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.     

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 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.     

On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. Discussion of the occurrence of an intermediate hydroxylation.

The last step of the biosynthesis of biotin, i.e. the conversion of dethiobiotin to biotin was studied using E. coli. The three dethiobiotin derivatives hydroxylated at C-2 or C-5 were synthesized and tested as potential precursors of biotin. It appears that none of these compounds is able to support the growth of E. coli C124, a mutant which does not synthesize dethiobiotin, but converts it into biotin. These results strongly disfavour the hypothesis of the activation of the saturated carbons by an hydroxylation process.     

Incorporation of the sulfur of L-[35S]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.     

Properties of the Biotin Transport System in Bifidobacterium breve N4

Binding of biotin to resting cells of Bifidobacterium breve N4, which grew in a biotin-deficient medium, was independent of pH from 1 to 9 and of temperature below 50 C. It was not inhibited by metabolic inhibitors including sulfhydryl reagents, but it was inhibited by treatment with 80% ethanol or 5% trichloroacetic acid. It was also competitively inhibited by biotin-sulfone, but not by tetrahydrothiophene nor dethiobiotin. The binding constant was calculated to be 3.3 × 10⁸m^?1. The amount of biotin unextractable with hot water, representing part of the transported biotin, increased gradually for 20 min, this increase was inhibited by NaF, hydroxylamine and low temperature. ¹⁴C-biotin on the cells was displaced by cold biotin and biotin-sulfone; the displacement was not inhibited by metabolic inhibitors, but it was dependent on temperature. A few minutes after binding, the biotin was released to the medium. The release was dependent on pH and temperature, was affected by energy sources and was inhibited by metabolic inhibitors, e.g. NaF, p-chloromercuribenzoic acid and hydroxylamine. It could be stopped at any time by cooling to 0 C or by adding NaF, and the amount of accumulated biotin did not increase under those conditions. These results suggest that the binding sites on the cell surface decreased in number or in their binding affinity for biotin through an energy-dependent process.     

Induction of Prodigiosin Biosynthesis after Shift-Down in Temperature of Nonproliferating Cells of Serratia marcescens

Nonpigmented bacteria obtained by growth of Serratia marcescens at 38 C synthesized prodigiosin at 25 C if certain individual amino acids were added to cultures of nonproliferating cells. In order of effectiveness, the amino acids were: DL-histidine, L-proline, L-hydroxyproline, DL-alanine, L-alanine, DL-aspartic acid, D-alanine, DL-proline, L-serine, L-ornithine, L-glutamic acid, and D-proline. DL-Histidine at its optimal concentration (20 mg/ml) induced formation of prodigiosin (198 mug of prodigiosin per mg of bacterial protein) after incubation of cultures for 54 hr. Lower concentrations (10 mg/ml) of the other amino acids usually were optimum but less prodigiosin was synthesized, and the maximal amount of pigment occurred between 36 and 48 hr. DL-Methionine was not effective alone but at a low concentration (40 mug/ml) enhanced and accelerated biosynthesis of prodigiosin in the presence of other suitable amino acids. Addition of 2 mg of L-proline per ml at 0 hr induced formation of only 30 mug of prodigiosin after incubation for 42 hr, but addition at 36 hr of 5 mg more of L-proline per ml increased synthesis to 120 mug at 42 hr. Again, DL-methionine markedly augmented prodigiosin biosynthesis in these cultures. Synthesis of prodigiosin ceased if cultures were shifted from 25 to 38 C. Prodigiosin biosynthesis by the nonproliferating cells was maximum when cultures were aerated, the amount of bacterial protein was about 2.0 mg/ml, and amino acids were added at 0 hr. Bacteria synthesized prodigiosin most efficiently when they were harvested from aerated cultures grown at 38 C for 24 hr in a complete medium in a fermentor.     

A continuous fluorescence displacement assay for BioA: an enzyme involved in biotin biosynthesis

Cofactor biosynthetic pathways represent a rich source of potential antibiotic targets. The second step in biotin biosynthesis is performed by BioA, a pyridoxal 5′-phosphate (PLP)-dependent enzyme. This enzyme has been confirmed as a candidate target in Mycobacterium tuberculosis; however, the current bioassay used to measure BioA activity is cumbersome and low throughput. Here we describe the design, development, and optimization of a continuous coupled fluorescence displacement assay to measure BioA activity. In this coupled assay, BioD converts the product of the BioA-catalyzed reaction into dethiobiotin, which is subsequently detected by displacement of a fluorescently labeled dethiobiotin probe from streptavidin. The assay was further adapted to a high-throughput screening format and validated against the LOPAC¹²⁸⁰ library.     

Enzymatic Conversion of Dethiobiotin to Biotin in Cell-free Extracts of a Bacillus sphaericus bioB Transformant

The activity of biotin synthase, responsible for biotin synthesis from dethiobiotin, was demonstrated in a completely defined reaction mixture with cell-free extracts of a Bacillus sphaericus bioB transformant. Among the sulfur compounds tested, only S-adenosyl-l-methionine was active, while l-methionine and l-cysteine had no significant effect. Protein concentrations higher than 15mg/ml in the reaction mixture were needed to detect biotin synthase activity. When dialyzed cell-free extracts were used for the reaction, NADH, NADPH, or FAD among the well-known cofactors tested enhanced the activity, and Fe²⁺, Mn²⁺, and Ca²⁺ among the metal ions tested also had some effects.     

Conversion of Dethiobiotin to Biotin in Cell-free Extracts of Escherichia coli

We constructed the plasmid pTTB151 in which the E. coli bio B gene was expressed under the control of the tac promoter. Conversion of dethiobiotin to biotin was demonstrated in cell-free extracts of E. coli carrying this plasmid. The requirements for this biotin-forming reaction included fructose-1,6-bisphosphate, Fe²⁺, S-adenosyl-L-methionine, NADPH, and KCl, as well as dethiobiotin as the substrate. The enzymes were partially purified from cell-free extracts by a procedure involving ammonium sulfate fractionation. Our results suggest that an unidentified enzyme(s) besides the bioB gene product is obligatory for the conversion of dethiobiotin to biotin.     

Synthesis of (R)-2-phenylpropanoic acid from its racemate through an isomerase-involving reaction by Nocardia diaphanozonaria

(R)-2-Phenylpropanoic acid was synthesized from the racemic acid through an isomerization reaction involving resting cells of Nocardia diaphanozonaria JCM3208. The isomerization activity of the cells was enhanced 25-fold by adding 5.5 mM racemic 2-phenylpropanoic acid to the culture medium. When 5 mM racemic 2-phenylpropanoic acid was included in the reaction mixture (4 ml) containing resting cells (100 mg dry cell wt) in 25 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.0) at 30 °C for 8 h, 4.56 mM (R)-2-phenylpropanoic acid (95.8% e.e.) was formed with a 91% molar conversion yield.     

Macromolecular syntheses during biosynthesis of prodigiosin by Serratia marcescens.

Amino acids that were utilized as sole sources of carbon and nitrogen for growth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin in non-proliferating bacteria. Addition of alanine, proline, or histidine to non-proliferating cells incubated at 27 C increased the rate of protein synthesis and also caused biosynthesis of prodigiosin. No increase in the rate of protein synthesis was observed upon the addition of amino acids that did not stimulate prodigiosin biosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) (a small amount) also occurred after addition of amino acids that resulted in biosynthesis of prodigiosin. After incubation of 24 h, the total amount of protein in suspensions of bacteria to which alanine or proline was added increased 67 and 98%, respectively. Total amounts of DNA and of RNA also increased before synthesis of prodigiosin. The amounts of these macromolecules did not increase after addition of amino acids that did not induce biosynthesis of progidiosin. However, macromolecular synthesis was not related only to prodigiosin biosynthesis because the rates of DNA, RNA, and protein synthesis also increased in suspensions of bacteria incubated with proline at 39 C, at which temperature no prodigiosin was synthesized. The quantities of DNA, RNA, and protein synthesized were lower in non-proliferating cells than in growing cells. The data indicated that amino acids causing biosynthesis of prodigiosin in non-proliferating cells must be metabolized and serve as sources of carbon and of nitrogen for synthesis of macromolecules and intermediates. Prodigiosin was synthesized secondarily to these primary metabolic events.     

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.     

Determination of the metabolic origins of the sulfur and 3'-nitrogen atoms in biotin of Escherichia coli by mass spectrometry

Two steps in the biosynthesis of biotin in Escherichia coli, incorporation of the nitrogen atom of methionine into 7-keto-8-aminopelargonic acid and of the sulfur atom into dethiobiotin, were examined. Sulfur and nitrogen metabolism were monitored by gas chromatography-mass spectrometry of volatile derivatives of internal (protein-bound) amino acids and excreted biotin. We were able to show that internal cysteine and excreted biotin were labeled to the same extent with 34S from either of two exogenous sulfur sources, 34SO4(2)-or L-[sulfane-34S]thiocystine. Internal methionine was eliminated from consideration, while cysteine, or possibly a closely related intermediate, was implicated as providing the sulfur atom for biotin biosynthesis. Also, in experiments designed to follow the metabolism of the nitrogen atom of methionine, it was found that biotin excreted into the culture medium by this organism grown with 95 atom % [15N]methionine contained greater than 70 atom % excess 15N in one of the nitrogens over that obtained from cultures grown with methionine of natural abundance 15N. These results provide evidence for the direct transfer of the methionine nitrogen as the role of S-adenosylmethionine in the conversion of 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid.     

Pretreatment of Cell Suspensions as a Method to Increase the Protoplast Yield of Haplopappus gracilis

A low yield of free protoplasts was obtained when fast-growing cell suspensions of Haplopappus gracilis were treated with Driselase, an active mixture of cellulase and pectinase. If the cells in the suspension culture were pretreated by increased levels of auxin, reduced sugar concentration, addition of sulphur-containing amino acids, or by the reducing agent mercaptoethanol, protoplasts were released from a higher proportion of the cells. These pretreatments did not adversely effect division of the protoplasts.     

Action of 5-(2-thienyl) valeric acid as a biotin antagonist

5-(2-Thienyl)valeric acid (TVA), a biotin analogue which can be easily prepared through chemical process, inhibited the growth of a biotin synthesizing Rhodotorula glutinis. The growth inhibition was reversed by the addition of biotin. Among biotin intermediates, dethiobiotin and 7,8-diaminopelargonic acid reversed the inhibition by TVA, while 7-keto-8-amino-pelargonic acid and pimelic acid did not. From these results, it was concluded that TVA is a biotin antagonist which probably acts as an inhibitor of biotin biosynthesis.     

Possible explanation for the metabolic radioprotective effect of cysteine on Escherichia coli B

The growth of logarithmic-phase cultures of E. coli B cells on glucose-mineral medium was partially inhibited by 0.2 mM L-cysteine-HCl. Twenty other amino acids failed to show a similar inhibiting effect even at a concentration of 10 mM. After incubation of the log-phase culture in the presence of 2.0 mM cysteine for 30 minutes (inhibition about 70 %) the cells were centrifuged, resuspended, and diluted 200-fold in cysteine-free phosphate buffer. These cells exhibited increased resistance to the effect of X-rays as measured by the number of colony-forming units (DRF = 2.1). Both the growth-inhibiting and the radioprotective effects of cysteine could be diminished by the presence of 0.5 mg of casein hydrolyzate per milliliter during the 30-minute preirradiation culture period. This "anti-cysteine" effect was caused mainly by L-leucine, L-isoleucine, L-valine, and L-threonine from among the amino acids of casein hydrolyzate. It is suggested that cysteine inhibits the biosynthesis of some amino acids, thereby blocking protein synthesis, which may result in increased radioresistance.     

Electron-microscopic localization of acetyl coenzyme A carboxylase in cells of Claviceps purpurea

Summary The ultrastructural localization of acetyl-CoA carboxylase activity was studied in two strains of the ascomycetous fungus Claviceps purpurea differing in the ergot alkaloid synthesis. Mycelia were harvested by centrifugation of saprophytic submerged cultures, fixed in cold 3% glutaraldehyde in 0.05 M cacodylate buffer pH 7.2 and washed repeatedly in the same buffer. The incubation medium of Yates et al. (1969) had to be modified in the molarity of ATP. The best results were obtained with a medium of the following composition: 50 mM cacodylate buffer pH 7.2, 4 mM ATP, 3.5 mM lead nitrate, 13.5 mM sodium citrate, 3.75 mM sodium bicarbonate, 1.25 mM manganese chloride, 0.4 mM acetyl-CoA and 2 mM biotin. The fixation is a prerequisite for a distinct localization. The enzyme activity was detected only in cells producing high amount of clavine alkaloids. It was confined to the membranes of endoplasmic reticulum and their derivatives: tonoplast of vacuoles, tiny vesicles and amorphous material inside vacuoles. The reaction product was very fine and localized in both leaflets of the membranes. The specificity of the reaction was confirmed by negative results in control preparations: boiled cells incubated in the complete medium, cells incubated in the medium supplemented with avidin or in the media from which either ATP, or acetyl CoA, or sodium bicarbonate, or biotin were omitted. It is suggested that the activity of acetyl-CoA carboxylase is linked to the synthesis of clavine alkaloid precursors which occurs in the endoplasmic reticulum and its derivatives.With technical assistance of J. Martínková     

Improved biotin,thiamine, and lipoic acid biosynthesis by engineering the global regulator IscR

Biotin, thiamine, and lipoic acid are industrially important molecules naturally synthesized by microorganisms via biosynthetic pathways requiring iron-sulfur (FeS) clusters. Current production is exclusively by chemistry because pathway complexity hinders development of fermentation processes. For biotin, the main bottleneck is biotin synthase, BioB, a S-adenosyl methionine-dependent radical enzyme that converts dethiobiotin (DTB) to biotin. BioB overexpression is toxic, though the mechanism remains unclear. We identified single mutations in the global regulator IscR that substantially improve cellular tolerance to BioB overexpression, increasing Escherichia coli DTB-to-biotin biocatalysis by more than 2.2-fold. Based on proteomics and targeted overexpression of FeS-cluster biosynthesis genes, FeS-cluster depletion is the main reason for toxicity. We demonstrate that IscR mutations significantly affect cell viability and improve cell factories for de novo biosynthesis of thiamine by 1.3-fold and lipoic acid by 1.8-fold. We illuminate a novel engineering target for enhancing biosynthesis of complex FeS-cluster-dependent molecules, paving the way for industrial fermentation processes.