A conserved and seemingly redundant Escherichia coli biotin biosynthesis gene expressed only during anaerobic growth.

Biotin is an essential metabolic cofactor and de novo biotin biosynthetic pathways are widespread in microorganisms and plants. Biotin synthetic genes are generally found clustered into bio operons to facilitate tight regulation since biotin synthesis is a metabolically expensive process. Dethiobiotin synthetase (DTBS) catalyzes the penultimate step of biotin biosynthesis, the formation of 7,8-diaminononanoate (DAPA). In Escherichia coli, DTBS is encoded by the bio operon gene bioD. Several studies have reported transcriptional activation of ynfK a gene of unknown function, under anaerobic conditions. Alignments of YnfK with BioD have led to suggestions that YnfK has DTBS activity. We report that YnfK is a functional DTBS, although an enzyme of poor activity that is poorly expressed. Supplementation of growth medium with DAPA or substitution of BioD active site residues for the corresponding YnfK residues greatly improved the DTBS activity of YnfK. We confirmed that FNR activates transcriptional level of ynfK during anaerobic growth and identified the FNR binding site of ynfK. The ynfK gene is well conserved in γ-proteobacteria.

Riemerella anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence.

Riemerella anatipestifer is a bacterial pathogen responsible for major economic losses within the duck industry. Recent studies have revealed that biotin biosynthesis is critical for the bacterium's survival and virulence. We previously found that R. anatipestifer AS87_RS09170, a putative bioF gene, is important for bacterial virulence. In the present study, we characterized the AS87_RS09170 gene in R. anatipestifer strain Yb2. Sequence analysis indicated that the AS87_RS09170 gene is highly conserved among R. anatipestifer strains; the deduced protein harbored the conserved pyridoxal 5'-phosphate binding pocket of 8-amino-7-oxononanoate synthase. Western blot analysis demonstrated that the biotin-dependent enzyme was present in smaller quantities in the mutant strain Yb2ΔbioF compared to that of the wide-type strain Yb2, suggesting that the biotin biosynthesis was defective. The mutant strain Yb2ΔbioF displayed a decreased growth rate at the exponential phase in tryptic soy broth culture and in BeaverBeads Streptavidin treated tryptic soy broth culture, but recovered when biotin was supplemented. In addition, the mutant strain Yb2ΔbioF showed an enhanced biofilm formation, as well as increased adhesion and invasion capacities to duck embryo fibroblasts. Moreover, the mutant strain Yb2ΔbioF exhibited irregular shapes with budding vegetations and relatively thickened cell walls under scanning and transmission electron microscope observation, as well as a reduced capacity to establish systemic infection in a duck infection model. These results provide the first evidence that the R. anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence.

Pimelic acid, the first precursor of the Bacillus subtilis biotin synthesis pathway, exists as the free acid and is assembled by fatty acid synthesis.

Biotin synthetic pathways are readily separated into two stages, synthesis of the seven carbon α, ω-dicarboxylic acid pimelate moiety and assembly of the fused heterocyclic rings. The biotin pathway genes responsible for pimelate moiety synthesis vary widely among bacteria whereas the ring synthesis genes are highly conserved. Bacillus subtilis seems to have redundant genes, bioI and bioW, for generation of the pimelate intermediate. Largely consistent with previous genetic studies it was found that deletion of bioW caused a biotin auxotrophic phenotype whereas deletion of bioI did not. BioW is a pimeloyl-CoA synthetase that converts pimelic acid to pimeloyl-CoA. The essentiality of BioW for biotin synthesis indicates that the free form of pimelic acid is an intermediate in biotin synthesis although this is not the case in E. coli. Since the origin of pimelic acid in Bacillus subtilis is unknown, 13 C-NMR studies were carried out to decipher the pathway for its generation. The data provided evidence for the role of free pimelate in biotin synthesis and the involvement of fatty acid synthesis in pimelate production. Cerulenin, an inhibitor of the key fatty acid elongation enzyme, FabF, markedly decreased biotin production by B. subtilis resting cells whereas a strain having a cerulenin-resistant FabF mutant produced more biotin. In addition, supplementation with pimelic acid fully restored biotin production in cerulenin-treated cells. These results indicate that pimelic acid originating from fatty acid synthesis pathway is a bona fide precursor of biotin in B. subtilis.

Organic cofactors in the metabolism of Dehalococcoides mccartyi strains.

Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes.

Design and synthesis of potential mechanism-based inhibitors of the aminotransferase BioA involved in biotin biosynthesis.

BioA, a pyridoxal 5'-phosphate (PLP) dependent aminotransferase, catalyzes the second step of biotin biosynthesis, converting 7-keto-8-aminopelargonic acid (KAPA) into 7,8-diaminopelargonic acid (DAPA). Amiclenomycin (ACM) isolated from cultures of different Streptomyces strains is a potent mechanism-based inhibitor of BioA that operates via an aromatization mechanism, irreversibly labeling the PLP cofactor. However, ACM is plagued by inherent chemical stability. Herein we describe the synthesis of four inhibitors, inspired by ACM but containing an allylic amine as the chemical warhead, designed to both improve stability and operate via a complementary Michael addition-pathway upon enzymatic oxidation of the allylic amine substrate to an enimine. Acyclic analogue M-1 contains a terminal olefin as the pro-Michael acceptor. The synthesis of M-1 features an alkyne-zipper reaction and the Overman rearrangement as key synthetic operations. The cyclic analogues M-2/3/4 contain either an endocyclic or exocyclic olefin as the pro-Michael acceptor. These were all prepared using a common strategy employing DIBAL reduction of a precursor bicyclic lactam, followed by in situ Horner-Wadsworth-Emmons (HWE) olefination as the key synthetic steps.

Peroxisomes are involved in biotin biosynthesis in Aspergillus and Arabidopsis.

Among the eukaryotes only plants and a number of fungi are able to synthesize biotin. Although initial events leading to the biosynthesis of biotin remain largely unknown, the final steps are known to occur in the mitochondria. Here we deleted the Aopex5 and Aopex7 genes encoding the receptors for peroxisomal targeting signals PTS1 and PTS2, respectively, in the filamentous fungus Aspergillus oryzae. In addition to exhibiting defects in the peroxisomal targeting of either PTS1 or PTS2 proteins, the deletion strains also displayed growth defects on minimal medium containing oleic acid as the sole carbon source. Unexpectedly, these peroxisomal transport-deficient strains also exhibited growth defects on minimal medium containing glucose as the sole carbon source that were remediated by the addition of biotin and its precursors, including 7-keto-8-aminopelargonic acid (KAPA). Genome database searches in fungi and plants revealed that BioF protein/KAPA synthase, one of the biotin biosynthetic enzymes, has a PTS1 sequence at the C terminus. Fungal ΔbioF strains expressing the fungal and plant BioF proteins lacking PTS1 still exhibited growth defects in the absence of biotin, indicating that peroxisomal targeting of KAPA synthase is crucial for the biotin biosynthesis. Furthermore, in the plant Arabidopsis thaliana, AtBioF localized to the peroxisomes through recognition of its PTS1 sequence, suggesting involvement of peroxisomes in biotin biosynthesis in plants. Taken together we demonstrate a novel role for peroxisomes in biotin biosynthesis and suggest the presence of as yet unidentified peroxisomal proteins that function in the earlier steps of biotin biosynthesis.

MMAR_2770, a new enzyme involved in biotin biosynthesis, is essential for the growth of Mycobacterium marinum in macrophages and zebrafish.

Biotin, which functions as an essential cofactor for certain carboxylases and decarboxylases, is synthesized by a multistep pathway in microorganisms and plants. Biotin biosynthesis has not been studied in detail in mycobacteria. In this study, we isolated a mutant of Mycobacterium marinum in which MMAR_2770, a previously uncharacterized gene encoding a predicted short-chain dehydrogenase/reductase, was inactivated. We found that this mutant is a biotin auxotroph that cannot grow in a minimal medium (Sauton) unless biotin is supplemented. Complementation of the mutant with an intact MMAR_2770 or its homolog Rv1882c of Mycobacterium tuberculosis restored the growth of the mutant, suggesting that MMAR_2770 is involved in biotin biosynthesis. We further showed that the mutant was unable to grow in cultured macrophages and was attenuated in zebrafish. Taken together, our results demonstrate that biotin biosynthesis is essential for the growth of mycobacteria in vitro and in vivo and have provided validation for targeting biotin biosynthetic enzymes for antimycobacterial drug development. The potential role of MMAR_2770 in mycobacterial biotin biosynthesis is discussed.

Engineering of biotin-prototrophy in Pichia pastoris for robust production processes.

Biotin plays an essential role as cofactor for biotin-dependent carboxylases involved in essential metabolic pathways. The cultivation of Pichia pastoris, a methylotrophic yeast that is successfully used as host for the production of recombinant proteins, requires addition of high dosage of biotin. As biotin is the only non-salt media component used during P. pastoris fermentation (apart from the carbon source), nonconformities during protein production processes are usually attributed to poor quality of the added biotin. In order to avoid dismissed production runs due to biotin quality issues, we engineered the biotin-requiring yeast P. pastoris to become a biotin-prototrophic yeast. Integration of four genes involved in the biotin biosynthesis from brewing yeast into the P. pastoris genome rendered P. pastoris biotin-prototrophic. The engineered strain has successfully been used as production host for both intracellular and secreted heterologous proteins in fed-batch processes, employing mineral media without vitamins. Another field of application for these truly prototrophic hosts is the production of biochemicals and small metabolites, where defined mineral media leads to easier purification procedures.

Recombinant Candida utilis for the production of biotin.

Biotin is an important nutritional supplement but is difficult to manufacture effectively. Here we present a trial of biotin production using the food yeast Candida utilis. In this system, we cloned the C. utilis biotin synthase (BIO2) gene, the gene of the rate-limiting enzyme for biotin biosynthesis, and assembled it under the control of a strong promoter. A series of plasmids were constructed to direct the integration of the BIO2 gene, either high-copy integration with 18S rDNA fragment or low-copy integration with URA3 or HIS3 fragment. The BIO2 gene can be successfully integrated into the C. utilis chromosome and can drive biotin production using these plasmids. The biotin yield in this system can reach 100-fold above the endogenous level in a small-scale culture. Although the biotin production is not stable if the selection pressure is removed, this system has the potential to produce biotin-rich feed or food additives directly without the requirement of further purification.

Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae.

Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.

Biotinylated dextran amine as a marker for fetal hypothalamic homografts and their efferents.

We have explored the use of biotinylated dextran amine (BDA) as a marker for labeling fetal brain grafts and their connections with the host. As a model system we used transplantation of the hamster suprachiasmatic nucleus, the site of an endogenous biological clock governing circadian rhythms. Similar transplants into arrhythmic hosts have been shown to restore behavioral function with a period specific to the donor. For locomotor rhythms, efferent connections are not necessary. For other responses, including endocrine rhythms, efferent connections may be necessary. In order to visualize homografts and their efferents, injections of BDA, an anterograde tracer, were made into the anterior hypothalamic (AH) region containing the SCN or into the dorsal cortex (CTX) of fetal hamster brains. The fetal AH or CTX was microdissected out and stereotaxically implanted into the third ventricle of intact, adult hamsters. After 2, 4, 8, or 12 weeks, hosts were sacrificed and their brains were processed for detection of BDA by either histochemistry or immunofluorescence. BDA intensely labeled graft neurons, their dendrites, and axons with minimal or no spread to the adjacent host brain. Labeled graft axons could be followed for long distances (>1 mm) into the host brain and graft-derived varicosities formed close contacts with host neurons. BDA-labeled graft neurons, located at the perimeter of the graft, also extended dendrite-like processes into the host parenchyma. We conclude that BDA is a useful marker for fetal homografts and their efferents for survival times of less than 2 months.

Biochemical characterization of the Arabidopsis biotin synthase reaction. The importance of mitochondria in biotin synthesis.

Biotin synthase, encoded by the bio2 gene in Arabidopsis, catalyzes the final step in the biotin biosynthetic pathway. The development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein. Under optimized conditions, the turnover number of the reaction was >2 h(-1) with this in vitro system. Purified Bio2 protein was not efficient by itself in supporting biotin synthesis. However, heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yielded a functional biotin synthase complex. Biotin synthase in this heterologous system obeyed Michaelis-Menten kinetics with respect to dethiobiotin (K(m) = 30 microM) and exhibited a kinetic cooperativity with respect to S-adenosyl-methionine (Hill coefficient = 1.9; K(0.5) = 39 microM), an obligatory cofactor of the reaction. In vitro inhibition of biotin synthase activity by acidomycin, a structural analog of biotin, showed that biotin synthase reaction was the specific target of this inhibitor of biotin synthesis. It is important that combination experiments using purified Bio2 protein and extracts from pea (Pisum sativum) leaf or potato (Solanum tuberosum) organelles showed that only mitochondrial fractions could elicit biotin formation in the plant-reconstituted system. Our data demonstrated that one or more unidentified factors from mitochondrial matrix (pea and potato) and from mitochondrial membranes (pea), in addition to the Bio2 protein, are obligatory for the conversion of dethiobiotin to biotin, highlighting the importance of mitochondria in plant biotin synthesis.

Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies.

8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using ultraviolet/visible spectroscopy, and the data were consistent with the kinetic data (K = 4.2 microm). Among the other compounds tested, two potential transition state analogs, 4-carboxybutyl(1-amino-1-carboxyethyl)phosphonate (4) and 2-amino-3-hydroxy-2-methylnonadioic acid (5) were found to be competitive inhibitors with respect to l-alanine with Ki of 68 microm and 80 microm, respectively.

Characterization of the cDNA and gene coding for the biotin synthase of Arabidopsis thaliana.

Biotin, an essential cofactor, is synthesized de novo only by plants and some microbes. An Arabidopsis thaliana expressed sequence tag that shows sequence similarity to the carboxyl end of biotin synthase from Escherichia coli was used to isolate a near-full-length cDNA. This cDNA was shown to code for the Arabidopsis biotin synthase by its ability to complement a bioB mutant of E. coli. Site-specific mutagenesis indicates that residue threonine-173, which is highly conserved in biotin synthases, is important for catalytic competence of the enzyme. The primary sequence of the Arabidopsis biotin synthase is most similar to biotin synthases from E. coli, Serratia marcescens, and Saccharomyces cerevisiae (about 50% sequence identity) and more distantly related to the Bacillus sphaericus enzyme (33% sequence identity). The primary sequence of the amino terminus of the Arabidopsis biotin synthase may represent an organelle-targeting transit peptide. The single Arabidopsis gene coding for biotin synthase, BIO2, was isolated and sequenced. The biotin synthase coding sequence is interrupted by five introns. The gene sequence upstream of the translation start site has several unusual features, including imperfect palindromes and polypyrimidine sequences, which may function in the transcriptional regulation of the BIO2 gene.

Biotin synthesis in higher plants: isolation of a cDNA encoding Arabidopsis thaliana bioB-gene product equivalent by functional complementation of a biotin auxotroph mutant bioB105 of Escherichia coli K12.

Biotin synthase is involved in the conversion of dethiobiotin to biotin in bacteria, yeast and higher plants. We isolated a complete cDNA (1.3 kb) encoding A. thaliana bioB-gene product by functional complementation of the bioB105 biotin auxotroph mutant of Escherichia coli K12 using an A. thaliana cDNA library. The open reading frame encodes a predicted protein of 378 amino acids and molecular mass of 41.634 Da. The bioB-gene product from A. thaliana shows specific regions of homology with the bioB-gene products of E. coli, Serratia marcescens, Bacillus sphaericus, and Saccharomyces cerevisiae. The predicted amino acid sequence of the plant protein contains the consensus region GXCXEDCXYCXQ involved in the [2Fe-2S] cluster binding. Furthermore, it does not present a transit peptide in its N-terminal region suggesting that the A. thaliana bioB-gene product reported in this paper is located in the cytosolic compartment.

Origin of carbon atoms of biotin. 13C-NMR studies on biotin biosynthesis in Escherichia coli.

The origin of the carbon atoms of pimeloyl-CoA, the earliest known precursor in the pathway of de novo biotin biosynthesis in Escherichia coli, was investigated by 13C-NMR spectroscopy. In fermentation of the biotin-overproducing DRK332/pXBA312 strain of Escherichia coli (a repressor mutant carrying a biotin operon fragment in the plasmid), a high dose of L-alanine (8 g/l) stimulated dethiobiotin and biotin accumulation. Although L-alanine is a known precursor of 7-keto-8-aminopelargonic acid in biotin biosynthesis, the 13C-NMR spectrum of dethiobiotin showed that the C-3 of L-[3-13C]alanine was incorporated into not only the methyl carbon (C-9) but also alternate carbons (C-2, C-4, C-6) of the side chain, and these latter positions are the same as those labeled with D-[1-13C]glucose. These data indicate that L-alanine can act as an alternative carbon source, suggesting that acetyl-CoA is a possible precursor for pimeloyl-CoA synthesis. In accordance with this hypothesis, the C-1 of sodium (1-13C)acetate and the C-2 of sodium (2-13C)acetate were incorporated into alternate carbons in the side chain of dethiobiotin, i.e., (C-1, C-3, C-5, C-7) and (C-1, C-2, C-4, C-6), respectively. These results suggested firstly that in E. coli pimeloyl-CoA is biosynthesized from L-alanine and/or acetate via acetyl-CoA, but not via pimelic acid, which has been suggested as a biotin precursor in other species, and secondly that the carboxyl group of biotin originates from carbon dioxide produced through the tricarboxylic acid cycle.

Biotin studies in pigs. 3. Biotin absorption and synthesis.

Eight pigs were given a semi-purified diet based on maize flour and casein containing 10 micrograms biotin/kg. The diet was given ad lib. with or without a supplement of 70 micrograms biotin/kg diet from 5 to 94 d of age. The flow of biotin in the stomach was similar to the biotin intake (13.5 and 112 micrograms/d) for the unsupplemented and biotin-supplemented pigs respectively. The flow of biotin through the small intestine decreased for the biotin-supplemented pigs from 39 micrograms/d in the first quarter of the small intestine to 7.9 micrograms/d in the last quarter. The flows of biotin in the caecum, large intestine and colon were similar for both the unsupplemented and biotin-supplemented pigs, with values of 17-54 micrograms/d, indicating the synthesis of biotin in the hind-gut.