The Controlling Action of Actithiazic Acid on the Biosynthesis of Biotin-vitamers by Various Microorganisms

By the addition of actithiazic acid, or acidomycin (ACM), to culture media, the accumulation of desthiobiotin by various microorganisms was enhanced from two-fold to about seventyfold, while that of biotin was markedly reduced. Especially, Bacillus sphaericus accumulated 350 μg per ml of biotin-vitamers assayed with Saccharomyces cerevisiae. ACM was not incorporated into the desthiobiotin molecule by resting cells of B. sphaericus. The amount of biotin-vitamers assayed with S. cerevisiae which was synthesized from pimelic acid by the resting cells grown with ACM was twice as great as that synthesized by the cells grown without ACM. From these results, the mechanism of the controlling action of ACM on biotin biosynthesis was discussed.     

Studies on Production of Biotin by Microorganisms

The utilization of hydrocarbons by microorganisms was studied in many fields, but the production of biotin vitamers by hydrocarbon-utilizing bacteria has never been reported.

We have screened many hydrocarbon-utilizing bacteria which produce biotin vitamers in the culture broth. The effects of cultural conditions on biotin vitamers production by strain 5–2, tentatively assigned to the genus Pseudomonas, were studied.

More than 98% of biotin vitamers produced from hydrocarbons by strain 5–2 was chromatographically determined as desthiobiotin. As nitrogen source, natural nutrients were more effective than inorganic nitrogen sources. The production of biotin vitamers was increased under the condition of good aeration. Exogenous pimelic or azelaic acid enhanced biotin vitamers production by strain 5–2.

The production of biotin vitamers from n-alkanes, n-alkenes or glucose by an isolated bacterium, strain 5-2, tentatively assigned to the genus Pseudomonas, was investigated. Among these carbon sources, n-undecane was the most excellent for biotin vitamers production.

The biosynthetic pathway of biotin vitamers, especially desthiobiotin, from n-undecane was also studied. It was found by thin-layer and gas-liquid chromatographical methods that pimelic and azelaic acids were the main acid components in n-undecane culture.

This result, together with previously reported enhancement of biotin vitamers production by these acids, suggests that pimelic and azelaic acids may be the intermediates of biotin vitamers biosynthesis from n-undecane.     

Studies on Biosynthesis of Biotin by Microorganisms

During the course of the study on the production of biotin from desthiobiotin by microorganisms, the present authors have found that some strains of molds produced an unknown biotin-vitamer (BS-factor) from desthiobiotin. The present investigation was undertaken to clarify the characteristics of the unknown vitamer. The unknown vitamer produced from desthiobiotin was isolated in crystalline form from culture filtrate of Aspergillus oryzae. The compound isolated was identified as 4-methyl-5-(ω-carboxybutyl)-imidazolidone-2 by the physico-chemical procedures.

The biosynthesis of biotin-vitamers by resting cell system of Bacillus sphaericus was studied.

It was found that pimelic acid was essential substrate in biosynthesis of biotin-vitamers and that some amino acids and organic acids stimulated the biosynthesis of biotin-vitamers from pimelic acid. Alanine was found to be most effective. It was assumed that, in the presence of pimelic acid, some amino acids, especially alanine, and some organic acids play an important role in the biosynthesis of biotin-vitamers.

The main component of the biotin-vitamers synthesized by the resting cell system was identified as desthiobiotin. The existence of a small amount of unknown biotin-vitamer, an avidin-uncombinable substance, which was assumed to be 7-keto-8-amino-pelargonic acid, was also observed. True biotin was hardly observed in any conditions tested.     

Studies on Biosynthesis on Biotin by Microorganisms

The accumulation of biotin-vitamers in the culture media of a large number of microorganisms (about 700 strains) was studied. The contents of the biotin-vitamers were quantitatively determined by microbiological assays with Lactobacillus arabinosus and Saccharomyces cerevisiae.

It was found that large amounts of biotin-vitamers were accumulated by various microorganisms such as Streptomyces, molds and bacteria, and that the yield of biotin-vitamers was enhanced by the addition of pimelic acid or azelaic acid to the media. It was also found that the main portion of the vitamers accumulated by many microorganisms did not support the growth of Lactobacillus arabinosus, while it did support that of Saccharomyces cerevisiae. The small amounts of true biotin were observed in the culture media of various Streptomyces and molds, but hardly in the culture media of bacteria.

The identification of biotin-vitamers accumulated by various microorganisms is described, and the distribution of the vitamers in microorganisms is also described.

The results presented in this paper show that the main component of the vitamers accumulated by many microorganisms is identified as desthiobiotin by anion exchange column chromatography, paper chromatography and chemical analysis. Small amounts of fraction B (unidentified vitamers) and Fraction D (biotin) were also detected in the culture media of various molds and Streptomyces. However, these fractions were not observed in the culture media of any bacteria tested.

It was also found that large amounts of an unknown biotin-vitamer was accumulated by various bacteria. The vitamer was avidin-uncombinable, and, from the paper electrophoretic studies, it was assumed that the vitamer might be an analogue of pelargonic acid.     

Biosynthesis of Biotin-vitamers from Glutaric Acid,a New Biotin Precursor

Biotin-vitamers were synthesized from glutaric acid by resting cells of certain strains of Agrobacterium. Pimelic acid, which has been known as a biotin precursor in many microorganisms, was not effective at all to this species. Optimum conditions for the biosynthesis of the vitamers by resting cells of Agrobacterium radiobacter IAM 1526 were investigated. L-Lysine was also effective, but the rate of the biosynthesis of biotin-vitamers from L-lysine was one-half that from glutaric acid. The vitamer synthesized was bioautographically identified as desthiobiotin. It was confirmed that ¹⁴C-labelled glutaric acid was incorporated into the desthiobiotin molecule.     

Studies on Production of Biotin by Microorganisms

In a preceding paper we reported that Rhodotorula flava 194 effectively converted biotin to biotinamide. In a present paper the metabolism of desthiobiotin by R. flava 194 was studied under the same condition as in the conversion of biotin to biotinamide. Two desthiobiotin derivatives (Vitamer I and II) were isolated. Vitamer II (crystalline) was identified as bisnordesthiobiotin and Vitamer I was chromatographically determined as desthiobiotinamide.     

Biosynthesis of Biotin in Microorganisms VI. Further Evidence for Desthiobiotin as a Precursor in Escherichia coli

Biotin auxotrophs were isolated from Escherichia coli K-12. One of the mutants was unable to grow on desthiobiotin and accumulated a large amount of a vitamer in medium when growing on an optimal concentration of biotin. The production of the vitamer was inhibited in the presence of an excess amount of biotin. The vitamer was identified as desthiobiotin on the basis of biological activities, avidin combinability, and chromatographic characteristics. The mutant lacked the ability to convert desthiobiotin to biotin. These results further support the hypothesis that desthiobiotin is a normal intermediate in the biosynthesis of biotin in E. coli.     

Further Characterization of Ureido Ring Synthetase from Pseudomonas graveolens

Detailed enzymatic properties of the ureido ring synthetase purified from Pseudomonas graveolens were investigated. Nucleotide specificity studies indicated that CTP, UTP, GTP, and ITP were each tenth to one-fifth as active as ATP. The effect of substrate concentration was examined. The Km values for 7,8-diaminopelargonic acid, biotin diaminocarboxylic acid, NaHCO₃, ATP, and MgCl₂ were 1 × 10^?4 M, 4 × 10^?5 M, 1 × 10^?2 m, 5 × 10^?5 M, and 3 × 10^?3 M, respectively. It was elucidated that only ADP was produced from ATP in both the reaction of desthiobiotin synthesis from 7,8-diaminopelargonic acid and biotin synthesis from biotin diaminocarboxylic acid. The reaction was remarkably inhibited by Ni²⁺, Cd²⁺, Cu²⁺, Ag⁺, and As³⁺, while Mn²⁺ remarkably enhanced the enzyme reaction. The reaction was remarkably inhibited by metal-chelating reagents. It was elucidated that ADP had a competitively inhibiting effect on this enzyme reaction. 7,8-DiaminopeIargonic acid, which is the substrate for the desthiobiotin synthesis, competitively inhibited the biotin synthesis from biotin diaminocarboxylic acid. The stoichiometry of the desthiobiotin synthesis indicated that the formation ratio of desthiobiotin to ADP was 1 to 1.     

Biosynthesis of Desthiobiotin in Cell-free Extracts of Escherichia coli

Cell-free extracts of Escherichia coli were active in catalyzing the synthesis of a biotin vitamer from 7,8-diaminopelargonic acid. The vitamer was identified as desthiobiotin on the basis of its chromatographic and electrophoretic characteristics and its biotin activities for a variety of microorganisms. The reaction was stimulated five-fold by bicarbonate, suggesting that an "active CO(2)" was incorporated into the carbonyl carbon of desthiobiotin. The enzyme was demonstrable in a wild-type (K-12) and in all biotin mutants of E. coli that were tested, with the exception of a strain which was able to grow on desthiobiotin but not on diaminopelargonic acid. Furthermore, the enzyme was repressible by biotin in all of the strains tested. These results are consistent with the hypothesis that the biosynthesis of desthiobiotin from 7,8-diaminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.     

Metabolism of biotin and analogues of biotin by microorganisms. IV. Degradation of biotin, oxybiotin, and desthiobiotin by Lactobacillus casei

Birnbaum, Jerome (University of Cincinnati, Cincinnati, Ohio), and Herman C. Lichstein. Metabolism of biotin and analogues of biotin by microorganisms. IV. Degradation of biotin, oxybiotin, and desthiobiotin by Lactobacillus casei. J. Bacteriol. 92:925-930. 1966.-Lactobacillus casei degrades biotin when it is present in excess to products not utilizable for growth by L. plantarum or Saccharomyces cerevisiae. Degrading activity was initiated in the early stationary phase and was controlled by the pH of the medium. Nonproliferating cells, grown previously in excess biotin for 40 hr, metabolized oxybiotin and desthiobiotin as well as biotin. Cells grown in low biotin, or in excess biotin for 20 hr, did not degrade either analogue. Oxybiotin was 50% as active as biotin for growth, whereas desthiobiotin acted as a competitive inhibitor. Cells grown in excess biotin for 40 hr, but not 20 hr, overcame the inhibitory effect of desthiobiotin, when subcultured to media containing a normally inhibitory concentration of the analogue. Moreover, the level of desthiobiotin dropped rapidly during the first 4 to 6 hr before growth ensued. The data indicate that growth in excess biotin enables L. casei to degrade desthiobiotin and, thereby, to overcome the inhibitory effect of the analogue.     

Synthesis of Desthiobiotin from 7,8-Diaminopel-argonic Acid in Biotin Auxotrophs of Escherichia coli K-12

The synthesis of desthiobiotin from 7,8-diaminopelargonic acid (DAP) was demonstrated in resting cell suspensions of Escherichia coli K-12 bioA mutants under conditions in which the biotin locus was derepressed. The biosynthetically formed desthiobiotin was identified by chromatography, electrophoresis, and by its ability to support the growth of yeast and those E. coli biotin auxotrophs that are blocked earlier in the biotin pathway. Optimal conditions for desthiobiotin synthesis were determined. Desthiobiotin synthetase activity was repressed 67% when partially derepressed resting cells were incubated in the presence of 3 ng of biotin per ml. Serine, bicarbonate, and glucose stimulated desthiobiotin synthesis apparently by acting as sources of CO(2). The results of this study are consistent with an earlier postulated pathway for biotin biosynthesis in E. coli: pimelic acid --> 7-oxo-8-aminopelargonic acid --> DAP --> desthiobiotin --> biotin.     

Metabolism of biotin and analogues of biotin by microorganisms. 3. Degradation of oxybiotin and desthiobiotin by Lactobacillus plantarum

Birnbaum, Jerome (University of Cincinnati, Cincinnati, Ohio), and Herman C. Lichstein. Metabolism of biotin and analogues of biotin by microorganisms. III. Degradation of oxybiotin and desthiobiotin by Lactobacillus plantarum. J. Bacteriol 92:920-924. 1966.-Lactobacillus plantarum growing in excess oxybiotin degraded a portion to products not utilizable by Saccharomyces cerevisiae. The loss of activity for the yeast suggested that no vitamers of oxybiotin accumulated during the degradation. The initiation of degrading activity was controlled by the pH of the growth medium and appeared during early stationary phase. Only cells grown in excess oxybiotin could degrade this biotin analogue. Nonproliferating cells grown previously in excess oxybiotin were able to convert biotin to vitamers (active for the yeast) as well as to degrade oxybiotin. Those grown in excess biotin also developed the ability to degrade oxybiotin as well as to convert biotin; however, in this case, the enzymes degenerated more rapidly. Cells grown with excessive amounts of either material were able to degrade desthiobiotin to products not available for the yeast. Both biotin conversion and oxybiotin degradation were found to have the same requirements for Mg and Mn ions. It was concluded that conversion of biotin to vitamers, and the degradation of oxybiotin or desthiobiotin are functions of the same on closely related enzyme systems.     

Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in <Emphasis Type="Italic">E. coli</Emphasis>

Background  

Biotin is an essential enzyme cofactor that acts as a CO₂ carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for ³⁵S labeling of biotin.     

Studies on the biosynthesis of biotin. Production of biotin and biotin-like compounds by a pseudomonad

1. Filtrates from cultures of a strain of Pseudomonas aeruginosa, grown in a basal glucose-ammonium chloride-vitamins-salts medium, possessed biotin activity as detected by microbiological assays. Exponential-phase culture filtrates contained biotin and desthiobiotin in the approximate ratio 1:3, with smaller amounts of biotin sulphoxide and three unidentified compounds with biotin activity. 2. The addition of malonate, adipate or pimelate to the basal medium stimulated the production of compounds with biotin activity; this effect was enhanced when these compounds were included in the medium as the major carbon source. Succinate, glutarate, suberate, fumarate or oxaloacetate did not stimulate the production of compounds with biotin activity. The ratio of biotin to desthiobiotin in filtrates from cultures grown in medium containing malonate as the carbon source was about 1:1. Experiments in which mixtures of malonate and pimelate were included in the medium as the carbon sources showed that these acids probably make a similar contribution in biotin biosynthesis. 3. A number of heterocyclic compounds, including several containing the ureido group (-NH-CO-NH-), were included in the basal medium but none of them stimulated the production of compounds with biotin activity to any marked degree. 4. Several amino acids, particularly cysteine (or cystine) and lysine, when added individually as supplements to the basal medium, stimulated the production of compounds with biotin activity. Filtrates from cultures grown in medium supplemented with cysteine contained approximately equal proportions of biotin and desthiobiotin. A much greater stimulation in the production of compounds with biotin activity was obtained when certain amino acids were included in the medium as the major source of nitrogen or carbon and nitrogen; ornithine, citrulline and argininosuccinate had the most marked effect. The ratio of biotin to desthiobiotin in filtrates from these cultures was usually greater than in filtrates from cultures grown in basal medium. 5-Aminovalerate also caused some stimulation when used as the nitrogen source, but urea was inactive. The effect of binary mixtures of certain amino acids was also examined. 5. The results are discussed in relation to the possible role of the stimulatory compounds during biotin biosynthesis.     

Effect of water soluble vitamins and their analogues on the growth of candida albicans. I. Biotin,pyridoxamine, pyridoxine and fluorinated pyrimidines

Summary C. albicans showed an absolute dependency for biotin in shaker cultures in a basal mineral synthetic medium free of vitamin-precursors and vitamin-sparing amino acids. Diminished growth activity was observed with biotin sulfone and biotin diamine sulfate, but not with biocytin, N-biotinyl--alanine, N-biotinyl-L-aspartic ethyl ester, D-desthiobiotin or biotin-D-sulfoxide. The ability of the organism to utilize desthiobiotin indicates that its block in biosynthesis of biotin occurs at a step prior to desthiobiotin biosynthesis. Pyridoxamine and pyridoxine were both highly growth stimulatory at 1000 and 2056 µg/ml but not in the vitamin range at 1 to 10 µg/ml. Since desoxypyridoxine compounds failed to inhibit growth in the absence of B₆, it was concluded thatC. albicans has no dependency for vitamin B₆, although it actively metabolizes it. Pyridoxamine shortened the lag phase of the organism and reversed the toxicity of 5-fluorouracil and 5-fluoro-2-deoxyuridine, pointing to a new role of vitamin B₆ in nucleic acid metabolism of the organism. Inhibition indices for pyridoxamine and pyridoxine versus FU and FUDR were inconstant, indicating that the antagonism with the fluoropyrimidines was non-competitive in nature and the B₆ competes with these compounds at more than one site on the cell.     

Biotin chemoresponse in Paramecium

Paramecium tetraurelia locate their␣foodsource by detecting bacterial metabolites and altering swimming behavior to congregate near bacterial populations on which they feed. Several attractants, such as folate, glutamate, cAMP and acetate have been identified and various aspects of chemoreception, signal transduction and effector mechanisms have been described. Here we characterize the Paramecium chemoresponse to biotin. An essential enzymatic cofactor in all cells, biotin is secreted by a large number of bacterial species during growth phase. P. tetraurelia are strongly attracted to biotin with a half-maximal behavioral response at 0.3 mmol · 1⁻¹ in T-maze assays. Physiological recordings from whole cells show that cells hyperpolarize in a concentration-dependent manner in biotin. Whole-cell binding assays utilizing ³H-biotin identify a saturable and specific binding site with an apparent dissociation constant of 0.4 mmol · l⁻¹. The biotin analogs desthiobiotin and biotin methyl ester are also strong attractants. Diaminobiotin fails to attract P. tetraurelia at 1 mmol · l⁻¹, but does interfere with the biotin chemoresponse and displaces ³H-biotin from whole cells. We hypothesize that the keto group and/or fidelity of the ureido ring of biotin are necessary for biotin chemoresponse. Accepted: 23 April 1998     

The ISC [corrected] proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.     

Incorporation of the sulfur of L-[S]methionine into the biotin molecule by intact cells of Rhodotorula glutinis

The source of sulfur for biotin in microorganisms was studied. Using intact cells of Rhodotorula glutinis AKU 4847, L-methionine was much more effective for the synthesis of biotin from dethiobiotin than various other sulfur compounds tested. The reaction was carried out in the presence of L-[³⁵S]methionine. The radioactive biotin synthesized was isolated from the reaction mixture by a procedure involving cation- and anion-exchange column chromatographies, avidin treatment and membrane filtration, and then identified by radiochromatography and bioautography with Lactobacillus arabinosus. It was thus shown that the sulfur of methionine was incorporated into the biotin molecule by R. glutinis.     

Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling

Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres^® and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.     

Biosynthesis of biotin: synthesis of 7,8-diaminopelargonic acid in cell-free extracts of Escherichia coli

Cell-free extracts prepared from a biotin auxotroph of Escherichia coli were active in catalyzing the synthesis of 7,8-diaminopelargonic acid, an intermediate of the biotin pathway, from 7-oxo-8-aminopelargonic acid. The product was identified on the basis of its chromatographic characteristics and its biotin activities for biotin auxotrophs of E. coli. Enzyme activity was determined in a reaction coupled with the desthiobiotin synthetase system, which is required for the conversion of 7,8-diaminopelargonic acid to desthiobiotin, and by measuring the amount of desthiobiotin formed by microbiological assay. The reaction was stimulated by l-methionine and pyridoxal-5'-phosphate. l-Methionine could not be replaced by any other amino acids tested. Pyridoxamine and pyridoxamine-5'-phosphate were as active as pyridoxal phosphate. The enzyme, presumably an aminotransferase, was demonstrable in the parent strain of E. coli and all mutant strains tested with the exception of a strain which is able to grow on diaminopelargonic acid but not on 7-oxo-8-aminopelargonic acid. Furthermore, the enzyme was repressible by biotin. The results were consistent with the hypothesis that the biosynthesis of 7,8-diaminopelargonic acid from 7-oxo-8-aminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.