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.     

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.     

Biosynthesis of Biotin-vitamers from Pelargonie Acid by Pseudomonas sp. Strain 393

The biosynthesis of biotin-vitamers from pelargonic acid by Pseudomonas sp. strain 393 was investigated. The main product of biotin-vitamers from pelargonic acid was desthiobiotin. The addition of streptomycin or l-alanine enhanced accumulation of desthiobiotin in culture fluid. Propionic, pimelic and azelaic acids were identified as main metabolites from pelargonic acid. When propionic acid was incubated with resting cells, pelargonic and azelaic acids were formed. The biosynthetic pathway of pelargonic acid to pimelic acid was also studied.     

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.     

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.     

Studies on Biosynthesis of Biotin by Microorganisms

Conversion of desthiobiotin to biotin by various kinds of microorganisms such as molds, Streptomyces, bacteria and yeasts was studied. The results described in the present paper showed that various kinds of microorganisms converted desthiobiotin to biotin during the cultivation of these microorganisms.

The conversion product from desthiobiotin by these microorganisms was chromatographically identified as biotin. The relationship between the producibilities of desthiobiotin and biotin from pimelic acid, and biotin synthesis from desthiobiotin was also presented.     

Biotin-vitamer Formation from Salicylic Acid by Pseudomonas sp.

Biotin-vitamer formation from salicylic acid was investigated. Strains of Pseudomonas sp., No. 102 and No. 362, isolated from soil samples utilized well salicylic acid as a sole source of carbon, and formed biotin-vitamers in culture broth. The metabolites were partially purified by the methods of active carbon adsorption and anion-exchange column chromatography, and clarified as desthiobiotin, bisnordesthiobiotin and 7-keto-8-aminopelargonic acid.     

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.     

Formation of Desthiobiotin from Oleic Acid by Brevibacterium sp.

Microbial formation of biotin-vitamers from oleic acid was investigated. Many strains of bacteria which were able to utilize oleic acid as a sole carbon source were isolated from soils and other natural materials. Among these bacteria, some strains formed a biotin-vitamer from oleic acid in the culture broth during the cultivation. The vitamer was purified from the culture broth of strain No. 23, and identified as desthiobiotin by chromatographical and biological methods.

From the results of investigation on the taxonomical characteristics, the bacterial strain No. 23 was assumed to be Brevibacterium sp.     

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.     

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.     

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.     

Antitumor Activity of a Pterin Deaminase

A variety of bacteria and yeasts were examined for activities of biotin biosynthetic enzymes, including pimelyl-CoA synthetase, 7-keto-8-aminopelargonic acid (KAPA) synthetase, 7,8-diaminopelargonic acid (DAPA) aminotransferase and dethiobiotin (DTB) synthetase. Among the strains tested, only Bacillus sphaericus, a DTB producer, showed significant activities for all four enzymes. The bacterium also exhibited high activity of biotin synthesis from DTB in an intact cell system. Using cell-free extract and intact cells, some properties of DAPA aminotransferase, DTB synthetase and biotin synthesizing reaction were examined.

Based on these results of enzyme activities DTB productivity of B. sphaericus was discussed.     

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.     

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.     

Bioconversion of Dethiobiotin into Biotin by Resting Cells and Protoplasts of Bacillus sphaericus bioB Transformant

The reaction system for the bioconversion of dethiobiotin into biotin by resting cells and protoplasts of a Bacillus sphaericus bioB transformant was established. The reaction mixtures consisted of completely synthetic components, such as amino acids and metal salts. Among the sulfur compounds tested, L-CyS and L-cystine were effective in the biosynthesis of biotin from dethiobiotin both by resting cells and by protoplasts. The optimum concentrations of L-Cys were 2 to 3 mM and more than 0.25 mM for resting cell and protoplast systems, respectively. Vigorous shaking enhanced the biotin biosynthesis by protoplasts. The addition of yeast extract to the reaction mixture without a mixture of amino acids brought about a three-fold increase in the, amount of biotin synthesized by protoplasts when compard to the case with the reaction mixture containing the amino acid mixture. The amount of biotin synthesized by protoplasts increased with the incubation time up to 6 h and reached about 2 μg/ml. There was a clear correlation between the number of remaining protopiasts and their biotin-biosynthesizing activity during the incubation.     

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.     

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.     

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.     

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.