维生素B₁₂和粪卟啉Ⅲ代谢合成的影响,并在120吨发酵罐中进行了发酵过程优化控制。结果表明:在发酵过程产物的合成期控制适当的供氧水平、控制二氧化碳浓度在8.6±0.8%、维持pH值在7.0±0.12能明显抑制卟啉Ⅲ的生物合成,同时使维生素B₁₂产量提高15%。     

One pathway can incorporate either adenine or dimethylbenzimidazole as an alpha-axial ligand of B12 cofactors in Salmonella enterica

Corrinoid (vitamin B₁₂-like) cofactors contain various α-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form “complete” corrinoids aerobically by importing an “incomplete” corrinoid, such as cobinamide (Cbi), and adding appropriate α- and β-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B₁₂, but it can make B₁₂ from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B₁₂ cofactors (having adenine as an α-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an α-ligand. When DMB is provided to these mutants, synthesis of pseudo-B₁₂ cofactors ceases and B₁₂ cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an α-ligand. Wild-type cells make pseudo-B₁₂ cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B₁₂ for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B₁₂ cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B₁₂. Models are described for the mechanism and control of α-axial ligand installation.     

Role of amino acids, betaine and choline in vitamin B12 biosynthesis by strains of Propionibacterium

This paper reports the role of amino acids, betaine and choline on vitamin B₁₂ biosynthesis in Propionibacterium shermanii 566, P. shermanii and Propionibacterium arl AKU 1251. l-Glutamic acid supplemented at the 0.05% (w/v) level in whey permeate stimulated vitamin B₁₂ production in the three organisms, whereas the influence of other amino acids differed in the three strains. A uniform increase in product formation in Propionibacterium cultures with increasing doses of betaine and choline was recorded, but with variable relative effectiveness. However, no significant difference at the 0.50 and 0.75% (w/v) levels of these two compounds was observed. The addition of betaine at 0.5% (w/v) concentration was considered optimal for maximum fermentation efficiency in the cultures. An increase of 2.8–25.7% and 5.1–40.8% in vitamin B₁₂ yield as compared to the control was observed by supplementing whey permeate medium with l-glutamic acid and betaine, respectively, at their optimum values in the organisms studied.     

Biochemical Studies on Vitamin B12 Part XI

In Part X, the authors published studies on vitamin B₁₂-like activity of cobalt-porphyrin-derivative prepared from radish-leaves, and the physiological significance of the compound as an active component in the intermediary metabolism of vitamin B₁₂ was described. , Afterward, pure chlorophyll a and b were isolated by column chromatography and the vitamin B₁₂-like activities of Co-porphyrin a and b were repeatedly confirmed.

Recently, effect of Co-porphyrin on the biosynthesis of real vitamin B₁₂ has been tested and the confirmation was also successfully completed by bioautography and paper electrophoresis.

In the present communication, it is discussed as of special importance that cobalt-porphyrin may be the active principle for the formation in vivo of vitamin B₁₂.     

Bacillus megaterium Has Both a Functional BluB Protein Required for DMB Synthesis and a Related Flavoprotein That Forms a Stable Radical Species

Despite the extensive study of the biosynthesis of the complex molecule B₁₂ (cobalamin), the mechanism by which the lower ligand 5,6-dimethylbenzimidazole (DMB) is formed has remained something of a mystery. However, recent work has identified and characterized a DMB-synthase (BluB) responsible for the oxygen-dependent, single enzyme conversion of FMN to DMB. In this work, we have identified BluB homologs from the aerobic purple, nonsulfur, photosynthetic bacterium Rhodobacter capsulatus and the aerobic soil bacterium Bacillus megaterium and have demonstrated DMB synthesis by the use of a novel complementation assay in which a B₁₂ deficient strain, substituted with the precursor cobinamide is recovered either by the addition of DMB or by the recombinant expression of a bluB gene. The DMB-synthetic activity of the purified recombinant BluB enzymes was further confirmed in vitro by providing the enzyme with FMNH₂ and oxygen and observing the formation of DMB by HPLC. The formation of a 4a-peroxyflavin intermediate, the first step in the oxygen dependent mechanism of DMB biosynthesis, is reported here and is the first intermediate in the enzyme catalysed reaction to be demonstrated experimentally to date. The identification and characterization of an FMN-binding protein found on the cobI operon of B. megaterium, CbiY, is also detailed, revealing an FMN-containing enzyme which is able to stabilize a blue flavin semiquinone upon reduction with a 1-electron donor.     

PRODUCTION OF VITAMIN B12, THIAMINE,AND BIOTIN BY PHYTOPLANKTON1

Some ecologically important phytoplankters released vitamins into culture medium during growth. Skeletonema costatum and Stephanopyxis turris (vitamin B₁₂-requirers) produced both thiamine (vitamin B₁) and biotin when growing with either 12 or 2 ng vitamin B₁₂/liter. Gonyaulax polyedra (vitamin B₁₂-requirer) produced thiamine with 12 ng vitamin B₁₂/liter, and Coccolithus huxleyi (thiamine-requirer) produced vitamin B₁₂ and biotin with 120 ng thiamine/liter, but only biotin with 10 ng thiamine/liter. The amount of vitamin produced by an alga and rate at which it was produced varied with the phytoplankter, the concentration of the required vitamin, and incubation time. Vitamins produced during early and exponential growth were due to excretions, and those produced at stationary growth resulted from excretion and release due to cell lysis. Uptake of the required vitamin by all phytoplankters was greatest during the first few days of incubation. On continued incubation the rate of uptake/cell decreased. In the sea phytoplankters may contribute a major portion of the amount of dissolved vitamins.     

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.     

Enhanced anaerobic biotransformation of carbon tetrachloride with precursors of vitamin B12 biosynthesis

Relatively low concentrations of Vitamin B₁₂ are known to accelerate the anaerobic biotransformation of carbon tetrachloride (CT) and chloroform (CF). However, the addition of vitamin B₁₂ for field-scale bioremediation is expected to be costly. The present study considered a strategy to generate vitamin B₁₂ by addition of biosynthetic precursors. One of the precursors, porphobilinogen (PB) involved in the formation of the corrin ring, significantly increased the CT biotransformation rates by 2.7−, 8.8- and 10.9-fold when supplemented at 160, 500 and 900 μM, respectively. A positive control with 10 μM of vitamin B₁₂ resulted in a 5.9-fold increase in the CT-bioconversion rate. PB additions provided high molar yields of inorganic chloride (57% of CT organochlorine), comparable to that obtained with vitamin B₁₂ supplemented cultures. The primary substrate, methanol, known to induce vitamin B₁₂ production in methanogens and acetogens, was required for PB to have a significant impact on CT conversion. The observation suggests that PB’s role was due to stimulating vitamin B₁₂ biosynthesis. The present study therefore provides insights on how to achieve vitamin B₁₂ enhanced rates of CT bioremediation through the use of less complex compounds that are precursors of vitamin B₁₂. Although PB is a costly chemical, its large impact points to corrin ring formation as the rate-limiting step.     

Mouse transcobalamin II metabolism: The effects of antibiotics on the clearance of vitamin B12 from the serum transcobalamin II-vitamin B12 complex and the reappearance of the free serum transcobalamin II in the mouse

The mechanism of the clearance of vitamin B₁₂ from the serum transcobalamin II-vitamin B₁₂ (Tc-II-B₁₂) complex and the reappearance of free Tc-II in mouse have been studied. When a saturating dose of vitamin B₁₂ is given parenterally to normal mice, a portion of the Tc-II-bound vitamin B₁₂ is rapidly cleared and free Tc-II promptly reappears until it reaches a constant level in 6–8 h. The remaining vitamin B₁₂ is cleared slowly from the rest of the Tc-II-B₁₂ complex. In cycloheximide or puromycin-treated mice, free Tc-II fails to reappear and the bound Tc-IIdecreases. Treatment with actinomycin D has no effect on the reappearance of free Tc-II. The probable mechanism of this inhibition is discussed. The results suggest that mouse serum Tc-II has a stable messenger RNA template and a fast turnover. The free Tc-II which reappears in the serum after Tc-II has been saturated with vitamin B₁₂, appears to be newly synthesized.     

Probiotic lactic acid bacterium from <Emphasis Type="Italic">kanjika</Emphasis> as a potential source of vitamin B<Subscript>12</Subscript>: evidence from LC-MS,immunological and microbiological techniques

Vitamin B₁₂ was produced by probiotic Lactobacillus plantarum in submerged fermentation (96 h) with successive anaerobic and aerobic phases of 48 h each to give 13 ng vitamin B₁₂/g dry biomass. Sodium cyanide-mediated cell lysis, followed by benzyl alcohol/chloroform/water extraction, improved the release of intracellular vitamin B₁₂ for analysis. The presence of the K⁺ adduct of cyanocobalamin (m/z of 1394) was established using electron spray ionization–mass spectra; growth of a mutant of Escherichia coli in the presence of cyanocobalamin ascertained its bioavailability.     

Role of Third Serum Vitamin B12 Binding Protein in Vitamin B12 Transport

A third vitamin B₁₂ binding protein present in normal serum has been shown to participate in transport of labelled vitamin B₁₂ absorbed from the gut. All three vitamin B₁₂ binding proteins in serum were labelled at the same time after oral administration of vitamin B₁₂, implying that “free” vitamin B₁₂ reached the portal blood from the gut mucosa.     

Enhancement of vitamin B6 levels in rice expressing Arabidopsis vitamin B6 biosynthesis de novo genes

Vitamin B₆ (pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B₆ content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B₆ in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B₆ biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B₆ levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B₆ biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B₆ in leaves (up to 28.3‐fold) and roots (up to 12‐fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B₆ did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B₆ content could also be achieved in rice seeds (up to 3.1‐fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B₆‐enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.     

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.     

Biosynthesis of vitamin B12: Isobacteriochlorins,the link between corrins and sulphite reductases

The beautiful structure of vitamin B₁₂ has attracted great interest in its biosynthesis. This review briefly outlines the background and then covers very recent developments which have shown the importance of isobacteriochlorins in relation to the B₁₂ biosynthetic pathway. These isobacteriochlorin systems are also key materials for sulphite and nitrite reductase enzymes, and thus they act as a bridge, presumably an evolutionary bridge, between these enzymes and vitamin B₁₂. Determination of the structures of the isobacteriochlorins opens up the central part of the biosynthetic pathway to the corrin macrocycle.     

Effects of maternal vitamin B12 deficiency from end of gestation to weaning on the growth and haematological and immunological parameters in mouse dams and offspring

Vitamin B₁₂-deficiency may induce specific symptoms as neurological alterations and unspecific symptoms such as anaemia and growth retardation. In this study, maternal vitamin B₁₂ deficiency from end of gestation to weaning was evaluated in mouse dams, which was provoked by feeding a vitamin B₁₂-deficient diet. The animals were divided into two groups (control and deficient). The control group received the vitamin B₁₂-deficient diet supplemented with commercial vitamin B₁₂. Compared to the control, the vitamin B₁₂-deficient dams and their offspring showed a significant decrease of body weight (by 20 and 39%, respectively), serum vitamin B₁₂ concentration (by 61 and 67%, respectively), haematological values as haematocrit (25 and 26%, respectively), and IgA producer cells (by 36 and 54%, respectively). In both, vitamin B₁₂-deficient mouse dams and their offspring, histological alterations of small intestine were observed, whereas growth retardation occurred in the offspring only. This experimental murine model allows assessing the incidence of maternal cobalamin deficiency in offspring and would be useful for evaluating novel adjuncts such as functional foods to prevent vitamin B₁₂ deficiency.     

Biochemical Studies During the Development of the Chick Embryo

The days when the concentration of conjugated- and free- vitamin B₁₂ per wet weight g increased or decreased agreeded with that of nucleic acids in the chick embryo from 3rd through 18th day of incubation. It is intereresting that the day of increasing or decreasing of those compounds concentration corresponds with the important stages through the growth of embryo. The changes of these compounds in the egg contents free of embryo were estimated. Although a considerable large amount of free vitamin B₁₂ was contained in the embryo, the most vitamin B₁₂ in the egg content except the embryo existed in the conjugated state. It seems that the amount of total vitamin B₁₂ in the egg during the incubation is relatively constant.     

Effect of nutrient limitation and two-stage continuous fermentor design on productivities during "Clostridium ragsdalei" syngas fermentation

The effect of three limiting nutrients, calcium pantothenate, vitamin B₁₂ and cobalt chloride (CoCl₂), on syngas fermentation using “Clostridium ragsdalei” was determined using serum bottle fermentation studies. Significant results from the bottle studies were translated into single- and two-stage continuous fermentor designs. Studies indicated that three-way interactions between the three limiting nutrients, and two-way interactions between vitamin B₁₂ and CoCl₂ had a significant positive effect on ethanol and acetic acid formation. In general, ethanol and acetic acid production ceased at the end of 9 days corresponding to the production of 2.01 and 1.95 g L⁻¹ for the above interactions. Reactor studies indicated the three-way nutrient limitation in two-stage fermentor showed improved acetic acid (17.51 g g⁻¹ cells) and ethanol (14.74 g g⁻¹ cells) yield compared to treatments in single-stage fermentors. These results further support the hypothesis that it is possible to modulate the product formation by limiting key nutrients during C. ragsdalei syngas fermentation.