Clinical, pathological and genetic features and follow-up of 110 patients with late-onset MADD: a single-center retrospective study.

To observe a long-term prognosis in late-onset multiple acyl-coenzyme-A dehydrogenation deficiency (MADD) patients and to determine whether riboflavin should be administrated in the long-term and high-dosage manner, we studied the clinical, pathological and genetic features of 110 patients with late-onset MADD in a single neuromuscular center. The plasma riboflavin levels and a long-term follow-up study were performed. We showed that fluctuating proximal muscle weakness, exercise intolerance and dramatic responsiveness to riboflavin treatment were essential clinical features for all 110 MADD patients. Among them, we identified 106 cases with ETFDH variants, 1 case with FLAD1 variants and 3 cases without causal variants. On muscle pathology, fibers with cracks, atypical ragged red fibers (aRRFs) and diffuse decrease of SDH activity were the distinctive features of these MADD patients. The plasma riboflavin levels before treatment were significantly decreased in these patients as compared to healthy controls. Among 48 MADD patients with a follow-up of 6.1 years on average, 31 patients were free of muscle weakness recurrence, while 17 patients had episodes of slight muscle weakness upon riboflavin withdrawal, but recovered after retaking a small-dose of riboflavin for a short-term. Multivariate Cox regression analysis showed vegetarian diet and masseter weakness were independent risk factors for muscle weakness recurrence. In conclusion, fibers with cracks, aRRFs and diffuse decreased SDH activity could distinguish MADD from other genotypes of lipid storage myopathy. For late-onset MADD, increased fatty acid oxidation and reduced riboflavin levels can induce episodes of muscle symptoms, which can be treated by short-term and small-dose of riboflavin therapy.

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.

BACKGROUND: Riboflavin 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. RESULTS: Through 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. CONCLUSIONS: Our 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.

A comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene.

Lipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders predominantly affecting skeletal muscle by triglyceride accumulation in muscle fibers. Riboflavin therapy has been shown to ameliorate symptoms in some LSM patients who are essentially concerned with multiple acyl-CoA dehydrogenation deficiency (MADD). It is proved that riboflavin responsive LSM caused by MADD is mainly due to ETFDH gene variant (ETFDH-RRMADD). We described here a case with riboflavin responsive LSM and MADD resulting from FLAD1 gene variants (c.1588 C > T p.Arg530Cys and c.1589 G > C p.Arg530Pro, FLAD1-RRMADD). And we compared our patient together with 9 FLAD1-RRMADD cases from literature to 106 ETFDH-RRMADD cases in our neuromuscular center on clinical history, laboratory investigations and pathological features. Furthermore, the transcriptomics study on FLAD1-RRMADD and ETFDH-RRMADD were carried out. On muscle pathology, both FLAD1-RRMADD and ETFDH-RRMADD were proved with lipid storage myopathy in which atypical ragged red fibers were more frequent in ETFDH-RRMADD, while fibers with faint COX staining were more common in FLAD1-RRMADD. Molecular study revealed that the expression of GDF15 gene in muscle and GDF15 protein in both serum and muscle was significantly increased in FLAD1-RRMADD and ETFDH-RRMADD groups. Our data revealed that FLAD1-RRMADD (p.Arg530) has similar clinical, biochemical, and fatty acid metabolism changes to ETFDH-RRMADD except for muscle pathological features.

Metabolic and physiological adaptations of microalgal growth-promoting bacterium Azospirillum brasilense growing under biogas atmosphere: a microarray-based transcriptome analysis.

Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24 h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72 h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24 h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.

Identification of the riboflavin cofactor-binding site in the Vibrio cholerae ion-pumping NQR complex: A novel structural motif in redox enzymes.

The ion-pumping NQR complex is an essential respiratory enzyme in the physiology of many pathogenic bacteria. This enzyme transfers electrons from NADH to ubiquinone through several cofactors, including riboflavin (vitamin B2). NQR is the only enzyme reported that is able to use riboflavin as a cofactor. Moreover, the riboflavin molecule is found as a stable neutral semiquinone radical. The otherwise highly reactive unpaired electron is stabilized via an unknown mechanism. Crystallographic data suggested that riboflavin might be found in a superficially located site in the interface of NQR subunits B and E. However, this location is highly problematic, as the site does not have the expected physiochemical properties. In this work, we have located the riboflavin-binding site in an amphipathic pocket in subunit B, previously proposed to be the entry site of sodium. Here, we show that this site contains absolutely conserved residues, including N200, N203, and D346. Mutations of these residues decrease enzymatic activity and specifically block the ability of NQR to bind riboflavin. Docking analysis and molecular dynamics simulations indicate that these residues participate directly in riboflavin binding, establishing hydrogen bonds that stabilize the cofactor in the site. We conclude that riboflavin is likely bound in the proposed pocket, which is consistent with enzymatic characterizations, thermodynamic studies, and distance between cofactors.

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

Iron (Fe) is an essential micronutrient, and deficiency in available Fe is one of the most important limiting factors for plant growth. In some species including Medicago truncatula, Fe deficiency results in accumulation of riboflavin, a response associated with Fe acquisition. However, how the plant's Fe status is integrated to tune riboflavin biosynthesis and how riboflavin levels affect Fe 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 Fe accumulation and improve photosynthetic efficiency, whereas overexpression 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 Fe acquisition by suppressing accumulation of riboflavin and its derivatives in roots, which in turn influences ferric reduction activity by riboflavin-dependent electron transport under Fe deficiency. Our findings uncover a new regulatory mechanism by which CIPK12 regulates riboflavin biosynthesis and Fe-deficiency responses in plants.

Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.

The Musa troglodytarum L. genome provides insights into the mechanism of non-climacteric behaviour and enrichment of carotenoids.

BACKGROUND: Karat (Musa troglodytarum L.) is an autotriploid Fe'i banana of the Australimusa section. Karat was domesticated independently in the Pacific region, and karat fruit are characterized by a pink sap, a deep yellow-orange flesh colour, and an abundance of ß-carotene. Karat fruit showed non-climacteric behaviour, with an approximately 215-day bunch filling time. These features make karat a valuable genetic resource for studying the mechanisms underlying fruit development and ripening and carotenoid biosynthesis. RESULTS: Here, we report the genome of M. troglodytarum, which has a total length of 603 Mb and contains 37,577 predicted protein-coding genes. After divergence from the most recent common ancestors, M. troglodytarum (T genome) has experienced fusion of ancestral chromosomes 8 and 9 and multiple translocations and inversions, unlike the high synteny with few rearrangements found among M. schizocarpa (S genome), M. acuminata (A genome) and M. balbisiana (B genome). Genome microsynteny analysis showed that the triplication of MtSSUIIs due to chromosome rearrangement may lead to the accumulation of carotenoids and ABA in the fruit. The expression of duplicated MtCCD4s is repressed during ripening, leading to the accumulation of α-carotene, ß-carotene and phytoene. Due to a long terminal repeat (LTR)-like fragment insertion upstream of MtERF11, karat cannot produce large amounts of ethylene but can produce ABA during ripening. These lead to non-climacteric behaviour and prolonged shelf-life, which contributes to an enrichment of carotenoids and riboflavin. CONCLUSIONS: The high-quality genome of M. troglodytarum revealed the genomic basis of non-climacteric behaviour and enrichment of carotenoids, riboflavin, flavonoids and free galactose and provides valuable resources for further research on banana domestication and breeding and the improvement of nutritional and bioactive qualities.

Molecular Mechanism Biomarkers Predict Diagnosis in Schizophrenia and Schizoaffective Psychosis, with Implications for Treatment.

Diagnostic uncertainty and relapse rates in schizophrenia and schizoaffective disorder are relatively high, indicating the potential involvement of other pathological mechanisms that could serve as diagnostic indicators to be targeted for adjunctive treatment. This study aimed to seek objective evidence of methylenetetrahydrofolate reductase MTHFR C677T genotype-related bio markers in blood and urine. Vitamin and mineral cofactors related to methylation and indolamine-catecholamine metabolism were investigated. Biomarker status for 67 symptomatically well-defined cases and 67 asymptomatic control participants was determined using receiver operating characteristics, Spearman's correlation, and logistic regression. The 5.2%-prevalent MTHFR 677 TT genotype demonstrated a 100% sensitive and specific case-predictive biomarkers of increased riboflavin (vitamin B2) excretion. This was accompanied by low plasma zinc and indicators of a shift from low methylation to high methylation state. The 48.5% prevalent MTHFR 677 CC genotype model demonstrated a low-methylation phenotype with 93% sensitivity and 92% specificity and a negative predictive value of 100%. This model related to lower vitamin cofactors, high histamine, and HPLC urine indicators of lower vitamin B2 and restricted indole-catecholamine metabolism. The 46.3%-prevalent CT genotype achieved high predictive strength for a mixed methylation phenotype. Determination of MTHFR C677T genotype dependent functional biomarker phenotypes can advance diagnostic certainty and inform therapeutic intervention.

WHITE AND LESION-MIMIC LEAF1, encoding a lumazine synthase, affects reactive oxygen species balance and chloroplast development in rice.

The riboflavin derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential cofactors for enzymes in multiple cellular processes. Characterizing mutants with impaired riboflavin metabolism can help clarify the role of riboflavin in plant development. Here, we characterized a rice (Oryza sativa) white and lesion-mimic (wll1) mutant, which displays a lesion-mimic phenotype with white leaves, chlorophyll loss, chloroplast defects, excess reactive oxygen species (ROS) accumulation, decreased photosystem protein levels, changes in expression of chloroplast development and photosynthesis genes, and cell death. Map-based cloning and complementation test revealed that WLL1 encodes lumazine synthase, which participates in riboflavin biosynthesis. Indeed, the wll1 mutant showed riboflavin deficiency, and application of FAD rescued the wll1 phenotype. In addition, transcriptome analysis showed that cytokinin metabolism was significantly affected in wll1 mutant, which had increased cytokinin and δ-aminolevulinic acid contents. Furthermore, WLL1 and riboflavin synthase (RS) formed a complex, and the rs mutant had a similar phenotype to the wll1 mutant. Taken together, our findings revealed that WLL1 and RS play pivotal roles in riboflavin biosynthesis, which is necessary for ROS balance and chloroplast development in rice.

Riboflavin integrates cellular energetics and cell cycle to regulate maize seed development.

Riboflavin is the precursor of essential cofactors for diverse metabolic processes. Unlike animals, plants can de novo produce riboflavin through an ancestrally conserved pathway, like bacteria and fungi. However, the mechanism by which riboflavin regulates seed development is poorly understood. Here, we report a novel maize (Zea mays L.) opaque mutant o18, which displays an increase in lysine accumulation, but impaired endosperm filling and embryo development. O18 encodes a rate-limiting bifunctional enzyme ZmRIBA1, targeted to plastid where to initiate riboflavin biosynthesis. Loss of function of O18 specifically disrupts respiratory complexes I and II, but also decreases SDH1 flavinylation, and in turn shifts the mitochondrial tricarboxylic acid (TCA) cycle to glycolysis. The deprivation of cellular energy leads to cell-cycle arrest at G1 and S phases in both mitosis and endoreduplication during endosperm development. The unexpected up-regulation of cell-cycle genes in o18 correlates with the increase of H3K4me3 levels, revealing a possible H3K4me-mediated epigenetic back-up mechanism for cell-cycle progression under unfavourable circumstances. Overexpression of O18 increases riboflavin production and confers osmotic tolerance. Altogether, our results substantiate a key role of riboflavin in coordinating cellular energy and cell cycle to modulate maize endosperm development.

Cofactor F420, an emerging redox power in biosynthesis of secondary metabolites.

Cofactor F420 is a low-potential hydride-transfer deazaflavin that mediates important oxidoreductive reactions in the primary metabolism of archaea and a wide range of bacteria. Over the past decade, biochemical studies have demonstrated another essential role for F420 in the biosynthesis of various classes of natural products. These studies have substantiated reports predating the structural determination of F420 that suggested a potential role for F420 in the biosynthesis of several antibiotics produced by Streptomyces. In this article, we focus on this exciting and emerging role of F420 in catalyzing the oxidoreductive transformation of various imine, ketone and enoate moieties in secondary metabolites. Given the extensive and increasing availability of genomic and metagenomic data, these F420-dependent transformations may lead to the discovery of novel secondary metabolites, providing an invaluable and untapped resource in various biotechnological applications.

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.

Coupling riboflavin de novo biosynthesis and cytochrome expression for improving extracellular electron transfer efficiency in Shewanella oneidensis.

Shewanella oneidensis MR-1, as a model exoelectrogen with divergent extracellular electron transfer (EET) pathways, has been widely used in microbial fuel cells (MFCs). The electron transfer rate is largely determined by riboflavin (RF) and c-type cytochromes (c-Cyts). However, relatively low RF production and inappropriate amount of c-Cyts substantially impede the capacity of improving the EET rate. In this study, coupling of riboflavin de novo biosynthesis and c-Cyts expression was implemented to enhance the efficiency of EET in S. oneidensis. First, the upstream pathway of RF de novo biosynthesis was divided into four modules, and the expression level of 22 genes in above four modules was fine-tuned by employing promoters with different strengths. Among them, genes zwf*, glyA, and ybjU which exhibited optimal RF production were combinatorially overexpressed, leading to the enhancement of maximum output power density by 166%. Second, the diverse c-Cyts genes were overexpressed to match high RF production, and omcA was selected for further combination. Third, RF de novo biosynthesis and c-Cyts expression were combined, resulting in 2.34-fold higher power output than the parent strain. This modular and combinatorial manipulation strategy provides a generalized reference to advance versatile practical applications of electroactive microorganisms.

Impact of the MTHFR C677T polymorphism on blood pressure and related central haemodynamic parameters in healthy adults.

BACKGROUND: The C677T polymorphism in the gene-encoding methylenetetrahydrofolate reductase (MTHFR) is associated with an increased risk of hypertension and cardiovascular disease. Riboflavin, the MTHFR cofactor, is an important modulator of blood pressure (BP) in adults homozygous for this polymorphism (TT genotype). The effect of this genetic variant on BP and related central haemodynamic parameters in healthy adults has not been previously investigated and was examined in this study. METHODS: Brachial BP, central BP and pulse wave velocity (PWV, SphygmoCor XCEL) were measured in adults aged 18-65 years prescreened for MTHFR genotype. Riboflavin status was assessed using the erythrocyte glutathione reductase activation coefficient assay. RESULTS: Two hundred and forty-two adults with the MTHFR 677TT genotype and age-matched non-TT (CC/CT) genotype controls were identified from a total cohort of 2546 adults prescreened for MTHFR genotype. The TT genotype was found to be an independent determinant of hypertension (p = 0.010), along with low-riboflavin status (p = 0.002). Brachial systolic and diastolic BP were higher in TT versus non-TT adults by 5.5 ± 1.2 and 2.4 ± 0.9 mmHg, respectively (both p < 0.001). A stronger phenotype was observed in women, with an almost 10 mmHg difference in mean systolic BP in TT versus non-TT genotype groups: 134.9 (95% confidence interval [CI] 132.1-137.6) versus 125.2 (95% CI 122.3-128.0) mmHg; p < 0.001. In addition, PWV was faster in women with the TT genotype (p = 0.043). CONCLUSION: This study provides the first evidence that brachial and central BP are significantly higher in adults with the variant MTHFR 677TT genotype and that the BP phenotype is more pronounced in women.

A case report of sudden-onset auditory neuropathy spectrum disorder associated with Brown-Vialetto-Van Laere syndrome (riboflavin transporter deficiency).

OBJECTIVE: The purpose of this paper is to describe a child with auditory neuropathy spectrum disorder (ANSD) associated with Brown-Vialetto-Van Laere (BVVL) syndrome, which is a rare, inherited, neurodegenerative disorder that is caused by defects in riboflavin transporter genes. DESIGN: We report the audiological and clinical profile of a child who presented with a complaint of sudden loss of speech understanding associated with an atypical form of ANSD. He was later diagnosed with BVVL. STUDY SAMPLE: An 11-year-old boy with ANSD associated with BVVL. RESULTS: The patient's severe neurological symptoms improved within a year of supplementation with high doses of riboflavin. His fluctuating hearing loss and 0% WDS remained unchanged. The patient was able to use hearing aids without any discomfort after treatment initiation, but he stopped using them again due to a lack of benefit in speech understanding. Although cochlear implantation was recommended, the patient and his family decided not to consider it for another year since they still had hope for complete recovery. CONCLUSIONS: Sudden-onset ANSD can be the earliest sign of undetected BVVL syndrome. Early detection of BVVL is crucial since all symptoms can be reversible with an early intervention of high doses of riboflavin supplementation.

The novel phosphatase RosC catalyzes the last unknown step of roseoflavin biosynthesis in Streptomyces davaonensis.

The bacterium Streptomyces davaonensis produces the antibiotic roseoflavin, which is a riboflavin (vitamin B2 ) analog. The key enzyme of roseoflavin biosynthesis is the 8-demethyl-8-amino-riboflavin-5'-phosphate (AFP) synthase RosB which synthesizes AFP from riboflavin-5'-phosphate. AFP is not a substrate for the last enzyme of roseoflavin biosynthesis the N, N-dimethyltransferase RosA, which generates roseoflavin from 8-demethyl-8-amino-riboflavin (AF). Consequently, the roseoflavin biosynthetic pathway depends on a phosphatase, which dephosphorylates AFP to AF. Here, we report on the identification and characterization of such an AFP phosphatase which we named RosC. The gene rosC is located immediately downstream of rosA and both genes are part of a cluster comprising 10 genes. Deletion of rosC from the chromosome of S. davaonensis led to reduced roseoflavin levels in the corresponding recombinant strain. In contrast to wild-type S. davaonensis, cell-free extracts of the rosC deletion strain did not catalyze dephosphorylation of AFP. RosC was purified from an overproducing Escherichia coli strain. RosC is the fastest enzyme of roseoflavin biosynthesis (kcat 31.3 ± 1.4 min-1 ). The apparent KM for the substrate AFP was 34.5 µM. Roseoflavin biosynthesis is now completely understood--it takes three enzymes (RosB, RosC, and RosA) to convert the flavin cofactor riboflavin-5'-phosphate into a potent antibiotic.

A copper-specific microbial fuel cell biosensor based on riboflavin biosynthesis of engineered Escherichia coli.

Copper pollution poses a serious threat to the aquatic environment; however, in situ analytical methods for copper monitoring are still scarce. In the current study, Escherichia coli Rosetta was genetically modified to express OprF and ribB with promoter Pt7 and PcusC , respectively, which could synthesize porin and senses Cu2+ to produce riboflavin. The cell membrane permeability of this engineered strain was increased and its riboflavin production (1.45-3.56 µM) was positively correlated to Cu2+ (0-0.5 mM). The biosynthetic strain was then employed in microbial fuel cell (MFC) based biosensor. Under optimal operating parameters of pH 7.1 and 37°C, the maximum voltage (248, 295, 333, 352, and 407 mV) of the constructed MFC biosensor showed a linear correlation with Cu2+ concentration (0.1, 0.2, 0.3, 0.4, 0.5 mM, respectively; R2 = 0.977). The continuous mode testing demonstrated that the MFC biosensor specifically senses Cu2+ with calculated detection limit of 28 µM, which conforms to the common Cu2+ safety standard (32 µM). The results obtained with the developed biosensor system were consistent with the existing analytical methods such as colorimetry, flame atomic absorption spectrometry, and inductively coupled plasma optical emission spectrometry. In conclusion, this MFC-based biosensor overcomes the signal conversion and transmission problems of conventional approaches, providing a fast and economic analytical alternative for in situ monitoring of Cu2+ in water.

Enhancement of riboflavin production in Bacillus subtilis via in vitro and in vivo metabolic engineering of pentose phosphate pathway.

OBJECTIVES: The production of riboflavin with Bacillus subtilis, is an established process, however it is yet to be fully optimized. The aim of this study was to explore how riboflavin yields can be improved via in vitro and in vivo metabolic engineering modification of the pentose phosphate pathway (PPP). RESULTS: In vitro, glucose was replaced with sodium gluconate to enhance PPP. Flask tests showed that the riboflavin titer increased from 0.64 to 0.87 g/L. The results revealed that the direct use of sodium gluconate could benefit riboflavin production. In vivo, gntP (encoding gluconate permease) was overexpressed to improve sodium gluconate uptake. The riboflavin titer reached 1.00 g/L with the mutant B. subtilis RF01. Ultimately, the fermentation verification of the engineered strain was carried out in a 7-L fermenter, with the increased riboflavin titer validating this approach. CONCLUSIONS: The combination of metabolic engineering modifications in vitro and in vivo was confirmed to promote riboflavin production efficiently by increasing PPP and has great potential for industrial application. This work is aimed to explore how to improve the riboflavin yield by the rational renovation of the pentose phosphate pathway (PPP). In vitro, metabolic engineering mainly uses sodium gluconate as a carbon source instead of glucose, and in vivo, metabolic engineering mainly includes the overexpression of sodium gluconate utility-related genes. The effect of sodium gluconate on cell growth, riboflavin production was investigated in the flasks and fermenter scale.

Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis.

Leuconostoccitreum, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine-Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of ribF and folE genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the rib operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.