Riboflavin Synthesis Research Articles

Improving riboflavin production by modifying related metabolic pathways in Bacillus subtilis.

Riboflavin is a feed additive, food additive and clinical drug, with a significant annual demand of nearly 8000 t. Fermentation using recombinant Bacillus subtilis is currently one of the most important industrial production method for riboflavin. First, a suitable medium was selected and the expression of the ureABC operon was modified. The ykgB gene was overexpressed in B. subtilis RX10, the production of the derivative strain RX20 was increased to 4·61 g l-1 riboflavin, and the yield was increased to 52 mg riboflavin g-1 glucose. The relative transcription level of pyr operon in RX20 was reduced to 71%, the production of the derivative strain RX21 was increased to 5·82 g l-1 riboflavin, and the yield was 76 mg riboflavin g-1 glucose. The start codon of the pyrE gene in RX21 was modified to 'TTG', the production of the derivative strain RX22 was increased to 7·01 g l-1 riboflavin, and the yield was 89 mg riboflavin g-1 glucose. These results indicated that overexpression of the ykgB gene and reduction of the metabolic flux of de novo synthesis of pyrimidine nucleotides were beneficial to the synthesis of riboflavin.

Lactose Inducible Expression of Transcription Factor Gene SEF1 Increases Riboflavin Production in the Yeast Candida famata

Riboflavin (vitamin B2) is required for synthesis of the flavin coenzymes: riboflavin-5’-phosphate (flavin mononucleotide) and flavin adenine dinucleotide. Riboflavin is important biotechnological commodity with annual market around 250 million US dollars. It is mostly used as component of feed premixes for animals (80%), in food industry as food colorant, in medicine and component of multivitamin mixtures and as drug for treatment of some diseases. Over the past two decades, the microbial production of riboflavin by fermentation completely replaces the chemical synthetic route. The main producers of riboflavin in industry are engineered strains of the bacterium Bacillus subtilis and of the mycelial fungus Ashbya gossypii. Flavinogenic yeast Candida famata has great biosynthetic potential. Using combination of classical selection and metabolic engineering (overexpression of SEF1, RIB1 and RIB7 genes coding the positive regulator, the first and the last structural enzymes of riboflavin synthesis) resulted in the construction of genetically stable strain of C. famata that produces 16 gram of riboflavin per liter in bioreactor. However, the productivity of riboflavin biosynthesis remains still insufficient for industrial production of this vitamin. Studies of transcriptional regulation of genes involved in riboflavin synthesis and using of strong promoters of C. famata for construction of efficient producers of vitamin B2 are areas of both scientific and industrial interest. Aim. The aim of the current work was to improve riboflavin oversynthesis by the available C. famata strains in synthetic and natural lactose-containing media. Methods. The plasmid DNA isolation, restriction, ligation, electrophoresis in agarose gel, electrotransformation, and PCR were carried out by the standard methods. Riboflavin was assayed fluorometrically using solution of synthetic riboflavin as a standard. The cultivation of yeasts was carried out in YNB or YPD media containing different source of carbon and on whey. Results. The strains of C. famata expressed additional copy of central regulatory gene SEF1 under control of the promoter of LAC4 gene (coding for β–galactosidase) C. famata were constructed. The influence of SEF1 gene expression under control of lactose inducible promoter of CfLAC4 gene on riboflavin production was studied. It was shown that the C. famata strains containing “pLAC4_cf-SEF1_cf” expression cassette revealed 1.6-2.1-fold increase in riboflavin yield on lactose when compared to the parental strain. The riboflavin production constructed strains on whey reached 1.69 gram per liter in flask batch culture. Conclusions. The constructed strains containing additional copy of SEF1 gene under the control of LAC4 promoter is a perfect platform for development of industrial riboflavin production on by-product of dairy industry, whey.

Engineering of salt-tolerant Shewanella aquimarina XMS-1 for enhanced pollutants transformation and electricity generation

Saline wastewater poses a challenge during bio-treatment process due to salinity affecting the physiological activity of microorganisms and inhibiting their growth and metabolism. Thus, screening and engineering the salt-tolerant strains with stronger performances are urgent. Shewanella aquimarina XMS-1, a salt-tolerant dissimilated metal reducing bacteria (DMRB), was isolated from seawater environment. Its ability for reducing pollutants and generating electricity was enhanced by overexpression of riboflavin synthesis pathway encoding genes from S. oneidensis MR-1 under salt stress. Furthermore, upon contact with graphene oxide (GO), the engineered strain XMS-1/pYYDT-rib with enhanced flavins synthesis could reduce GO and self-assemble to form a three-dimensional (3D) biohybrid system named XMS-1/flavins/rGO. This 3D biohybrid system significantly enhanced the EET efficiency of S. aquimarina XMS-1. Our findings provide a feasible strategy for treatment of salt-containing industrial wastewater contaminated by metal and organic pollutants.

Varicella Zoster Virus Impairs Expression of the Nonclassical Major Histocompatibility Complex Class I-Related Gene Protein (MR1).

The antigen presentation molecule MR1 (major histocompatibility complex, class I-related) presents ligands derived from the riboflavin (vitamin B) synthesis pathway, which is not present in mammalian species or viruses, to mucosal-associated invariant T (MAIT) cells. In this study, we demonstrate that varicella zoster virus (VZV) profoundly suppresses MR1 expression. We show that VZV targets the intracellular reservoir of immature MR1 for degradation, while preexisting, ligand-bound cell surface MR1 is protected from such targeting, thereby highlighting an intricate temporal relationship between infection and ligand availability. We also identify VZV open reading frame (ORF) 66 as functioning to suppress MR1 expression when this viral protein is expressed during transient transfection, but this is not apparent during infection with a VZV mutant virus lacking ORF66 expression. This indicates that VZV is likely to encode multiple viral genes that target MR1. Overall, we identify an immunomodulatory function of VZV whereby infection suppresses the MR1 biosynthesis pathway.

Characteristics of mucosal-associated invariant T cells and their roles in immune diseases.

Mucosal-associated invariant T (MAIT) cells are a subset of innate-like T cells that express a semi-invariant T-cell receptor and are restricted by the major histocompatibility complex class I-related molecule 1 (MR1). MAIT cells recognize biosynthetic derivatives of the riboflavin synthesis pathway present in microbes. MAIT cells have attracted increased interest related to various immune responses because of their unique features including their abundance in humans, non-peptidic antigens and ability to respond to antigenic and non-antigenic stimuli. The numbers of circulating MAIT cells are decreased in many immune diseases such as multiple sclerosis, systemic lupus erythematosus and inflammatory bowel diseases. However, the remaining MAIT cells have an increased cytokine-producing capacity and activated status, which are related to disease activity. Additionally, MAIT cells have been observed at sites of inflammation including the kidneys, synovial fluid and intestinal mucosa. These findings suggest their involvement in the pathogenesis of immune diseases. In this mini-review, we summarize the recent findings of MAIT cells in human immune diseases and animal models, and discuss their role and potential as a therapeutic target.

Generation of Fluorescent Bacteria with the Genes Coding for Lumazine Protein and Riboflavin Biosynthesis.

Lumazine protein is a member of the riboflavin synthase superfamily and the intense fluorescence is caused by non-covalently bound to 6,7-dimethyl 8-ribityllumazine. The pRFN4 plasmid, which contains the riboflavin synthesis genes from Bacillus subtilis, was originally designed for overproduction of the fluorescent ligand of 6,7-dimethyl 8-ribityllumazine. To provide the basis for a biosensor based on the lux gene from bioluminescent bacteria of Photobacterium leiognathi, the gene coding for N-terminal domain half of the lumazine protein extending to amino acid 112 (N-LumP) and the gene for whole lumazine protein (W-LumP) from P. leiognathi were introduced by polymerase chain reaction (PCR) and ligated into pRFN4 vector, to construct the recombinant plasmids of N-lumP-pRFN4 and W-lumP-pRFN4 as well as their modified plasmids by insertion of the lux promoter. The expression of the genes in the recombinant plasmids was checked in various Escherichia coli strains, and the fluorescence intensity in Escherichia coli 43R can even be observed in a single cell. These results concerning the co-expression of the genes coding for lumazine protein and for riboflavin synthesis raise the possibility to generate fluorescent bacteria which can be used in the field of bio-imaging.

Activating Mucosal-Associated Invariant T Cells Induces a Broad Antitumor Response.

Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. Although in vitro-activated MAIT cells show antitumor activity, the in vivo role of MAIT cells in cancer is still unclear. Here, we have shown that MAIT cells have antitumor function in vivo when activated by a combination of the synthetic riboflavin synthesis pathway-derived antigen 5-OP-RU [5-(2-oxopropylideneamino)-6-D-ribitylaminouracil] and the Toll-like receptor 9 (TLR9) agonist CpG. Coadministration of 5-OP-RU and CpG induced strong systemic in vivo expansion and activation of MAIT cells with high CD69 expression, pronounced effector memory phenotype, and upregulated levels of effector molecules including IFNγ, granzyme B, and perforin. Activated and expanded MAITs induced a potent and broad antitumor immune response in murine models of liver metastasis and hepatocellular carcinoma, lung metastasis, and subcutaneous tumors in two different mouse strains. Such tumor inhibition was absent in MAIT-deficient Mr1 -/- mice. CRISPR/Cas9-mediated MR1 knockout in tumor cells did not affect efficacy of this MAIT-directed immunotherapy, pointing toward an indirect mechanism of action. Our findings suggest that MAIT cells are an attractive target for cancer immunotherapy.See related Spotlight by Lantz, p. 996.

Complete Genome Sequence and Function Gene Identify of Prometryne-Degrading Strain Pseudomonas sp. DY-1.

The genus Pseudomonas is widely recognized for its potential for environmental remediation and plant growth promotion. Pseudomonas sp. DY-1 was isolated from the agricultural soil contaminated five years by prometryne, it manifested an outstanding prometryne degradation efficiency and an untapped potential for plant resistance improvement. Thus, it is meaningful to comprehend the genetic background for strain DY-1. The whole genome sequence of this strain revealed a series of environment adaptive and plant beneficial genes which involved in environmental stress response, heavy metal or metalloid resistance, nitrate dissimilatory reduction, riboflavin synthesis, and iron acquisition. Detailed analyses presented the potential of strain DY-1 for degrading various organic compounds via a homogenized pathway or the protocatechuate and catechol branches of the β-ketoadipate pathway. In addition, heterologous expression, and high efficiency liquid chromatography (HPLC) confirmed that prometryne could be oxidized by a Baeyer-Villiger monooxygenase (BVMO) encoded by a gene in the chromosome of strain DY-1. The result of gene knock-out suggested that the sulfate starvation-induced (SSI) genes in this strain might also involve in the process of prometryne degradation. These results would provide the molecular basis for the application of strain DY-1 in various fields and would contribute to the study of prometryne biodegradation mechanism as well.

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.

Biotechnological Strategies of Riboflavin Biosynthesis in Microbes

Riboflavin is an essential micronutrient for humans and must be obtained exogenously from foods or supplements. Numerous studies have suggested a major role of riboflavin in the prevention and treatment of various diseases. There are mainly three strategies for riboflavin synthesis, including total chemical synthesis, chemical semi-synthesis, and microbial fermentation, the latter being currently the most promising strategy. In recent years, flavinogenic microbes have attracted increasing attention. Fungi, including Eremothecium ashbyii and Ashbya gossypii , and bacteria, including Bacillus subtilis , Escherichia coli , and lactic acid bacteria, are ideal cell factories for riboflavin overproduction. Thus they are good candidates for enhancing the level of riboflavin in fermented foods or designing novel riboflavin bio-enriched foods with improved nutritional value and/or beneficial properties for human health. This review briefly describes the role of riboflavin in human health and the historical process of its industrial production, and then highlights riboflavin biosynthesis in bacteria and fungi, and finally summarizes the strategies for riboflavin overproduction based on both the optimization of fermentation conditions and the development of riboflavin-overproducing strains via chemical mutagenesis and metabolic engineering. Overall, this review provides an updated understanding of riboflavin biosynthesis and can promote the research and development of fermented food products rich in riboflavin.

Metabolic engineering of Bacillus subtilis for enhancing riboflavin production by alleviating dissolved oxygen limitation

Riboflavin, an essential vitamin for animals, is used widely in the pharmaceutical industry and as a food and feed additive. The microbial synthesis of riboflavin requires a large amount of oxygen, which limits the industrial-scale production of the vitamin. In this study, a metabolic engineering strategy based on transcriptome analysis was identified as effective in increasing riboflavin production. First, transcriptional profiling revealed that hypoxia affects purine, and nitrogen metabolism. Next, the precursor supply pool was increased by purR knockout and tnrA and glnR knockdown to balance intracellular nitrogen metabolism. Finally, increased oxygen utilization was achieved by dynamically regulating vgb. Fed-batch fermentation of the engineered strain in a 5-liter bioreactor produced 10.71 g/l riboflavin, a 45.51% higher yield than that obtained with Bacillus subtilis RF1. The metabolic engineering strategy described herein is useful for alleviating the oxygen limitation of bacterial strains used for the industrial production of riboflavin and related products.

Human liver-derived MAIT cells differ from blood MAIT cells in their metabolism and response to TCR-independent activation.

Mucosal associated invariant T (MAIT) cells are anti-microbial innate-like T cells that are abundant in blood and liver. MAIT cells express a semi-invariant T-cell receptor (TCR) that recognizes a pyrimidine ligand, derived from microbial riboflavin synthesis, bound to MR1. Both blood and liver derived (ld)-MAIT cells can be robustly stimulated via TCR or by cytokines produced during bacterial or viral infection. In this study, we compared the functional and transcriptomic response of human blood and ld-MAIT cells to TCR signals (Escherichia coli or the pyrimidine ligand) and cytokines (IL-12 + IL-18). While the response of blood and ld-MAIT cells to TCR signals were comparable, following cytokine stimulation ld-MAIT cells were more polyfunctional than blood MAIT cells. Transcriptomic analysis demonstrated different effector programmes of ld-MAIT cells with the two modes of activation, including the enrichment of a tissue repair signature in TCR-stimulated MAIT cells. Interestingly, we observed enhancement of IL-12 signaling and fatty acid metabolism in untreated ld-MAIT cells compared with blood MAIT cells. Additionally, MAIT cells from blood and liver were modulated similarly by TCR and cytokine signals. Therefore, we report that blood and ld-MAIT cells are fundamentally different but undergo conserved changes following activation via TCR or by cytokines.

Martelella alba sp. nov., isolated from mangrove rhizosphere soil within the Beibu Gulf

Strain BGMRC 2036T was isolated from rhizosphere soil of Bruguiear gymnorrhiza collected from the Beibu Gulf of China. Optimum growth occurred at 28 °C, pH 7.0, and under the conditions of 3–5% (w/v) NaCl. The phylogenetic comparisons of 16S rRNA gene sequences displayed that strain BGMRC 2036T was closely related to Martelella limonii NBRC109441T (96.6% sequence similarity), M. mediterranea CGMCC 1.12224T (96.5%), M. lutilitoris GH2-6T (96.5%), M. radicis BM5-7T (96.2%), and M. mangrove BM9-1T (95.9%), M. suaedae NBRC109440T (95.8%). The phylogenomic tree based on the up-to-date bacterial core gene set indicated that the strain BGMRC 2036T form a clade formed with members of the genera Martelella. The major polar lipids include phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphotidylinositol, two unidentified phospholipids, and three unidentified ninhydrin positive phospholipids. The major respiratory quinone is Q-10, which is similar to those of genera Martelella. The main cellular fatty acids are C18:1ω7c, C16:0, and C12:0 aldehyde. Genome sequencing revealed a genome size of 4.99 Mbp and a G + C content of 62.3 mol%. Pairwise comparison of the genomes of the new strain BGMRC 2036T and the three reference strains M. endophytica YC 6887T, M. mediterranea CGMCC 1.12224T, and M. mangrovi USBA-857 indicated that gANI value was lower than 81% and a digital DNA–DNA hybridization value was lower than 27%. The strain BGMRC 2036T possessed genes putatively encoding riboflavin synthesis and flavodoxin A polyphasic taxonomic study suggested that strain BGMRC 2036T represented a novel species belonging to the genus Martelella, and it was named Martelella alba sp. nov. The type strain is BGMRC 2036T (=KCTC 52121T =NBRC 111908T).

Bruguierivorax albus gen. nov. sp. nov. Isolated from Mangrove Sediment and Proposal of Bruguierivoracaceae fam. nov

A novel Gram-negative, motile, aerobic rod-shaped bacterium designated BGMRC 2031T was isolated from mangrove sediment collected from Guangxi Province, China. Optimal growth occurred at 28 °C and pH 7.0–8.0 in the presence of 1% (w/v) NaCl. Alignment based on 16S rRNA gene sequences indicated that strain BGMRC 2031T is most closely related to Sodalis praecaptivus HS1T (95.6%, sequence similarity), followed by Biostraticola tofi DSM 19580T (95.5%), Sodalis glossinidius DSM 16929T (95.4%), and Brenneria goodwinii FRB141T (94.9%) sequence similarity. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BGMRC 2031T formed a distinct branch in a robust cluster and revealed that strain BGMRC 2031T, genera Biostraticola and Sodalis, formed a novel family-level clade in the order Enterobacterales. The novel strain showed an average nucleotide similarity of 74.7%, 74.2%, and 73.1% for S. praecaptivus HS1T, S. glossinidius DSM 16929T, and B. tofi DSM 19580T, respectively. The genomes of the BGMRC 2031T shared the presence of a riboflavin synthesis gene cluster. The menaquinones of strain BGMRC 2031T were MK-8 and Q-8, which were similar to those of genus Biostraticola. The major fatty acids (> 10%) were C16:0 (19.9%), summed feature 2 (iso-C16:1 and/or C14:0 3-OH, 18.10%), summed feature 3 (C16:1ω7c and/or C16:1ω6c, 15.3%), C12:0 (13.9%), C17:0 cyclo (11.4%), and C14:0 (10.4%). The main polar lipids were phosphatidyl methylethanolamine, phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl inositol, one unidentified phospholipid, and one unknown polar lipid. The G+C content of strain BGMRC 2031T was 55.4%. Strain BGMRC 2031T could extend the mean lifespan and maximum lifespan of Caenorhabditis elegans by 4.5% and 12.5%, respectively. Overall, the results of this study indicate that BGMRC 2031T is a novel species in a new genus, for which the name Bruguierivorax albus gen. nov. sp. nov. is proposed, and the type of strain is designated as BGMRC 2031T (= NBRC 111907T = KCTC 52119T). In addition, a novel family, Bruguierivoracaceae fam. nov., is proposed to accommodate the genera Bruguierivorax, Biostraticola, and Sodalis.

Quantitative proteomic analysis of Xanthoceras sorbifolium Bunge seedlings in response to drought and heat stress

Yellowhorn (Xanthoceras sorbifolium Bunge) is a woody oil species that is widely distributed in northwestern China. To investigate the molecular mechanisms underlying the drought and heat tolerance response of yellowhorn seedlings, changes in protein abundance were analyzed via comparative proteomics. Drought and heat treatment of seedlings was applied in growth chamber, and the leaves were harvested after 7 days of treatment. The total protein was extracted, and comparative proteomic analysis was performed via isobaric tag for relative and absolute quantitation (iTRAQ). The abundance of most of the proteins associated with oxidative phosphorylation, NADH dehydrogenase and superoxide dismutase (SOD) was reduced. The differential proteins associated with photosynthesis enzymes indicated that stress had different effects on photosystem I (PSI) and photosystem II (PSII). After comprehensively analyzing the results, we speculated that drought and heat stress could hinder the synthesis of riboflavin, reducing NADH dehydrogenase content, which might further have an impact on energy utilization. Yellowhorn seedlings relied on Fe–Mn SOD enzymes rather than Cu/Zn SOD enzymes to remove reactive oxygen species (ROS). In addition, heat-shock proteins (HSPs) had significant increase and played a key role in stress response, which could be divided into two categories according to their transcription and translation efficiency. Over all, the results can provide a basis for understanding the molecular mechanism underlying resistance to drought and heat stress in yellowhorn and for subsequent research of posttranslational modification-related omics of key proteins.

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.

Overexpression of Riboflavin Excretase Enhances Riboflavin Production in the Yeast Candida famata.

Many microorganisms are capable of riboflavin oversynthesis and accumulation in a medium, suggesting that they efficiently excrete riboflavin. The mechanisms of riboflavin efflux in microorganisms remain elusive. Candida famata are representatives of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. The riboflavin overproducers of this yeast species have been obtained by classical mutagenesis and metabolic engineering. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The appropriate protein and gene have not been identified in yeasts till recently. At the same time, the gene BCRP (breast cancer resistance protein) has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in the flavinogenic yeasts Debaryomyces hansenii and C. famata. Here we evaluate the yeast homologs of BCRP with respect to improvement of a riboflavin production by C. famata. The closest homologs from D. hansenii or C. famata were expressed under the control of TEF1 promoter of these yeasts in the wild-type and riboflavin-overproducing strains of C. famata. Resulted transformants overexpressed the corresponding genes (designated as DhRFE and CfRFE) and produced 1.4- to 6-fold more riboflavin as compared to the corresponding parental strains. They also were characterized by overexpression of RIB1 and RIB6 genes which encode the first and the last structural enzymes of riboflavin synthesis and exhibited elevated specific activity of GTP cyclohydrolase II. Thus, overexpression of yeast homolog of mammal gene BCRP may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant overproducing C. famata strain or other flavinogenic microorganisms.

Synthesis of new riboflavin modified ODNs: Effect of riboflavin moiety on the G-quadruplex arrangement and stability

In the panorama of modified G-quadruplexes (G4s) with interesting proprieties, here, it has been reported the synthesis of new modified d(TGGGAG) sequences forming G-quadruplexes, with the insertion of a riboflavin unit (Rf, vitamin B2). Exploiting the flavin similarity with the hydrogen bond pattern of guanine and aiming at mimic a typical nucleoside scaffold, the synthesis of the riboflavin building block 3 it has been efficiently carried out. The effect of insertion of riboflavin mimic nucleoside on the G-quadruplex properties has been here, for the first time investigated. A biophysical characterization of Rf-modified sequences (A-D) has been carried out by circular dichroism (CD), fluorescence spectroscopy, differential scanning calorimetry (DSC) and native gel electrophoresis. CD and electrophoresis data have suggested that Rf-modified sequences are able to form parallel tetramolecular G4 structures similar to that of the unmodified sequence. Analysis of the DSC thermograms has revealed that all modified G-quadruplexes have a higher thermal stability compared with the natural sequence, particularly the stabilisation is higher when the Rf residue is introduced at the 3′-end. Further, DSC analysis has revealed that the Rf residues introduced at the 3′-end are able to form additional stabilising interactions, energetically almost comparable to the enthalpic contribution of a G-tetrad. Fluorescence measurement are consistent with this result showing that the Rf residues introduced at 3′-end are able to form stacking interactions with the adjacent bases within the G-quadruplex structure. The whole of data suggested that the introduction of Rf unit can stabilize G-quadruplex structures and can be a promising candidate for future theranostic applications.

Complete genome sequence of an Arctic Ocean bacterium Shewanella sp. Arc9-LZ with capacity of synthesizing silver nanoparticles in darkness.

Shewanella sp. Arc9-LZ is a bacterium capable of synthesizing silver nanoparticles in darkness. It was isolated from the marine sediment from the Arctic Ocean (158°01'12"W; 84°28'38"N) collected during the 9th Chinese National Arctic Expedition in 2018. Here, we describe the complete genome of Shewanella sp. Arc9-LZ. The complete genome of Shewanella sp. Arc9-LZ is composed of a circular chromosome of 4,911,031bp with G+C content of 41.61mol%. The genome encodes 4040 protein-coding genes (CDSs), 104 tRNAs, and 35 rRNAs. The rRNAs contain 14 copies of 5S rRNA gene, 11 copies of 16S rRNA gene, and 10 copies of 23S rRNA gene. Based on the KEGG, COG, NR, Swiss-Prot, TCDB, and CAZy analysis, a total of 64 genes belonging to 9 kinds are related to the AgNPs synthesis. These genes are involeved in the synthesis of riboflavin, b-type cytochrome, c-type cytochrome, coenzyme Q, NADPH dehydrogenase, cytochrome c reductase, cytochrome c oxidase, nitroreductase, and nitrate reductase.

MR1-Restricted T Cells in Cancer Immunotherapy.

Major histocompatibility complex class I-related (MR1) was first identified as a cell membrane protein involved in the development and expansion of a unique set of T cells expressing an invariant T-cell receptor (TCR) α-chain. These cells were initially discovered in mucosal tissues, such as the intestinal mucosa, so they are called mucosal-associated invariant T (MAIT) cells. MR1 senses the presence of intermediate metabolites of riboflavin and folic acid synthesis that have been chemically modified by the side-products of glycolysis, glyoxal or methylglyoxal. These modified metabolites form complexes with MR1 and translocate from the endoplasmic reticulum to the plasma membrane where MAIT cells’ TCRs recognize them. Recent publications report that atypical MR1-restricted cytotoxic T cells, differing from MAIT cells in TCR usage, antigen, and transcription factor profile, recognize an as yet unknown cancer-specific metabolite presented by MR1 in cancer cells. This metabolite may represent another class of neoantigens, beyond the neo-peptides arising from altered tumor proteins. In an MR1-dependent manner, these MR1-restricted T cells, while sparing noncancerous cells, kill many cancer cell lines and attenuate cell-line-derived and patient-derived xenograft tumors. As MR1 is monomorphic and expressed in a wide range of cancer tissues, these findings raise the possibility of universal pan-cancer immunotherapies that are dependent on cancer metabolites.