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.     

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.     

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.     

Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

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.     

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 Utilization of Hydrocarbons by Yeasts

The production of microbial cell substances from hydrocarbons has been attracting attention of people for many years. Production of bacterial cell from hydrocarbons is disadvantageous because of the difficulty in separating cell from the broth.

We have tested hydrocarbon-utilizing yeasts isolated from garden soil for cell production. The effect of medium composition on yeast growth and the utilization of individual hydrocarbon by yeast, strain Y-3, were investigated.

As a nitrogen source, urea was more effective than ammonium nitrate. When a very smal! amount of corn steep liquor was added, yeast growth was very improved. Aliphatic series of hydrocarbon lower than C₉ were not or very slightly assimilated by this yeast.

Generally speaking, series of even-number hydrocarbons were more effective than those of odd-number hydrocarbons.

We found that the yeast Y-3 strain reported in the previous paper¹⁾ has a diterminal oxidation system of hydrocarbon.

This yeast capable of growing in mineral-salts solution with hydrocarbons as sole source of carbon produced a series of dioic acid from n-undecane. These acids are 1,11-undecane dioic acid, 1,9-nonane dioic acid (azelaic acid), 1,7-heptane dioic acid (pimelic acid) and 1,5-pentane dioic acid (glutaric acid). 1,10-Decane dioic acid (sebacic acid) was also isolated from n-decane cultures.

Azelaic acid was partially transformed into pimelic acid and glutaric acid by treating it with resting cells of this yeast.

1,11-Undecane dioic was also transformed into azelaic acid pimelic acid, and glutaric acid by the same treatment as described above.     

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.     

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.     

Metabolism of deuterium-labeled nonanoic acids in the riboflavin-deficient rat model of multiple acyl-CoA dehydrogenase deficiency

Riboflavin-deficient rats are used to study the metabolism of deuterium-labeled nonanoic acids under conditions mimicking the human disorder of multiple acyl-CoA dehydrogenase deficiency in which large amounts of ethyl-malonic, glutaric, adipic, suberic, 4-octenedioic, sebacic and 4-decenedioic acids are excreted. Both control and deficient rats convert the nonanoic acids to labeled azelaic and pimelic acids. The labeling pattern in pimelic acid is consistent with the omega-oxidation of nonanoic acids to azelaic acid followed by beta-oxidation to pimelic acid.     

Oleic acid synthesis in the fruit of Persea americana MILL

The tissue slices from the mesocarp of avocado could incorporateradioactive acetate into lipids. Oleic, palmitic and stearicacids were the most labeled fatty acids found to accumulatein triglycerides. The conclusions that oleic acid was formedby way of chain elongation of already unsaturated short-chainfatty acids, and that there was no evidence for the desaturationof stearic acid were based on the following observations: 1)Stearic acid-¹⁴C was incorporated into triglycerides by thetissue slices without formation of oleic acid. 2) The oleicacid synthesized from specifically labeled acetate was not randomlylabeled. The specific radioactivity of azelaic acid moiety ofoleic acid was rapidly increased while that of pelargonic acidmoiety was gradually increased. 3) An unexpected rise of stearicacid was observed among commonly occurring fatty acids in thetissue slices. This was accentuated by anaerobiosis which prevailedduring vacuum infiltration of labeled acetate. ¹Present address: Department of Botany, Faculty of Agriculture,Hokkaido University, Sapporo, Japan. (Received January 13, 1969; )     

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.     

On the effect of aliphatic saturated dicarboxylic acids on anaerobic glycolysis in chicken embryo

Oxalic, succinic, glutaric and pimelic acid (5 mM) had no effect on lactate formation from glucose if added to a crude extract of chicken embryo at the same time as substrate and cofactors; conversely malonic, adipic, suberic, azelaic and sebacic acid had an inhibitory effect ranging from 20% to 35%. When the enzyme preparation was pre-incubated with the dicarboxylic acids for one hour before beginning the experiments, all compounds tested, with the exception of succinate, inhibited anaerobic glycolysis. Hexokinase activity was significantly reduced by saturated dicarboxylic acids from C3 to C10, but not by oxalic acid. Phosphofructokinase was inhibited only by oxalic, pimelic and suberic acid. Pyruvate kinase appeared sensitive only to oxalic acid (64% inhibition).     

Fatty Acid Hydroxamate Formation by Intact Cells of Micrococcus sp.

When azelaic acid was used as a sole carbon source on the growth of Micrococcus sp. which was isolated from soil, intact cells of the organism catalyzed the enzymic condensation of fatty acids with hydroxylamine. Some of the characteristics of fatty acid hydroxamate formation were investigated.

The enzyme activity was tested with azelaic acid compared to other fatty acids. Azelylhydroxamate formation was activated with the addition of reduced glutathione or 2-mercaptoethanol. The reaction was inhibited by p-chloromercuribenzoate (PCMB), ethylene diamine tetraacetate (EDTA), NaF and benzoate.     

Fatty Acid Metabolism in Microorganisms

During the investigation on the metabolism of azelaic acid by Micrococcus sp., it was found that the bacterium produced a large amount of keto acid (α-ketoglutaric acid) under the restricted condition for nitrogen source. The acid was identified as α-ketoglutaric acid by physico-chemical and biological methods. The mechanism of the production of α-ketoglutaric acid from azelaic acid was investigated. From the result, it was suggested that α-ketoglutaric acid production proceeded thrpugh the further oxidation of acetic acid produced from azelaic acid and that the production might be functioned by TCA cycle enzymes of the bacterium. Similarly, α-ketoglutaric acid was found to be produced remarkably from other various fatty acids.     

Gas chromatography-mass spectrometry profile of urinary organic acids of Wistar rats orally treated with ozonized unsaturated triglycerides and ozonized sunflower oil

The main products in the ozonolysis of unsaturated triglycerides or vegetable oils are peroxides, aldehydes, Criegee ozonides and carboxylic acids. Some of these compounds are present in different concentrations in the biological fluids. The aim of this work is to study, using gas chromatography-mass spectrometry (GC-MS), the organic acid excretion in urine of rats orally treated with ozonized sunflower oil (OSO), ozonized triolein or ozonized trilinolein. Oral administration of OSO to Wistar rats has produced changes in the urinary content of dicarboxylic organic acids. Among others heptanedioic (pimelic acid) and nonanedioic acids (azelaic acid) were the major increased dicarboxylic acids found. The urinary dicarboxylic acid profiles of rats which received ozonized triolein only showed an increase in heptanedioic and nonanedioic acids. However, when ozonized trilinolein is applied, the profile is similar to that obtained when OSO is administered. A biochemical mechanism is proposed to explain the formation of dicarboxylic acids from ozonated unsaturated triglycerides.     

Fatty Acid Hydroxamate Formation and Ester Hydrolysis by Cell-free Extracts of Micrococcus sp.

When Micrococcus sp. which was isolated from soil assimilated azelaic acid as a sole carbon source, cell-free extract of the organism catalyzed enzymic fatty acid hydroxamate formation. The enzyme was effective only for mono-carboxylic acid, but not for di-carboxylic acids such as azelaic acid. The activity was high with higher fatty acid such as oleic acid. Some of the properties of higher fatty acid hydroxamate formation were investigated.

Olelylhydroxamate was formed with the high concentration of hydroxylamine. The reaction was inhibited by PCMB, but recovered by the addition of SH-compounds (such as cysteine).

On the other hand, when methylacetate was used as a sole carbon source and cell-free extract of Micrococcus sp. hydrolyzed several fatty acid esters. The fatty acid hydroxamate degradation by esterolysis are also discussed.     

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.     

Carboxylic Acids in Cultured Broth of Sporobolomyces odorus

Carboxylic acids found in the cultured broth of Sporobolomyces odorus AHU 3246 which produces γ-lactones as principles of the aromatic flavor, were analyzed. The concentrate of methylated acids was steam-distilled and in the residue, succinic acid, nonanedioic acid (azelaic acid), undecanedioic acid and 2-hydroxy-3-phenylpropionic acid (β-phenyllactic acid) were identified as their methyl esters by GLC and spectroscopic methods. Phthalic acid and its mono-n-butyl ester were also found, but these compounds were thought to arise from di-n-butyl phthalate, one of impurities of deionized water.