Riboflavin Biosynthesis Pathway Research Articles

Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway

BackgroundRiboflavin is the precursor of several cofactors essential for normal physical and cognitive development, but only plants and some microorganisms can produce it. Humans thus rely on their dietary intake, which at a global level is mainly constituted by cereals (> 50%). Understanding the riboflavin biosynthesis players is key for advancing our knowledge on this essential pathway and can hold promise for biofortification strategies in major crop species. In some bacteria and in Arabidopsis, it is known that RibA1 is a bifunctional protein with distinct GTP cyclohydrolase II (GTPCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) domains. Arabidopsis harbors three RibA isoforms, but only one retained its bifunctionality. In rice, however, the identification and characterization of RibA has not yet been described.ResultsThrough mathematical kinetic modeling, we identified RibA as the rate-limiting step of riboflavin pathway and by bioinformatic analysis we confirmed that rice RibA proteins carry both domains, DHBPS and GTPCHII. Phylogenetic analysis revealed that OsRibA isoforms 1 and 2 are similar to Arabidopsis bifunctional RibA1. Heterologous expression of OsRibA1 completely restored the growth of the rib3∆ yeast mutant, lacking DHBPS expression, while causing a 60% growth improvement of the rib1∆ mutant, lacking GTPCHII activity. Regarding OsRibA2, its heterologous expression fully complemented GTPCHII activity, and improved rib3∆ growth by 30%. In vitro activity assays confirmed that both OsRibA1 and OsRibA2 proteins carry GTPCHII/DHBPS activities, but that OsRibA1 has higher DHBPS activity. The overexpression of OsRibA1 in rice callus resulted in a 28% increase in riboflavin content.ConclusionsOur study elucidates the critical role of RibA in rice riboflavin biosynthesis pathway, establishing it as the rate-limiting step in the pathway. By identifying and characterizing OsRibA1 and OsRibA2, showcasing their GTPCHII and DHBPS activities, we have advanced the understanding of riboflavin biosynthesis in this staple crop. We further demonstrated that OsRibA1 overexpression in rice callus increases its riboflavin content, providing supporting information for bioengineering efforts.

High fatty acid accumulation and coloration molecular mechanism of the elm mushroom (Pleurotus citrinopileatus).

Pleurotus citrinopileatus is a low-cholesterol, protein-rich, and high-nutrient food. The molecular mechanisms of the compounds and coloration have not been reported. Metabolome and transcriptome were used to clarify the molecular mechanisms of key compounds biosynthesis. K-means analysis identified 19 compounds in P. citrinopileatus, mainly lipids and alkaloids in class 8. In addition, 84 lipids were higher and that the different compounds were mainly enriched in linoleic acid metabolism. A total of 14 compounds detected in the linoleic acid metabolism pathway were significantly up-regulated, while 3 sterol regulatory element binding protein (SREBP) transcription factors were screened. Tryptophan metabolism and riboflavin biosynthesis pathway analysis indicated that 3 Unigenes had tryptophan decarboxylase similar elements, which belonged to tyrosine decarboxylase 1. Moreover, CL15618.Contig5_All had high homology with MFS. In conclusion, the expression of 3 SREBP, the synthesis of isobavachalcone D, and the regulation of riboflavin transport by MCH5 were the reasons for fatty acid accumulation and yellow cap formation in the P. citrinopileatus.

Site-specialization of human oral Gemella species

ABSTRACT Gemella species are core members of the human oral microbiome in healthy subjects and are regarded as commensals, although they can cause opportunistic infections. Our objective was to evaluate the site-specialization of Gemella species among various habitats within the mouth by combining pangenomics and metagenomics. With pangenomics, we identified genome relationships and categorized genes as core and accessory to each species. With metagenomics, we identified the primary oral habitat of individual genomes. Our results establish that the genomes of three species, G. haemolysans, G. sanguinis and G. morbillorum, are abundant and prevalent in human mouths at different oral sites: G. haemolysans on buccal mucosa and keratinized gingiva; G. sanguinis on tongue dorsum, throat, and tonsils; and G. morbillorum in dental plaque. The gene-level basis of site-specificity was investigated by identifying genes that were core to Gemella genomes at a specific oral site but absent from other Gemella genomes. The riboflavin biosynthesis pathway was present in G. haemolysans genomes associated with buccal mucosa but absent from the rest of the genomes. Overall, metapangenomics show that Gemella species have clear ecological preferences in the oral cavity of healthy humans and provides an approach to identifying gene-level drivers of site specificity.

Developing a PAM-Flexible CRISPR-Mediated Dual-Deaminase Base Editor to Regulate Extracellular Electron Transport in Shewanella oneidensis.

Shewanella oneidensis MR-1 is a promising electroactive microorganism in environmental bioremediation, bioenergy generation, and bioproduct synthesis. Accelerating the extracellular electron transfer (EET) pathway that enables efficient electron exchange between microbes and extracellular substances is critical for improving its electrochemical properties. However, the potential genomic engineering strategies for enhancing EET capabilities are still limited. Here, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-mediated dual-deaminase base editing system, named in situ protospacer-adjacent motif (PAM)-flexible dual base editing regulatory system (iSpider), for precise and high-throughput genomic manipulation. The iSpider enabled simultaneous C-to-T and A-to-G conversions with high diversity and efficiency in S. oneidensis. By weakening DNA glycosylase-based repair pathway and tethering two copies of adenosine deaminase, the A-to-G editing efficiency was obviously improved. As a proof-of-concept study, the iSpider was adapted to achieve multiplexed base editing for the regulation of the riboflavin biosynthesis pathway, and the optimized strain showed an approximately three-fold increase in riboflavin production. Moreover, the iSpider was also applied to evolve the performance of an inner membrane component CymA implicated in EET, and one beneficial mutant facilitating electron transfer could be rapidly identified. Taken together, our study demonstrates that the iSpider allows efficient base editing in a PAM-flexible manner, providing insights into the design of novel genomic tools for Shewanella engineering.

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol.

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L-1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.

Protein kinase MtCIPK12 modulates iron reduction in Medicago truncatula by regulating riboflavin biosynthesis.

Iron (Fe) is an essential micronutrient, and deficiency of available Fe is one of the most important limiting factors for plant growth. In some species including Medicago truncatula, iron deficiency results in accumulation of riboflavin, a response associated with iron acquisition. However, how the plant's Fe status is integrated to tune riboflavin biosynthesis and how riboflavin levels affect iron acquisition and utilization remains largely unexplored. We report that protein kinase CIPK12 regulates ferric reduction by accumulation of riboflavin and its derivatives in roots of M. truncatula via physiological and molecular characterization of its mutants and over-expressing materials. Mutations in CIPK12 enhance iron accumulation and improve photosynthetic efficiency, whereas over-expression of CIPK12 shows the opposite phenotypes. The Calcineurin B-like proteins CBL3 and CBL8 interact with CIPK12, which negatively regulates the expression of genes encoding key enzymes in the riboflavin biosynthesis pathway. CIPK12 negatively regulates iron acquisition by suppressing accumulation of riboflavin and its derivatives in roots, which in turn influences ferric reduction activity by riboflavin-dependent electron transport under iron deficiency. Our findings uncover a new regulatory mechanism by which CIPK12 regulates riboflavin biosynthesis and iron-deficiency responses in plants. This article is protected by copyright. All rights reserved.

RNA-Seq transcriptomic analysis reveals gene expression profiles of acetic acid bacteria under high-acidity submerged industrial fermentation process.

Acetic acid bacteria (AAB) are Gram-negative obligate aerobics in Acetobacteraceae family. Producing acetic acid and brewing vinegars are one of the most important industrial applications of AAB, attributed to their outstanding ability to tolerate the corresponding stresses. Several unique acid resistance (AR) mechanisms in AAB have been revealed previously. However, their overall AR strategies are still less-comprehensively clarified. Consequently, omics analysis was widely performed for a better understanding of this field. Among them, transcriptome has recently obtained more and more attention. However, most currently reported transcriptomic studies were conducted under lab conditions and even in low-acidity environment, which may be unable to completely reflect the conditions that AAB confront under industrialized vinegar-brewing processes. In this study, we performed an RNA-Seq transcriptomic analysis concerning AAB’s AR mechanisms during a continuous and periodical industrial submerged vinegar fermentation process, where a single AAB strain performed the fermentation and the acetic acid concentration fluctuated between ~8% and ~12%, the highest acidity as far we know for transcriptomic studies. Samples were directly taken from the initial (CK), mid, and final stages of the same period of the on-going fermentation. 16S rRNA sequence analysis indicated the participation of Komagataeibacter europaeus in the fermentation. Transcriptomic results demonstrated that more genes were downregulated than upregulated at both mid and final stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrich analysis reflected that the upregulated genes mainly carried out tricarboxylic acid cycle and oxidative phosphorylation processes, probably implying a considerable role of acetic acid overoxidation in AR during fermentation. Besides, upregulation of riboflavin biosynthesis pathway and two NAD+-dependent succinate-semialdehyde dehydrogenase-coding genes suggested a critical role of succinate oxidation in AR. Meanwhile, downregulated genes were mainly ribosomal protein-coding ones, reflecting that the adverse impact on ribosomes initiates at the transcription level. However, it is ambiguous whether the downregulation is good for stress responding or it actually reflects the stress. Furthermore, we also assumed that the fermentation stages may have a greater effect on gene expression than acidity. Additionally, it is possible that some physiological alterations would affect the AR to a larger extent than changes in gene expression, which suggests the combination of molecular biology and physiology research will provide deeper insight into the AR mechanisms in AAB.

Riboswitch-mediated regulation of riboflavin biosynthesis genes in prokaryotes.

Prokaryotic organisms frequently use riboswitches to quantify intracellular metabolite concentration via high-affinity metabolite receptors. Riboswitches possess a metabolite-sensing system that controls gene regulation in a cis-acting fashion at the initiation of transcriptional/translational level by binding with a specific metabolite and controlling various biochemical pathways. Riboswitch binds with flavin mononucleotide (FMN), a phosphorylated form of riboflavin and controls gene expression involved in riboflavin biosynthesis and transport pathway. The first step of the riboflavin biosynthesis pathway is initiated by the conversion of guanine nucleotide triphosphate (GTP), which is an intermediate of the purine biosynthesis pathway. An alternative pentose phosphate pathway of riboflavin biosynthesis includes the enzymatic conversion of ribulose-5-phosphate into 3, 4 dihydroxy-2-butanone-4-phosphates by DHBP synthase. The product of ribAB interferes with both GTP cyclohydrolase II as well as DHBP synthase activities, which catalyze the cleavage of GTP and converts DHBP Ribu5P in the initial steps of both riboflavin biosynthesis branches. Riboswitches are located in the 5' untranslated region (5' UTR) of messenger RNAs and contain an aptamer domain (highly conserved in sequence) where metabolite binding leads to a conformational change in an aptamer domain, which modulate the regulation of gene expression located on bacterial mRNA. In this review, we focus on how riboswitch regulates the riboflavin biosynthesis pathway in Bacillus subtilis and Lactobacillus plantarum.

High-yield production of coenzyme F420 in Escherichia coli by fluorescence-based screening of multi-dimensional gene expression space

Coenzyme F420 is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F420-dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F420 levels are produced in microorganisms, F420 availability is a serious bottleneck for research and application. Recent advances in our understanding of the F420 biosynthesis enabled heterologous overproduction of F420 in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F420 biosynthesis in E. coli. However, it still led to the production of low amounts of F420 and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module (“core”) addressed parts of the riboflavin biosynthesis pathway and FO synthase for the conversion of GTP to the stable F420 intermediate FO. The enzymes of the second module (“decoration”) were chosen to turn FO into F420. The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F420-derived fluorescence. This approach yielded the highest titer of coenzyme F420 produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F420-dependent bioprocesses as well as applications of F420-dependent enzymes in biocatalysis.

Identification and Functional Analysis of GTP Cyclohydrolase II in Candida glabrata in Response to Nitrosative Stress.

Reactive nitrogen species (RNS) are signal molecules involved in various biological events; however, excess levels of RNS cause nitrosative stress, leading to cell death and/or cellular dysfunction. During the process of infection, pathogens are exposed to nitrosative stress induced by host-derived RNS. Therefore, the nitrosative stress resistance mechanisms of pathogenic microorganisms are important for their infection and pathogenicity, and could be promising targets for antibiotics. Previously, we demonstrated that the RIB1 gene encoding GTP cyclohydrolase II (GCH2), which catalyzes the first step of the riboflavin biosynthesis pathway, is important for nitrosative stress resistance in the yeast Saccharomyces cerevisiae. Here, we identified and characterized the RIB1 gene in the opportunistic pathogenic yeast Candida glabrata. Our genetic and biochemical analyses indicated that the open reading frame of CAGL0F04279g functions as RIB1 in C. glabrata (CgRIB1). Subsequently, we analyzed the effect of CgRIB1 on nitrosative stress resistance by a growth test in the presence of RNS. Overexpression or deletion of CgRIB1 increased or decreased the nitrosative stress resistance of C. glabrata, respectively, indicating that GCH2 confers nitrosative stress resistance on yeast cells. Moreover, we showed that the proliferation of C. glabrata in cultures of macrophage-like cells required the GCH2-dependent nitrosative stress detoxifying mechanism. Additionally, an infection assay using silkworms as model host organisms indicated that CgRIB1 is indispensable for the virulence of C. glabrata. Our findings suggest that the GCH2-dependent nitrosative stress detoxifying mechanism is a promising target for the development of novel antibiotics.

Augmentation of the Riboflavin-Biosynthetic Pathway Enhances Mucosa-Associated Invariant T (MAIT) Cell Activation and Diminishes Mycobacterium tuberculosis Virulence.

ABSTRACTMucosa-associated invariant T (MAIT) cells play a critical role in antimicrobial defense. Despite increased understanding of their mycobacterial ligands and the clinical association of MAIT cells with tuberculosis (TB), their function in protection against Mycobacterium tuberculosis infection remains unclear. Here, we show that overexpressing key genes of the riboflavin-biosynthetic pathway potentiates MAIT cell activation and results in attenuation of M. tuberculosis virulence in vivo. Further, we observed greater control of M. tuberculosis infection in MAIThi CAST/EiJ mice than in MAITlo C57BL/6J mice, highlighting the protective role of MAIT cells against TB. We also endogenously adjuvanted Mycobacterium bovis BCG with MR1 ligands via overexpression of the lumazine synthase gene ribH and evaluated its protective efficacy in the mouse model of M. tuberculosis infection. Altogether, our findings demonstrate that MAIT cells confer host protection against TB and that overexpression of genes in the riboflavin-biosynthetic pathway attenuates M. tuberculosis virulence. Enhancing MAIT cell-mediated immunity may also offer a novel approach toward improved vaccines against TB.

MAIT and Vδ2 Unconventional T Cells Predict Favorable Outcome after Allogeneic HCT and Are Supported By a Diverse Intestinal Microbiome

MAIT and Vδ2 Unconventional T Cells Predict Favorable Outcome after Allogeneic HCT and Are Supported By a Diverse Intestinal Microbiome

Transcription-dependent confined diffusion of enzymes within subcellular spaces of the bacterial cytoplasm

BackgroundKnowledge on the localization and mobility of enzymes inside bacterial cells is scarce, but important for understanding spatial regulation of metabolism. The four central enzymes (Rib enzymes) of the riboflavin (RF) biosynthesis pathway in the Gram positive model bacterium Bacillus subtilis have been studied extensively in vitro, especially the heavy RF synthase, a large protein complex with a capsid structure formed by RibH and an encapsulated RibE homotrimer, which mediates substrate-channeling. However, little is known about the behavior and mobility of these enzymes in vivo.ResultsWe have investigated the localization and diffusion of the Rib enzymes in the cytoplasm of B. subtilis. By characterizing the diffusion of Rib enzymes in live cells using single particle tracking (SPT) we provide evidence for confined diffusion at the cell poles and otherwise Brownian motion. A majority of RibH particles showed clear nucleoid occlusion and a high degree of confined motion, which is largely abolished after treatment with Rifampicin, revealing that confinement is dependent on active transcription. Contrarily, RibE is mostly diffusive within the cell, showing only 14% encapsulation by RibH nanocompartments. By localizing different diffusive populations within single cells, we find that fast diffusion occurs mostly across the nucleoids located in the cell centers, while the slower, confined subdiffusion occurs at the crowded cell poles.ConclusionsOur results provide evidence for locally different motion of active enzymes within the bacterial cytoplasm, setting up metabolic compartmentalization mostly at the poles of cells.

Improving riboflavin production by knocking down ribF, purA and guaC genes using synthetic regulatory small RNA

Riboflavin is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. The down-regulation of expression levels of ribF, purA and guaC genes involved in the downstream or branch reactions of riboflavin biosynthesis pathway could direct more carbon flux to riboflavin accumulation. In this study, we made an attempt to fine-tune the expression levels of the 3 genes by using synthetic regulatory small RNA to enhance riboflavin production in Escherichia coli. Firstly, each of the 3 genes was knocking down by using 5 different sRNAs, respectively, and a highest increase of 50.2 % in riboflavin titer was achieved by using anti-ribF5 sRNA. Then this sRNA was further co-expressed with 5 anti-purA and 5 anti-guaC sRNAs to simultaneously knocking down 2 or 3 genes. Co-expression of anti-ribF5 and anti-guaC3 led to the highest riboflavin production of 1091.3 mg/L, which was further increased by 97.6 % compared to the base strain. Finally, the expression levels of anti-ribF5 and anti-guaC3 were further fine-tuned by using 4 different promoters. The best strain WY40, in which the two sRNAs were respectively expressed by PJ23100 and PJ23107 promoter, produced 1454.5 mg/L riboflavin with an increase of 163.4 % compared to the base strain. To our knowledge, it’s the first study to enhance riboflavin synthesis by simultaneously regulating the expression levels of ribF, purA and guaC genes, which led to a highest yield of 0.147 g/g glucose among all reported riboflavin-producing strains.

Structurally disordered C-terminal residues of GTP cyclohydrolase II are essential for its enzymatic activity

GTP cyclohydrolase II (GCHII) is one of the rate limiting enzymes in riboflavin biosynthesis pathway and is shown to be a potential drug target for most of the pathogens. Previous biochemical and structural studies have identified the active site residues and elucidated the steps involved in the catalytic mechanism of GCHII. However, the last ∼20–25 C-terminal residues of GCHII remains unstructured in all the crystal structures determined to date and their role in the catalytic activity, if any, remains elusive. Therefore, to understand the role of these unstructured C-terminal residues, a series of C-terminal deletion mutants of GCHII from Helicobacter pylori (hGCHII) were generated and their catalytic activity was compared with its wild-type. Surprisingly, none of the C-terminal deletion mutants shows any enzymatic activity indicating that these are essential for GCHII function. To get additional insights for such loss of activity, homology models of full-length and deletion mutants of hGCHII in complex with GTP, Mg2+, and Zn2+ were generated and subjected to molecular dynamics simulation studies. The simulation studies show that a conserved histidine at 190th position from the unstructured C-terminal region of hGCHII interacts with α-phosphate of GTP. We propose that His-190 may play a role in the hydrolysis of pyrophosphate from GTP and in releasing the product, DARP. In summary, we demonstrate that the unstructured C-terminal residues of GCHII are important for its enzymatic activity and must be considered during rational drug designing. Communicated by Ramaswamy H. Sarma

Immuno-antibiotics: targeting microbial metabolic pathways sensed by unconventional T cells.

SummaryHuman Vγ9/Vδ2 T cells, mucosal-associated invariant T (MAIT) cells, and other unconventional T cells are specialised in detecting microbial metabolic pathway intermediates that are absent in humans. The recognition by such semi-invariant innate-like T cells of compounds like (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), the penultimate metabolite in the MEP isoprenoid biosynthesis pathway, and intermediates of the riboflavin biosynthesis pathway and their metabolites allows the immune system to rapidly sense pathogen-associated molecular patterns that are shared by a wide range of micro-organisms. Given the essential nature of these metabolic pathways for microbial viability, they have emerged as promising targets for the development of novel antibiotics. Here, we review recent findings that link enzymatic inhibition of microbial metabolism with alterations in the levels of unconventional T cell ligands produced by treated micro-organisms that have given rise to the concept of ‘immuno-antibiotics’: combining direct antimicrobial activity with an immunotherapeutic effect via modulation of unconventional T cell responses.

Recent Advances in Construction of the Efficient Producers of Riboflavin and Flavin Nucleotides (FMN, FAD) in the Yeast Candida famata.

The approaches used by the authors to design the Candida famata strains capable to overproduce riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are described. The metabolic engineering approaches include overexpression of SEF1 gene encoding positive regulator of riboflavin biosynthesis, IMH3 (coding for IMP dehydrogenase) orthologs from another species of flavinogenic yeast Debaryomyces hansenii, and the homologous genes RIB1 and RIB7 encoding GTP cyclohydrolase II and riboflavin synthase, the first and the last enzymes of riboflavin biosynthesis pathway, respectively. Overexpression of the abovementioned genes in the genetically stable riboflavin overproducer AF-4 obtained by classical selection resulted in fourfold increase of riboflavin production in shake flask experiments.Overexpression of engineered enzymes phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase catalyzing the initial steps of purine nucleotide biosynthesis enhances riboflavin synthesis in the flavinogenic yeast C. famata even more.Recombinant strains of C. famata containing FMN1 gene from D. hansenii encoding riboflavin kinase under control of the strong constitutive TEF1 promoter were constructed. Overexpression of the FMN1 gene in the riboflavin-producing mutant led to the 30-fold increase of the riboflavin kinase activity and 400-fold increase of FMN production in the resulting recombinant strains which reached maximally 318.2mg/L.FAD overproducing strains of C. famata were also constructed. This was achieved by overexpression of FAD1 gene from D. hansenii in C. famata FMN overproducing strain. The 7- to 15-fold increase in FAD synthetase activity as compared to the wild-type strain and FAD accumulation into cultural medium were observed. The maximal FAD titer 451.5mg/L was achieved.

Synthesis, biological evaluation and molecular modelling of new potent clickable analogues of 5-OP-RU for their use as chemical probes for the study of MAIT cell biology.

MAIT cells are preset αβ T lymphocytes that recognize a series of microbial antigens exclusively derived from the riboflavin biosynthesis pathway, which is present in most bacteria. The most active known antigen is unstable 5-(2-oxopropylideneamino)-6-(d-ribitylamino)uracil (5-OP-RU) which is stabilized when bound and presented to MAIT cells by MHC-related protein 1 (MR1). Here we describe the chemical synthesis and biological evaluation of new chemical probes for the study of MAIT cell biology. The two probes were ethinyl functionalized analogues of 5-OP-RU able to react through CuAAC also called "click chemistry". The molecules up-regulated more MR1 than 5-OP-RU and they efficiently activated iVα19Vβ8 TCR transgenic murine MAIT cells but not iVα19 TCRα transgenic MAIT cells indicating a surprisingly strong impact of the TRCβ chain. Moreover, the use of these molecules as chemical probes was validated invitro by efficient and selective binding to MR1 revealed via fluorescence microscopy. This study was also complemented by molecular modelling investigation of the probes and the binary/ternary complexes they form with MR1 and the TCR. These new probes will be crucial to delineate the dynamics of 5-OP-RU at the cellular or whole organism level and to identify the cells presenting 5-OP-RU to MAIT cells invivo.

RibBX of Bradyrhizobium ORS285 Plays an Important Role in Intracellular Persistence in Various Aeschynomene Host Plants.

Bradyrhizobium ORS285 forms a nitrogen-fixating symbiosis with both Nod factor (NF)-dependent and NF-independent Aeschynomene spp. The Bradyrhizobium ORS285 ribBA gene encodes for a putative bifunctional enzyme with 3,4-dihydroxybutanone phosphate (3,4-DHBP) synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps in the riboflavin biosynthesis pathway. In this study, we show that inactivating the ribBA gene does not cause riboflavin auxotrophy under free-living conditions and that, as shown for RibBAs from other bacteria, the GTP cyclohydrolase II domain has no enzymatic activity. For this reason, we have renamed the annotated ribBA as ribBX. Because we were unable to identify other ribBA or ribA and ribB homologs in the genome of Bradyrhizobium ORS285, we hypothesize that the ORS285 strain can use unconventional enzymes or an alternative pathway for the initial steps of riboflavin biosynthesis. Inactivating ribBX has a drastic impact on the interaction of Bradyrhizobium ORS285 with many of the tested Aeschynomene spp. In these Aeschynomene spp., the ORS285 ribBX mutant is able to infect the plant host cells but the intracellular infection is not maintained and the nodules senesce early. This phenotype can be complemented by reintroduction of the 3,4-DHBP synthase domain alone. Our results indicate that, in Bradyrhizobium ORS285, the RibBX protein is not essential for riboflavin biosynthesis under free-living conditions and we hypothesize that its activity is needed to sustain riboflavin biosynthesis under certain symbiotic conditions.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of light-sensitive riboflavin (vitamin B2)

Due to their capability of photosynthesis and autotrophic growth, cyanobacteria are currently investigated with regard to the sustainable production of a wide variety of chemicals. So far, however, no attempt has been undertaken to engineer cyanobacteria for the biotechnological production of vitamins, which is probably due to the light-sensitivity of many of these compounds. We now describe a photoautotrophic bioprocess to synthesize riboflavin, a vitamin used as a supplement in the feed and food industry. By overexpressing the riboflavin biosynthesis genes ribDGEABHT from Bacillus subtilis in the marine cyanobacterium Synechococcus sp. PCC 7002 riboflavin levels in the supernatant of the corresponding recombinant strain increased 56-fold compared to the wild-type. Introduction of a second promoter region upstream of the heterologous ribAB gene – coding for rate-limiting enzymatic functions in the riboflavin biosynthesis pathway – led to a further increase of riboflavin levels (211-fold compared to the wild-type). Degradation of the light-sensitive product riboflavin was prevented by culturing the genetically engineered Synechococcus sp. PCC 7002 strains in the presence of dichromatic light generated by red light-emitting diodes (λ = 630 and 700 nm). Synechococcus sp. PCC 7002 naturally is resistant to the toxic riboflavin analog roseoflavin. Expression of the flavin transporter pnuX from Corynebacterium glutamicum in Synechococcus sp. PCC 7002 resulted in roseoflavin-sensitive recombinant strains which in turn could be employed to select roseoflavin-resistant, riboflavin-overproducing strains as a chassis for further improvement.