RESUMO
Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.
Assuntos
Alteromonadaceae , Ligantes , Rhodobacteraceae , Vitamina B 12 , Oceano Atlântico , Técnicas de Cocultura , Interações Microbianas , Prófagos/genética , Prófagos/crescimento & desenvolvimento , Prófagos/metabolismo , Vitamina B 12/biossíntese , Vitamina B 12/química , Vitamina B 12/metabolismo , Alteromonadaceae/crescimento & desenvolvimento , Alteromonadaceae/metabolismo , Rhodobacteraceae/citologia , Rhodobacteraceae/metabolismo , Rhodobacteraceae/virologia , Ribonucleosídeos/metabolismo , Cobamidas/metabolismo , EcossistemaRESUMO
Vitamin B12 (VB12) is a critical micronutrient that controls DNA metabolic pathways to maintain the host genomic stability and tissue homeostasis. We recently reported that the newly discovered commensal Propionibacterium, P. UF1, regulates the intestinal immunity to resist pathogen infection, which may be attributed in part to VB12 produced by this bacterium. Here we demonstrate that VB12 synthesized by P. UF1 is highly dependent on cobA gene-encoding uroporphyrinogen III methyltransferase, and that this vitamin distinctively regulates the cobA operon through its 5' untranslated region (5' UTR). Furthermore, conserved secondary structure and mutagenesis analyses revealed a VB12-riboswitch, cbiMCbl (140 bp), within the 5' UTR that controls the expression of downstream genes. Intriguingly, ablation of the cbiMCbl significantly dysregulates the biosynthesis of VB12, illuminating the significance of this riboswitch for bacterial VB12 biosynthesis. Collectively, our finding is an in-depth report underscoring the regulation of VB12 within the beneficial P. UF1 bacterium, through which the commensal metabolic network may improve gut bacterial cross-feeding and human health.
Assuntos
Regulação Bacteriana da Expressão Gênica , Propionibacterium/metabolismo , Riboswitch/genética , Vitamina B 12/biossíntese , Regiões 5' não Traduzidas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microbioma Gastrointestinal/fisiologia , Metiltransferases/genética , Metiltransferases/metabolismo , Mutagênese Sítio-Dirigida , Óperon/genética , Probióticos/metabolismo , Propionibacterium/genéticaRESUMO
Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.
Assuntos
Regulação Bacteriana da Expressão Gênica , Metionina/biossíntese , Mycobacterium smegmatis/metabolismo , Vitamina B 12/biossíntese , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Metiltransferases/genética , Metiltransferases/metabolismo , Mycobacterium smegmatis/genética , RiboswitchRESUMO
Sinorhizobium meliloti 320 is a vitamin B12 (VB12 ) high-producing strain that has been isolated and identified in our previous study. Because the regulatory toolbox for S. meliloti is limited, we searched for new genetic components and identified the two xylose-inducible promoters PA and PB based on a promoter-probe vector with a green fluorescent protein (GFP) as reporter. Compared with the ParaA promoter from S. meliloti, both promoters exhibited higher induced expression and lower basal expression. Subsequently, the influence of glucose or sucrose on the expression of GFP driven by these three promoters was assayed. Glucose repressed all three promoters, and the expression of ParaA was the lowest in the presence of glucose. Although sucrose repressed the expression of PA by 35% and improved the expression of ParaA by 16%, the expression level of PA was the highest and was 13% higher than that of ParaA . Lastly, we overexpressed the hemA gene in the C4 pathway using the PA promoter in S. meliloti 320, and the VB12 production of the engineered strain increased by 11%. The VB12 production was further increased by 11% by adding 0.1% sodium succinate to the culture medium.
Assuntos
Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas , Sinorhizobium meliloti , Vitamina B 12 , Xilose , Vetores Genéticos/genética , Plasmídeos/genética , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Vitamina B 12/biossíntese , Vitamina B 12/genética , Xilose/genética , Xilose/metabolismo , Xilose/farmacologiaRESUMO
The human microbiome is a rich factory for metabolite production and emerging data has led to the concept that orally administered microbial strains can synthesize metabolites with neuroactive potential. Recent research from ex vivo and murine models suggests translational potential for microbes to regulate anxiety and depression through the gut-brain axis. However, so far, less emphasis has been placed on the selection of specific microbial strains known to produce the required key metabolites and the formulation in which microbial compositions are delivered to the gut. Here, we describe a double-capsule technology to deliver high numbers of metabolically active cells derived from the 24-strain probiotic product SH-DS01 to the gastrointestinal tract, including the small intestine, where immune responses and adsorption of metabolites into the bloodstream occur. Based on its genome sequence, Limosilactobacillus reuteri SD-LRE2-IT was predicted to have the genetic capacity to de novo produce a specific metabolite of interest to brain health, vitamin B12, which could be confirmed in vitro. Taken together, our data conceptualizes the importance of rationally defined microbial strain characterization based on genomics and metabolomics data, combined with carefully designed capsule technology for delivery of live cells and concomitant functionality in and beyond the gut ecosystem.
Assuntos
Microbioma Gastrointestinal , Limosilactobacillus reuteri/metabolismo , Probióticos/administração & dosagem , Vitamina B 12/biossíntese , Genômica , Humanos , Análise de Sequência de DNARESUMO
Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.
Assuntos
Vitamina B 12/biossíntese , Vitamina B 12/genética , Vitamina B 12/metabolismo , Bactérias/metabolismo , Biotecnologia/métodos , Fermentação/genética , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Metagenoma/genética , Metagenômica/métodos , Estudos ProspectivosRESUMO
Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a "next-generation probiotic." However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B12) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B12 biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B12 production by the AmII phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome.IMPORTANCE There is significant interest in the therapeutic and probiotic potential of the common gut bacterium Akkermansia muciniphila However, knowledge of both the genomic and physiological diversity of this bacterial lineage is limited. Using a combination of genomic, molecular biological, and traditional microbiological approaches, we identified at least four species-level phylogroups with differing functional potentials that affect how these bacteria interact with both their human host and other members of the human gut microbiome. Specifically, we identified and isolated Akkermansia strains that were able to synthesize vitamin B12 The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated Akkermansia strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.
Assuntos
Genoma Bacteriano , Verrucomicrobia/genética , Vitamina B 12/biossíntese , Vitaminas/biossíntese , Criança , Pré-Escolar , Genômica , Humanos , Verrucomicrobia/metabolismo , Vitamina B 12/genética , Vitaminas/genéticaRESUMO
BACKGROUND: Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production. RESULTS: By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L-1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L-1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA. CONCLUSIONS: Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L-1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Engenharia Metabólica , Uroporfirinas/biossíntese , Vitamina B 12/biossíntese , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , ÓperonRESUMO
CblC is a chaperone that catalyzes removal of the ß-axial ligand of cobalamin (or B12), generating cob(II)alamin in an early step in the cofactor trafficking pathway. Cob(II)alamin is subsequently partitioned to support cellular needs for the synthesis of active cobalamin cofactor derivatives. In addition to the ß-ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and clinical R161G/Q variants of the human protein exhibit robust thiol oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O2 to H2O2. The chemical efficiency of the thiol oxidase side reaction during ceCblC-catalyzed dealkylation of alkylcobalamins is noteworthy in that it effectively scrubs ambient oxygen from the reaction mixture, leading to air stabilization of the highly reactive cob(I)alamin product. In this study, we report that the enhanced thiol oxidase activity of ceCblC requires the presence of KCl, which explains how the wasteful thiol oxidase activity is potentially curtailed inside cells where the chloride concentration is low. We have captured an unusual chlorocob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by electron paramagnetic resonance, magnetic circular dichroism, and computational analyses. The ability to form a chlorocob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, which is structurally related to bacterial B12-dependent reductive dehalogenases that have been proposed to form halogen cob(II)alamin intermediates in their catalytic cycle.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Glutationa Transferase/metabolismo , Oxirredutases/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Vitamina B 12/biossíntese , Biocatálise , Proteínas de Caenorhabditis elegans/química , Glutationa Transferase/química , Modelos Moleculares , Proteínas Proto-Oncogênicas c-cbl/química , Vitamina B 12/químicaRESUMO
Vitamin B12 is a crucial fine chemical that is widely used in the pharmaceutical, food and chemical industries, and its production solely dependents on microbial fermentation. We previously constructed an artificial vitamin B12 biosynthesis pathway in Escherichia coli, but the yield of the engineered strains was low. Here, we removed metabolic bottlenecks of the vitamin B12 biosynthesis pathway in engineered E. coli strains. After screening cobB genes from different sources, optimizing the expression of cobN and customizing the ribosome binding sites of cobS and cobT, the vitamin B12 yield increased to 152.29 µg/g dry cell weight (DCW). Optimization of the downstream module, which converts co(II)byrinic acid a,c-diamide into adenosylcobinamide phosphate, elevated the vitamin B12 yield to 249.04 µg/g DCW. A comparison of a variety of equivalent components indicated that glucose and corn steep liquor are optimal carbon and nitrogen sources, respectively. Finally, an orthogonal array design was applied to determine the optimal concentrations of glucose and nitrogen sources including corn steep liquor and yeast extract, through which a vitamin B12 yield of 530.29 µg/g DCW was obtained. The metabolic modifications and optimization of fermentation conditions achieved in this study offer a basis for further improving vitamin B12 production in E. coli and will hopefully accelerate its industrial application.
Assuntos
Vias Biossintéticas , Meios de Cultura/química , Escherichia coli , Engenharia Metabólica , Vitamina B 12 , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Vitamina B 12/biossíntese , Vitamina B 12/genéticaRESUMO
Only a few cobalamin-producing bacterial species are known which are suitable for food fermentations. The strain of Acetobacter pasteurianus DSM 3509 was found to have the capability to synthesize cobalamin. A survival test and a preliminary genetic study of the gene of uroporphyrinogen-III synthase indicated the ability to synthesize cobalamin. By a modified microbiological assay based on Lactobacillus delbrueckii ssp. lactis DSM 20355, 4.57 ng/mL of cyanocorrinoids and 0.75 ng/mL of noncorrinoid growth factors were detected. The product extracted and isolated by immunoaffinity chromatography in its cyanide form had the similar UV spectrum as standard cyanocobalamin and Coα-[α-(7-adenyl)]-(Coß-cyano) cobamide also known as pseudovitamin B12 produced by Lactobacillus reuteri DSM 20016. The chromatographically separated product of A. pasteurianus was subjected to mass spectrometrical analysis. There, its fragmentation pattern turned out to be equivalent to that of cyanocobalamin also produced by Propionibacterium freudenreichii ssp. freudenreichii DSM 20271 and clearly differs from pseudovitamin B12. Due to the presence of this species in several food applications, there might be cobalamin residues in food fermented with these bacteria.
Assuntos
Acetobacter/metabolismo , Vitamina B 12/biossíntese , Acetobacter/química , Cobamidas/metabolismo , Limosilactobacillus reuteri/metabolismo , Espectrometria de Massas , Propionibacterium/metabolismo , Vitamina B 12/químicaRESUMO
The in vivo physiological role of the gene cobZ, which encodes precorrin-3B synthase, which catalyzes the initial porphyrin ring contraction step of cobalamin biosynthesis via the cob pathway, has been demonstrated here for the first time. Cobalamin is known to be essential for an early step of bacteriochlorophyll biosynthesis in anoxygenic purple bacteria. The cobZ (cobZRR) gene of the purple bacterium Rhodospirillum rubrum was localized to a 23.5 kb insert of chromosomal DNA contained on the cosmid pSC4. pSC4 complemented several mutants of bacteriochlorophyll and carotenoid biosynthesis, due to the presence of the bchCX and crtCDEF genes at one end of the cosmid insert, flanking cobZRR. A second gene, citB/tcuB, immediately downstream of cobZRR, shows homologies to both a tricarballylate oxidoreductase (tcuB) and a gene (citB) involved in signal transduction during citrate uptake. CobZRR shows extensive homology to the N-terminal domain of the bifunctional CobZ from Rhodobacter capsulatus, and the R. rubrum citB/tcuB gene is homologous to the CobZ C-terminal domain. A mutant, SERGK25, containing a terminatorless kanamycin interposon inserted into cobZRR, could not grow by anaerobic photosynthesis, but grew normally under dark, aerobic and microaerophilic conditions with succinate and fructose as carbon sources. The anaerobic in vivo activity of CobZ indicates that it does not require oxygen as a substrate. The mutant excreted large amounts of protoporphyrin IX-monomethylester, a brown precursor of bacteriochlorophyll biosynthesis. The mutant was complemented either by the cobZRR gene in trans, or when exogenous cobalamin was added to the medium. A deletion mutant of tcuB/citB did not exhibit the cob phenotype. Thus, a role for tcuB/citB in cobalamin biosynthesis could not be confirmed.
Assuntos
Fotossíntese/fisiologia , Rhodospirillum rubrum , Vitamina B 12/biossíntese , Sequência de Aminoácidos , Bacterioclorofilas/biossíntese , Carotenoides/biossíntese , Cosmídeos/genética , DNA Bacteriano/genética , Deleção de Genes , Metiltransferases/genética , Oxirredutases/genética , Oxigênio/metabolismo , Porfirinas/metabolismo , Rhodospirillum rubrum/enzimologia , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismoRESUMO
BACKGROUND: As a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12-producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. RESULT: By the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5-2 was obtained and considered as a candidate for industrial applications. After 7 d's cultivation on a rotary shaker at 30 °C, the VB12 titer of S. meliloti MC5-2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5-2 was sequenced, and gene mutations were identified and analyzed. CONCLUSION: To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB12-yield strains.
Assuntos
Proteínas de Bactérias/genética , Engenharia Metabólica/métodos , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Vitamina B 12/biossíntese , Vitamina B 12/genética , Regulação Bacteriana da Expressão Gênica , Ensaios de Triagem em Larga Escala , Mutagênese , Mutação , RiboswitchRESUMO
Bacteria from the Propionibacterium genus consists of two principal groups: cutaneous and classical. Cutaneous Propionibacterium are considered primary pathogens to humans, whereas classical Propionibacterium are widely used in the food and pharmaceutical industries. Bacteria from the Propionibacterium genus are capable of synthesizing numerous valuable compounds with a wide industrial usage. Biomass of the bacteria from the Propionibacterium genus constitutes sources of vitamins from the B group, including B12, trehalose, and numerous bacteriocins. These bacteria are also capable of synthesizing organic acids such as propionic acid and acetic acid. Because of GRAS status and their health-promoting characteristics, bacteria from the Propionibacterium genus and their metabolites (propionic acid, vitamin B12, and trehalose) are commonly used in the cosmetic, pharmaceutical, food, and other industries. They are also used as additives in fodders for livestock. In this review, we present the major species of Propionibacterium and their properties and provide an overview of their functions and applications. This review also presents current literature concerned with the possibilities of using Propionibacterium spp. to obtain valuable metabolites. It also presents the biosynthetic pathways as well as the impact of the genetic and environmental factors on the efficiency of their production.
Assuntos
Propionatos/metabolismo , Propionibacterium/metabolismo , Vitamina B 12/biossíntese , Bacteriocinas/biossíntese , Biomassa , Vias Biossintéticas , Fermentação , Microbiologia Industrial , Trealose/metabolismoRESUMO
Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B12 itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism.
Assuntos
Benzimidazóis/metabolismo , Eubacterium/metabolismo , Vitamina B 12/biossíntese , Anaerobiose , Archaea/genética , Archaea/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corrinoides/biossíntese , DNA Recombinante/genética , Escherichia coli/metabolismo , Eubacterium/genética , Genes Arqueais , Genes Bacterianos , Geobacter/genética , Geobacter/metabolismo , Redes e Vias Metabólicas , Dados de Sequência Molecular , Estrutura Molecular , Moorella/genética , Moorella/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Riboswitch/genética , Salmonella typhimurium/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Amoebae are unicellular eukaryotes that consume microbial prey through phagocytosis, playing a role in shaping microbial food webs. Many amoebal species can be cultivated axenically in rich media or monoxenically with a single bacterial prey species. Here, we characterize heterolobosean amoeba LPG3, a recent natural isolate, which is unable to grow on unicellular cyanobacteria, its primary food source, in the absence of a heterotrophic bacterium, a Pseudomonas species coisolate. To investigate the molecular basis of this requirement for heterotrophic bacteria, we performed a screen using the defined nonredundant transposon library of Vibrio cholerae, which implicated genes in corrinoid uptake and biosynthesis. Furthermore, cobalamin synthase deletion mutations in V. cholerae and the Pseudomonas species coisolate do not support the growth of amoeba LPG3 on cyanobacteria. While cyanobacteria are robust producers of a corrinoid variant called pseudocobalamin, this variant does not support the growth of amoeba LPG3. Instead, we show that it requires cobalamin that is produced by the Pseudomonas species coisolate. The diversity of eukaryotes utilizing corrinoids is poorly understood, and this amoebal corrinoid auxotroph serves as a model for examining predator-prey interactions and micronutrient transfer in bacterivores underpinning microbial food webs.IMPORTANCE Cyanobacteria are important primary producers in aquatic environments, where they are grazed upon by a variety of phagotrophic protists and, hence, have an impact on nutrient flux at the base of microbial food webs. Here, we characterize amoebal isolate LPG3, which consumes cyanobacteria as its primary food source but also requires heterotrophic bacteria as a source of corrinoid vitamins. Amoeba LPG3 specifically requires the corrinoid variant produced by heterotrophic bacteria and cannot grow on cyanobacteria alone, as they produce a different corrinoid variant. This same corrinoid specificity is also exhibited by other eukaryotes, including humans and algae. This amoebal model system allows us to dissect predator-prey interactions to uncover factors that may shape microbial food webs while also providing insight into corrinoid specificity in eukaryotes.
Assuntos
Amoeba/fisiologia , Cianobactérias/metabolismo , Cadeia Alimentar , Pseudomonas/metabolismo , Vitamina B 12/biossíntese , Amoeba/crescimento & desenvolvimento , Cianobactérias/genética , Processos HeterotróficosRESUMO
Vitamin B12 is an essential vitamin that is widely used in medical and food industries. Vitamin B12 biosynthesis is confined to few bacteria and archaea, and as such its production relies on microbial fermentation. Rational strain engineering is dependent on efficient genetic tools and a detailed knowledge of metabolic pathways, regulation of which can be applied to improve product yield. Recent advances in synthetic biology and metabolic engineering have been used to efficiently construct many microbial chemical factories. Many published reviews have probed the vitamin B12 biosynthetic pathway. To maximize the potential of microbes for vitamin B12 production, new strategies and tools are required. In this review, we provide a comprehensive understanding of advances in the microbial production of vitamin B12, with a particular focus on establishing a heterologous host for the vitamin B12 production, as well as on strategies and tools that have been applied to increase microbial cobalamin production. Several worthy strategies employed for other products are also included.
Assuntos
Archaea/metabolismo , Bactérias/metabolismo , Vias Biossintéticas/genética , Vitamina B 12/biossíntese , Archaea/genética , Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Engenharia Metabólica/métodos , Engenharia Metabólica/tendências , Biologia Sintética/métodos , Biologia Sintética/tendênciasRESUMO
Vitamin B12 is an essential nutrient required for crucial metabolic processes in humans. Vitamin B12-producing lactic acid bacteria (LAB) have been attracting increased attentions currently because of the generally recognized as safe (GRAS) status. Most of recent studies focused on Lactobacillus, and little is known about B12-producing Enterococcus. In the present study, five Enterococcus strains isolated from infant feces were identified as vitamin B12 producers. Among them, Enterococcus faecium LZ86 had the highest B12 production (499.8 ± 83.7 µg/L), and the B12 compound from LZ86 was identified as the biological active adenosylcobalamin, using reversed phase high-performance liquid (RP-HPLC) chromatogram. We examined basic probiotic and safety properties of E. faecium LZ86 and found that it was able to survive harsh environmental conditions (hot temperature, cold temperature, ethanol and osmotic stresses), tolerate gastric acid (pH 2.0, 3 h) and bile salts (0.3%), and adhere to Caco-2 cells. We also showed that E. faecium LZ86 is devoid of transferable antibiotic resistance and potential virulence factors. Together, here we report a B12-producing E. faecium strain LZ86 firstly, which has desirable probiotic properties and may serve as a good candidate for vitamin B12 fortification in food industry.
Assuntos
Enterococcus/isolamento & purificação , Enterococcus/metabolismo , Probióticos/isolamento & purificação , Vitamina B 12/biossíntese , Antibacterianos/farmacologia , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Cobamidas/isolamento & purificação , Resistência Microbiana a Medicamentos , Enterococcus/efeitos dos fármacos , Enterococcus/crescimento & desenvolvimento , Fezes/microbiologia , Humanos , Lactente , Lactobacillus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Fatores de Virulência , Vitamina B 12/análiseRESUMO
Ribosome engineering has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking vitamin B12 production as a representative example. The introduction into Propionibacterium shermanii of mutations conferring resistance to rifampicin, gentamicin, and erythromycin, respectively, increased per cell production (µg/L/OD600) of vitamin B12 5.2-fold, although net production (µg/L) was unchanged, as the cell mass of the mutants was reduced. Real-time qPCR analysis demonstrated that the genes involved in vitamin B12 fermentation by P. shermanii were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of vitamin B12 by the mutants. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites.
Assuntos
Alquil e Aril Transferases/genética , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Propionibacterium/genética , Ribossomos/genética , Vitamina B 12/biossíntese , Alquil e Aril Transferases/metabolismo , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/genética , Eritromicina/farmacologia , Fermentação , Expressão Gênica , Engenharia Genética , Gentamicinas/farmacologia , Mutação , Propionibacterium/efeitos dos fármacos , Propionibacterium/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Rifampina/farmacologia , Análise de Sequência de DNA , Vitamina B 12/genéticaRESUMO
Euglena gracilis Z requires vitamins B1 and B12 for growth. It takes up and accumulates large amounts of these exogenous vitamins through energy-dependent active transport systems. Except for these essential vitamins, E. gracilis Z has the ability to synthesize all human vitamins. Euglena synthesizes high levels of antioxidant vitamins such as vitamins C and E, and, thus, are used as nutritional supplements for humans and domestic animals. Methods to effectively produce vitamins in Euglena have been investigated.Previous biochemical studies indicated that E. gracilis Z contains several vitamin-related novel synthetic enzymes and metabolic pathways which suggests that it is a highly suitable organism for elucidating the physiological functions of vitamins in comparative biochemistry and biological evolution. E. gracilis Z has an unusual biosynthetic pathway for vitamin C, a hybrid of the pathways found in animals and plants. This chapter presents up-to-date information on the biochemistry and physiological functions of vitamins in this organism.