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.     

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 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 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 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.     

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.     

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.     

The Pimelyl-CoA Synthetase Responsible for the First Step in Biotin Biosynthesis by Microorganisms

Cell-free extracts of various bacteria were active in catalyzing the synthesis of pimelyl-CoA from pimelic acid and CoA. The pimelyl-CoA formed was determined in a reaction coupled with the 7-keto-8-aminopelargonic acid (KAPA) synthetase system, which is required to convert pimely-CoA to KAPA, and by microbiologically assaying the amount of KAPA formed. The enzyme synthesizing pimelyl-CoA was named pimely-CoA synthetase and should belong to EC 6.2.1. The pimelyl-CoA synthetase reaction required pimelic acid, CoA, ATP and Mg²⁺. The enzyme was partially purified from a cell-free extract of Bacillus megaterium. Using purified enzyme, characterization of the enzyme was performed. The enzyme reaction was remarkably inhibited by typical metal-chelating agents. Mn²⁺ and ADP could replace Mg²⁺ and ATP, respectively. No feedback repression was observed even with the addition of 1.0 µg per ml of biotin to the culture medium.     

Conversion of Biotin Diaminocarboxylic Acid into Biotin and Bisnorbiotin by Resting Cell System of Microorganisms

The effects of dietary fat and protein levels on the conversion of Trp-Nam were investigated. In rats fed with 20% casein diets, the Trp-Nam conversion ratio [(urinary excretion of Nam + MNA + 2-Py + 4-Py in μmol/day)/(daily Trp intake during urine collection in μmol/day) × 100] was about 4.3% for the groups fed with the 20% corn oil and 20% soybean oil diets, 2.8% for the group fed with the 20% lard diet, and 2.1 % for the group fed with the no fat diet. In rats fed with 40% casein diets, a similar phenomenon was observed, but the ratios were 2.0%, 2.4%, 1.6%, and 0.8% for the groups fed with the 20% corn oil, 20% soybean oil, 20% lard, and no fat diets, respectively. From these results, it was found that an increase in fat intake elevated the conversion ratio regardless of the dietary protein level, while an increase in protein intake reduced it.     

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.     

Biosynthesis of Biotin from Dethiobiotin by Intact Cells of Biotin-producing Bacterium

Intact cells of a biotin-producing bacterium, KY–21–1–25, were found to synthesize biotin from dethiobiotin. Optimal conditions for the biosynthesis of biotin from dethiobiotin by intact cells were investigated. Intact cells harvested from adenine-supplemented medium showed intensive biosynthesis. However, the biosynthesis of biotin by intact cells was strongly inhibited by the addition of adenine or adenosine. The inhibitory activity of adenine was about 10-fold greater than that of adenosine. Formation of several unidentified biotin-vitamers was observed in both reaction mixtures incubated with and/or without addition of adenine.     

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 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 Kojic Acid Metabolism by Microorganisms

An enzyme, comenic aldehyde dehydrogenase, which catalyzes the oxidation of comenic aldehyde to comenic acid was partially purified from cell extract of Arthrobacter ureafaciens K-1.

The enzyme was purified 31-fold at Sephadex G-100 filtration step, 112-fold at DEAE-Sephadex A-50 fractionation step, and recovery of the activity was 73.3% and 38.5% respectively.

NADP and magnesium ion were essential for the oxidation. The enzyme shows optimum activity at pH 7.8. Enzyme activity was extremely sensitive to sulfhydryl reagents such as p-chloromercuribenzoate and monoiodoacetate. l-Cysteine or dithiothreitol protected the enzyme from p-chloromercuribenzoate inhibition. Carbonyl reagents, such as hydroxylamine and semicarbazide, inhibit the enzyme reaction by formation of addition compounds between carbonyl reagents and aldehyde group of the substrate. The enzyme was completely inactivated after heating for 5 min at 40°C The Km for 5-methoxy comenic aldehyde is 2.5×10^?6 m, and for NADP is 0.4×1O^?6 m. The reaction product, 5-methoxy comenic acid was identified by paperchromatography. The characterization of the enzyme has been carried out by using 5-methoxy comenic aldehyde as the substrate in stead of comenic aldehyde.     

Studies on Antibiotics Produced by Psychrophilic Microorganisms

A screening for antibacterial antibiotics was carried out with psychrophilic microorganisms. The most active microorganism, a soil actinomycete, was selected and characterized to be a facultative psychrophile, Streptomyces sp. No. 81. This strain was found to produce antibiotic(s) in the culture fluid only at low temperature cultivation below 20°C but not at moderate temperature. Mycelial growth at low temperature seemed to be indispensable for the antibiotic production. The antibiotic produced by Streptomyces sp. No. 81 was isolated and characterized. It appeared that the antibiotic had the selective toxicity against several Gram-positive bacteria. From the comparative studies with several known antibiotics, the antibiotic appears to be a new compound derived from the new metabolic routes involving temperature-sensitive mechanisms.     

Studies on Kojic Acid Metabolism by Microorganisms

An enzymatic oxidation of kojic acid to comenic aldehyde was found in the decomposition process of kojic acid by Arthrobacter ureafaciens strain (K-l), a kojic acid decomposing bacteria.

This enzyme was (probable a new type of non-heme iron protein) is assumed to catalyze the dehydrogenation of kojic acid, while the ferric ion contained in the enzyme is considered to serve as an acceptor of hydrogen released from kojic acid. The resulted ferrous ions are oxidized either by molecular oxygen under aerobic conditions or by NAD under anaerobic conditions, accompanying hydrogen peroxide in the former and reduced NAD in the latter. The enzyme was partially purified by using ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and column chromatography with DEAE-Sephadex A-50. The activity increased to 85 fold, compared with crude extracts and the recovery of the activity was 33.9%. The optimum pH of the reaction was 7.75. The enzyme was inactivated by PCMB, and unstable upon heat treatment. A loss of about 50% of the activity was caused by heating at 35%C for 5 min, but some reducing agents protected the enzyme from PCMB inhibition and the heat inactivation. Not only kojic acid, but also benzyl kojic acid or 5-methoxy kojic acid may be substrates. Km value for kojic acid was 1.43 × 10^?5m. The molecular weight of the enzyme was estimated to be about 55,000 and the enzyme contained about two atoms of iron in one molecule. The reaction mechanism for kojic acid oxidase is discussed.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Taxonomical studies on ten strains of hydrocarbon-utilizing bacteria reported in previous paper, which produced various kinds of amino acid, were carried out. They were Achromo-bacter cycloclastes, Achromobacter delmarvae, Bacillus species, Corynebacterium species, Micrococcus species. Many of them were not identical with the species which are described in Bergey’s Manual of 7th Edition.     

Studies on the Utilization of Hydrocarbon by Microorganisms

An uracil-requiring mutant (KY7122) of Arthrobacter paraffineus KY4303 (ATCC15591) was found to accumulate orotic acid and orotidine on n-paraffine as a sole carbon source.

Both substances were definitely indentified as orotic acid and orotidine, from the results on column and paper chromatography, UV and IR absorption spectra, elementary analysis and analyses of hydrolysate.

Cultural conditions for orotic acid and orotidine fermentation were then investigated. As the carbon source n-paraffines from C₁₄ to C₁₆ were the most suitable for the fermentation, and sorbitol, fructose and mannitol were best utilized for the growth, and orotidine produced from them were twice as much as those from hydrocarbon. The addition of 200 mg of uracil and 2 g of C. S. L to 1 liter of medium was most optimal for orotic acid and orotidine fermentation.

Orotic acid and orotidine accumulations were enhanced by the addition of either l-tyrosine, l-leusine, l-threonine, gluconate or meat extract.