Metabolic engineering of roseoflavin-overproducing microorganisms.

BACKGROUND: Roseoflavin, a promising broad-spectrum antibiotic, is naturally produced by the bacteria Streptomyces davaonensis and Streptomyces cinnabarinus. The key enzymes responsible for roseoflavin biosynthesis and the corresponding genes were recently identified. In this study we aimed to enhance roseoflavin production in S. davaonensis and to synthesize roseoflavin in the heterologous hosts Bacillus subtilis and Corynebacterium glutamicum by (over)expression of the roseoflavin biosynthesis genes. RESULTS: While expression of the roseoflavin biosynthesis genes from S. davaonensis was not observed in recombinant strains of B. subtilis, overexpression was successful in C. glutamicum and S. davaonensis. Under the culture conditions tested, a maximum of 1.6 ± 0.2 µM (ca. 0.7 mg/l) and 34.9 ± 5.2 µM (ca. 14 mg/l) roseoflavin was produced with recombinant strains of C. glutamicum and S. davaonensis, respectively. In S. davaonensis the roseoflavin yield was increased by 78%. CONCLUSIONS: The results of this study provide a sound basis for the development of an economical roseoflavin production process.

Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.

Light wavelengths that induce oxidation of oxymyoglobin in meat.

Light wavelengths that induce meat discoloration and the photoreceptors in the meat were studied. We investigated the effects of the light wavelength on the oxidation rate of myoglobin (Mb) by exposing Mb extracts or model solutions containing Mb to light at specific wavelengths with a bandwidth of 5 nm using a fluorescence spectrophotometer. The wavelengths examined comprised 385, 415, 445, 460, 490, 525, 555, 580, 605, 630,660, and 750 nm. In the Mb extracts, Mb oxidation was induced through exposure to the light at 445 and 580-605 nm; Mb was insensitive to light at 445 nm. Mitochondria, containing cytochrome a and cytochrome a3 with absorption peaks at 448 and 600 nm, and riboflavin with fluorescence at 450 nm were studied as 445 nm receptors. Mitochondria significantly oxidized Mb via cytochrome c oxidation through complex IV activity; however, no 445 nm-specific photo sensitivity effects were observed. In contrast, riboflavin increased the Mb oxidation rate induced via exposure to the light at 450 nm in a concentration-dependent manner (minimum concentration: 38.4 µg L-1). While native mitochondria did not show 445 nm-specific photosensitivity effects on Mb, supernatants of heated mitochondria conferred 445 nm-wavelength sensitivity to Mb. Riboflavin concentration in this supernatant was 182 ± 60 µg L-1. The Mb photosensitivity spectrum with 473 µg L-1 riboflavin had two peaks at 445 nm and 580 nm, which were similar to those of Mb extract. These results suggest that mitochondrial damage affects the meat discoloration through the release of cytochrome c and riboflavin.

Sensitive riboflavin sensing using silver nanoparticles deposited onto screen-printed electrodes via controlled-energy spark discharges.

In this work, we elaborated the graphite screen-printed electrodes (SPEs) modification with metal nanoparticles formed as a result of spark discharges produced between a metal wire electrode and SPE that are connected to an Arduino board-based DC high voltage power supply. This sparking device allows, on the one hand, the toposelective formation of NPs of controlled dimensions through a direct and liquid-free approach, and on the other hand, controls the number and energy of the discharges delivered to the electrode surface during a single spark event. This way, the potential damage to the SPE surface by the action of heat evolved during the sparking process is considerably minimized compared with the standard setup in which each spark event consists of multiple electrical discharges. Data demonstrated that the sensing properties of the resulting electrodes are significantly improved compared with those achieved when conventional spark generators are employed, as demonstrated for silver-sparked SPEs that exhibit enhanced sensitivity to riboflavin. Sparked AgNp-SPEs were characterized using scanning electron microscopy and voltammetric measurements in alkaline conditions. The analytical performance of sparked AgNP-SPEs was evaluated by various electrochemical techniques. Under optimum conditions, the detection range for DPV was from 1.9 (LOQ) to 100 nM riboflavin (R2 = 0.997), while a limit of detection (LOD, S/N 3) of 0.56 nM was achieved. The analytical utility is demonstrated for the determination of riboflavin in the real matrices of B-complex pharmaceutical preparation and an energy drink.

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.

Riboflavin Supplementation in Patients with Crohn's Disease [the RISE-UP study].

BACKGROUND AND AIMS: Crohn's disease [CD] is characterised by chronic intestinal inflammation and dysbiosis in the gut. Riboflavin [vitamin B2] has anti-inflammatory, antioxidant and microbiome-modulatory properties. Here, we analysed the effect of riboflavin on oxidative stress, markers of inflammation, clinical symptoms, and faecal microbiome in patients with CD. METHODS: In this prospective clinical intervention study, patients received 100 mg riboflavin [DSM, Nutritional Products Ltd] daily for 3 weeks. Clinical disease activity [Harvey-Bradshaw Index: HBI], serum biomarkers of inflammation and redox status [plasma free thiols], and faecal microbiome taxonomical composition and functionality [fluorescent in situ hybridisation: FISH; and metagenomic shotgun sequencing: MGS], were analysed before and after riboflavin intervention. RESULTS: In total, 70 patients with CD with varying disease activity were included. Riboflavin supplementation significantly decreased serum levels of inflammatory markers. In patients with low faecal calprotectin [FC] levels, IL-2 decreased, and in patients with high FC levels, C-reactive protein [CRP] was reduced and free thiols significantly increased after supplementation. Moreover, HBI was significantly decreased by riboflavin supplementation. Riboflavin supplementation led to decreased Enterobacteriaceae in patients with low FC levels as determined by FISH; however, MGS analysis showed no effects on diversity, taxonomy, or metabolic pathways of the faecal microbiome. CONCLUSIONS: Three weeks of riboflavin supplementation resulted in a reduction in systemic oxidative stress, mixed anti-inflammatory effects, and a reduction in clinical symptoms [HBI]. FISH analysis showed decreased Enterobacteriaceae in patients with CD with low FC levels, though this was not observed in MGS analysis. Our data demonstrate that riboflavin supplementation has a number of anti-inflammatory and anti-oxidant effects in CD.

Adequate vitamin B12 and riboflavin status from menus alone in residential care facilities in the Lower Mainland, British Columbia.

Older adults have potential increased risk of nutrient deficiencies because of age-related decreased dietary intake and malabsorption; it is important to ensure nutrient needs are met to avoid adverse health outcomes. B vitamins are of particular interest: vitamin B12 deficiency can cause irreversible neurodegeneration; there is mandatory folic acid fortification in Canada; and suboptimal riboflavin status has been reported among older adults in the United Kingdom. In this exploratory secondary analysis study we assessed vitamin B12 and riboflavin biochemical status (via microparticle enzyme immunoassay and erythrocyte glutathione reductase activity coefficient (EGRac), respectively), and the vitamin B12, riboflavin, and folate content of menus served to a convenience sample of older adults (≥65 years) from 5 residential care facilities within the Lower Mainland of British Columbia, Canada. Diet was assessed from customized 28-day cycle meal plans. Participants (n = 207; 53 men and 154 women) were aged 86 ± 7 years, largely of European descent (92%), and nonsmokers (95%). The menus served had a low prevalence of inadequacy for vitamin B12 and riboflavin (only 4% and 1% of menus contained less than the estimated average requirement (EAR), respectively), but 93% contained less than the EAR for folate. Mean ± SD serum total vitamin B12 concentration was 422 ± 209 pmol/L, and EGRac was 1.30 ± 0.19. The majority of older adults in residential care were provided with adequate vitamin B12 and riboflavin menu amounts, and only 5% were vitamin B12 deficient (<148 pmol/L). However, 26% were riboflavin deficient (EGRac ≥ 1.4), which may warrant further investigation.

Hierarchically-Designed 3D Flower-Like Composite Nanostructures as an Ultrastable, Reproducible, and Sensitive SERS Substrate.

Surface-enhanced Raman spectroscopy (SERS) is an attractive tool in the analytical sciences due to its high specificity and sensitivity. Because SERS-active substrates are only available as two-dimensional arrays, the fabrication of three-dimensional (3D) nanostructures allows for an increased number of hot spots in the focus volume, thus further amplifying the SERS signal. Although a great number of fabrication strategies for powerful SERS substrates exist, the generation of 3D nanostructures with high complexity and periodicity is still challenging. For this purpose, we report an easy fabrication technique for 3D nanostructures following a bottom-up preparation protocol. Enzymatically generated silver nanoparticles (EGNPs) are prepared, and the growth of hierarchically-designed 3D flower-like silica-silver composite nanostructures is induced by applying plasma-enhanced atomic layer deposition (PE-ALD) on the EGNPs. The morphology of these nanocomposites can be varied by changes in the PE-ALD cycle number, and a flower height of up to 10 µm is found. Moreover, the metallized (e.g., silver or gold) 3D nanostructures resulting from 135 PE-ALD cycles of silica creation provide highly reproducible SERS signals across the hydrophobic surface. Within this contribution, the morphological studies, optical properties, as well as the SERS response of these metallized silica-silver composite nanostructures applying vitamin B2 as a model analyte are introduced.

Thermodynamic and Probabilistic Metabolic Control Analysis of Riboflavin (Vitamin B2) Biosynthesis in Bacteria.

In this study, we applied a coupled in silico thermodynamic and probabilistic metabolic control analysis methodology to investigate the control mechanisms of the commercially relevant riboflavin biosynthetic pathway in bacteria. Under the investigated steady-state conditions, we found that several enzyme reactions of the pathway operate far from thermodynamic equilibrium (transformed Gibbs energies of reaction below about -17 kJ mol(-1)). Using the obtained thermodynamic information and applying enzyme elasticity sampling, we calculated the distributions of the scaled concentration control coefficients (CCCs) and scaled flux control coefficients (FCCs). From the statistical analysis of the calculated distributions, we inferred that the control over the riboflavin producing flux is shared among several enzyme activities and mostly resides in the initial reactions of the pathway. More precisely, the guanosine triphosphate (GTP) cyclohydrolase II activity, and therefore the bifunctional RibA protein of Bacillus subtilis because it catalyzes this activity, appears to mainly control the riboflavin producing flux (mean FCCs = 0.45 and 0.55, respectively). The GTP cyclohydrolase II activity and RibA also exert a high positive control over the riboflavin concentration (mean CCCs = 2.43 and 2.91, respectively). This prediction is consistent with previous findings for microbial riboflavin overproducing strains.