Studies on Degradation of d-Biotin by Microorganisms

During the course of our investigations on the metabolism of d-biotin by microorganism, it has been found that some strains of fungi belonging to the genera Rhodotorula, Penicillium and Endomycopsis, are able to degrade d-biotin oxidatively into various biotin vitamers. The present work was undertaken to characterize these vitamers. The vitamers formed were separated by the ion exchange column chromatography, into Fraction A (d-biotin sulfoxide), Fraction B (unknown vitamer II), Fraction C (d-biotin) and Fraction D (unknown vitamer I). Rf values of vitamer I and vitamer II were found to be different from those of the known biotin vitamers. The vitamers I and II did not support the growth of Lactobacillus arabinosus and Saccharomyces cerevisiae, but did support that of Bacillus subtilis. This degradation reaction occurred rather favorably in high aerobic condition.     

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.     

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 Degradation of d-Biotin by Microorganisms

During the course of investigations on the metabolism of d-biotin by microorganisms, the authors have found that a strain belonging to Endomycopsis effectively converted d-biotin into unknown biotin vitamers. The unknown biotin vitamers formed were isolated in crystalline form from the culture filtrate of a strain of Endomycopsis species and characterized as bisnorbiotin and bisnorbiotin sulfoxide by their physico-chemical and biological properties. The isolated vitamers were shown to support the growth of Bacillus subtilis, but not of Saccharomyces cerevisiae and of Lactobacillus arabinosus. The degradative pathway of d-biotin in microorganisms was also discussed.     

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 vitamers content of lactose and beet molasses

The biotin activity of beet and lactose molasses against the test strain Saccharomyces cerevisiae 225 by auxanographic method was evaluated. The level of lactose molasses biotin activity is almost twice as high as that obtained in the case of beet molasses. The results of bioautography with test strains Saccharomyces cerevisiae 225 and Lactobacillus arabinosus 17-5 indicate the qualitative composition of biotin derivatives (vitamers) in both molasses. Depending on the various technological steps e.g. sterilization or clarification one may find differences in the content and qualitative composition of biotin vitamers.     

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.     

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 by Yarrowia lipolytica

The hydrocarbon utilizing yeast Yarrowia lipolyyica NCYC 1421 produces biotin and its vitamers when grown on glucose in biotin-free media. Levels of production can be influenced by the medium composition. Growth in the presence of longchained fatty acids greatly increases biotin vitamer production. The biotin vitamers produced are normally dethiobiotin and 7-keto, 8-aminopelargonic acid. The addition of succinic acid at 0.5 g per litre causes the vitamer 7, 8-diaminopelargonic acid to be produced at high levels. The biotin antagonist α-dehydrobiotin inhibits the growth of Yarrowia lipolytica . Mutants can be readily isolated which show resistance to α-dehydrobiotin, but these do not produce greater amounts of biotin or its vitamers.     

Metabolism of biotin and analogues of biotin by microorganisms. II. Further studies on the conversion of D-biotin to biotin vitamers by Lactobacillus plantarum

Birnbaum, Jerome (University of Cincinnati, Cinncinati, Ohio), and Herman C. Lichstein. Metabolism of biotin and analogues of biotin by microorganisms. II. Further studies on the conversion of d-biotin to biotin vitamers by Lactobacillus plantarum. J. Bacteriol. 92:913-919. 1966.-Lactobacillus plantarum growing in excess biotin converts a portion to two vitamers (combinable and uncombinable with avidin) not utilizable for growth. These were detected by differential yeast-lactobacillus assay. In the present study, suspensions of 12- and 72-hr cells showed no converting activity. Vitamer formation by nonproliferating 24-hr cells required glucose and exhibited a lag; 17-hr cells showed neither a lag nor a glucose requirement. Iodoacetate and chloramphenicol inhibited vitamer formation by 24-hr cells, but had no effect on 17-hr cells. Addition of hydrolyzed casein or preincubation in biotin decreased the lag and enhanced vitamer formation in 24-hr cells, but had no effect in 17-hr cells. Apparently, 17-hr cells contain the converting enzymes which degenerate as growth proceeds; the lag exhibited by 24-hr cells represents the time necessary to reform the enzymes. Equal amounts of the two vitamers were formed in 17-hr cells; only the avidin-combinable form was produced initially by 24-hr cells, unless hydrolyzed casein was present. Electrophoresis revealed that the avidin-combinable vitamer has the same charge as biotin,whereas the uncombinable form possesses both positive and negative groups. Column chromatography was used to separate the avidin uncombinable material from biotin and the avidin-combinable form. L. plantarum was unable to accumulate the avidin-uncombinable vitamer under conditions permitting good biotin accumulation. It was concluded that L. plantarum sequentially converts biotin to avidin-combinable and -uncombinable vitamers, the latter being impermeable to the cells.     

Biosynthesis of biotin in microorganisms. V. Control of vitamer production

Use of a yeast-lactobacillus differential microbiological assay permitted investigation into the synthesis of biotin vitamers by a variety of bacteria. A major portion of the biotin activity was found extracellularly. The level of total biotin (assayable with yeast) greatly exceeded the level of true biotin (assayed with lactobacillus). Values for intracellular biotin generally showed good agreement between the assays, suggesting the presence of only true biotin within the cells. Bioautographic analysis of the medium after growth of each organism revealed the presence of large amounts of a vitamer which corresponded to dl-desthiobiotin on the basis of Rf value and biological activity. Biotin, when detected at all, was at very low concentrations. Also, an avidin-uncombinable vitamer was synthesized by a majority of the bacteria. Addition of d-biotin to the growth medium prevented completely the synthesis of both vitamers of biotin. d-Biotin-d-sulfoxide had no effect on the synthesis of desthiobiotin or the avidin-uncombinable vitamer. Addition of dl-desthiobiotin did not prevent its own synthesis nor that of the other vitamer. Control of vitamer synthesis is therefore highly specific for d-biotin. The avidin-uncombinable vitamer was produced only at repressed levels in the presence of high concentrations of both d-biotin and dl-desthiobiotin, which suggested that it is not a degradation product of these substances. A possible mechanism for the overproduction of the biosynthetic precursors of biotin is presented.     

Studies on Substances Active on the Behavior of Planarian

Substances which acted on the behavior of planarian were searched for, and two active components were isolated from culture broth of Streptomyces sp. 340. They were identified as trans-3-methylthioacrylic acid (MTAA) and 3-methylthiopropionic acid (MTPA).

It was found that many Streptomyces and some fungi accumulated MTAA, and many Streptomyces, fungi, bacteria and yeasts accumulated MTPA when the microorganisms were grown in the medium containing methionine.     

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.     

An early intermediate in the biosynthesis of biotin: Incorporation studies with [1,7-C(2)]pimelic acid

1. An unknown biotin vitamer was obtained in high yields in culture filtrates of Penicillium chrysogenum. 2. Production of this vitamer and desthiobiotin is controlled by the biotin concentration in the medium. 3. The unknown vitamer becomes labelled when the organism is grown in the presence of radioactive pimelic acid. 4. Chromatographic procedures were developed for the purification of the radioactive vitamer. 5. The vitamer is extremely stable in concentrated acid but gives rise to new vitamers under certain conditions. 6. The intermediate role of this vitamer in the synthesis of biotin is discussed.     

Studies on Production of Biotin by Microorganisms

During the course of the study on biotin vitamers production by a hydrocarbon-utilizing bacterium, strain 5–2 (Pseudomonas sp.), it was found that crude RNA-alkali-hydrolyzate from yeast increased the accumulaion of biotin vitamers, most of which was determined as desthiobion, and that adenine in the crude RNA-alkali-hydrolyzate was a potent stimulator. Effect of adenine on biotin vitamers accumulation was observed in the medium with either hydrocarbon or glucose as a sole carbon source. The accumulation of total biotin vitamers by some other bacteria was also increased by adenine but that of true biotin was scarcely increased or inhibited by adenine.

The role of adenine on the accumulation of biotin vitamers was investigated with non-proliferating cells of strain 5–2, and it was supposed that adenine would not only inhibit the accumulation of true biotin but, as a result, cause the large accumulation of biotin vitamers which might be intermediates of biotin synthesis. When the medium was supplemented with excess biotin, complete repression occurred even in the presence of adenine.     

Production of biotin by yeasts

A quantitative screening procedure for biotin and biotin vitamer production was conducted on 129 yeast strains able to grow in a biotin-free medium. Production of biotin and biotin vitamers varied considerably from strain to strain even within a species. The best producers of biotin were strains of Sporobolomyces roseus and Rhodotorula rubra whilst strains of Rhodotorula rubra and Yarrowia lipolytica produced the largest amounts of vitamers.     

Studies on Biosynthesis of Biotin by Microorganisms

A biotinless mutant (K-681-UV-134) accumulated a large amount of desthiobiotin and an unknown biotin-vitamer in the culture medium.

The parent strain (K-681) of this mutant isolated from soil was identified as Bacillus cereus.

The unknown vitamer was accumulated at the early stage of the incubation in comparison with desthiobiotin.

The unknown vitamer was purified by the paper- and column-chromatographic methods from the culture filtrate. The purified vitamer gave a single spot when spraying with the ninhydrin reagent after paper chromatographing and its R_F values in several solvent systems were identical with those of authentic 7-keto-8-aminopelargonic acid.     

Studies on Production of Biotin by Microorganisms

Biotin derivatives with biotin activity for some biotin-requiring microorganisms have been isolated in crystalline form from the culture filtrate of strain 194, identified as Rhodotorula flava. The crystalline vitamer was identified as d-biotinamide.     

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.     

Studies on the Lipoprotein Lipases of Microorganisms

About 2000 strains of microorganisms were examined for lipoprotein lipase producibilities and some microorganisms were found to produce lipases similar to animal lipoprotein lipases.

Microorganisms were cultured on solid media containing a serum-activated olive oil emulsion, and strains which formed a clear zone around the colony were collected. The collected microorganisms were cultured on liquid media containing 0.5% of olive oil by shaking and the culture filtrates were tested for lipoprotein lipase activity by a turbidity method. The superior lipoprotein lipase producers obtained belonged to genera of Serratia, Pseudomonas, Mucor, and Streptomyces.