Riboflavin Production Research Articles

Factors stimulating riboflavin produced by Lactobacillus plantarum CRL 725 grown in a semi-defined medium.

Riboflavin (vitamin B2 ) is one of the B-group water-soluble vitamins and is essential for energy metabolism of the cell. The aim of this study was to determine factors that affect riboflavin production by Lactobacillus (L.) plantarum CRL 725 grown in a semi defined medium and evaluate the expression of its rib genes. The factors found to enhance riboflavin production in this medium were incubation at 30 °C, and the addition of specific medium constituents, such as casamino acids (10 g L-1 ), guanosine (0.04 g L-1 ), and sucrose as carbon source (20 g L-1 ). In these conditions, higher riboflavin concentrations were directly associated with significant increases in the expression of ribA, ribB, and ribC genes. The culture conditions defined in this work and its application to a roseoflavin resistant mutant of L. plantarum allowed for a sixfold increase in riboflavin concentrations in our semi-defined medium which were also significantly higher than those obtained previously using the same strain to ferment soymilk. These conditions should thus be evaluated to increase vitamin production in fermented foods.

Some improved estimation strategies in high-dimensional semiparametric regression models with application to riboflavin production data

Due to advances in technologies, modern statistical studies often encounter linear models with high-dimension, where the number of explanatory variables is larger than the sample size. Estimation in these high-dimensional problems with deterministic covariates or designs is very different from those in the case of random covariates, due to the identifiability of the high-dimensional semiparametric regression parameters. In this paper, we consider ridge estimators and propose preliminary test, shrinkage and its positive rule ridge estimators in the restricted semiparametric regression model when the errors are dependent under a multicollinear setting, in high-dimension. The asymptotic risk expressions in addition to biases are exactly derived for the estimators under study. For our proposal, a real data analysis about production of vitamin B2 and a Monte–Carlo simulation study are considered to illustrate the efficiency of the proposed estimators. In this regard, kernel smoothing and cross-validation methods for estimating the optimum ridge parameter and nonparametric function are used.

In Situ Molecular Imaging of the Biofilm and Its Matrix.

Molecular mapping of live biofilms at submicrometer resolution presents a grand challenge. Here, we present the first chemical mapping results of biofilm extracellular polymeric substance (EPS) in biofilms using correlative imaging between super resolution fluorescence microscopy and liquid time-of-flight secondary ion mass spectrometry (TOF-SIMS). Shewanella oneidensis is used as a model organism. Heavy metal chromate (Cr2O72-) anions consisting of chromium Cr(VI) was used as a model environmental stressor to treat the biofilms. Of particular interest, biologically relevant water clusters have been first observed in the biofilms. Characteristic fragments of biofilm matrix components such as proteins, polysaccharides, and lipids can be spatially imaged. Furthermore, characteristic fatty acids (e.g., palmitic acid), quinolone signal, and riboflavin fragments were found to respond after the biofilm is treated with Cr(VI), leading to biofilm dispersal. Significant changes in water clusters and quorum sensing signals indicative of intercellular communication in the aqueous environment were observed, suggesting that they might result in fatty acid synthesis and inhibition of riboflavin production. The Cr(VI) reduction seems to follow the Mtr pathway leading to Cr(III) formation. Our approach potentially opens a new avenue for mechanistic insight of microbial community processes and communications using in situ imaging mass spectrometry and super resolution optical microscopy.

Draft Genome Sequence of Lactobacillus collinoides CUPV237, an Exopolysaccharide and Riboflavin Producer Isolated from Cider.

Lactobacillus collinoides CUPV237 is a strain isolated from a Basque cider. Lactobacillus collinoides is one of the most frequent species found in cider from Spain, France, or England. A notable feature of the L. collinoides CUPV237 strain is its ability to produce exopolysaccharides.

Isolation and screening lactic acid bacteria for riboflavin production and their use for bioenrichment of curd

As many as 47 lactic acid bacteria were isolated from various vegetables and fruits and raita collected from local households and characterized. All of them were Gram positive and catalase negative. The isolates were screened for riboflavin production. The riboflavin production varied from 0.86 to 10.90 mg L-1. The isolate Ra1 produced the highest riboflavin (10.90 ppm). Incidentally, it also produced 5.6 per cent lactic acid and 21.4 ppm exopolysaccharide (EPS). Similarly, N2 and F2 isolates produced 10.90 and 10.20 ppm riboflavin and 21.17 and 21.24 ppm EPS, respectively. These three selected isolates were used for preparing a functional curd andevaluated. The curd produced by inoculating N2 and Ra1 were of very good quality with excellent flavor, taste and texture and smooth cutting quality. Ra1 produced a functional curd with the highest riboflavin content (13.97 ppm). N2 and RA1 resulted in very high acceptability index of 95.37 and 94.44 per cent, respectively. The betterorganoleptic parameters of the functional curd may also be due to high lactic acid and exopolysaccharide production by these isolates. Thus, by inoculating riboflavin synthesizing LAB isolates to curd, riboflavin-enriched functional curd with enhanced consumer appeal, can be produced.

Increased riboflavin production by knockout of 6-phosphofructokinase I and blocking the Entner-Doudoroff pathway in Escherichia coli.

To construct an Escherichia coli strain capable of producing riboflavin with high titer and yield. A low copy number plasmid pLS01 containing a riboflavin operon under the control of a constitutive promoter was constructed and introduced into Escherichia coli MG1655. Subsequently, the pfkA, edd and ead genes were disrupted, and the resulting strain LS02T produced 667mg riboflavin/l in MSY medium supplied with 10g glucose/l in flask cultivation. In a fed-batch process, riboflavin production of the strain reached 10.4g/l with a yield of 56.8mg riboflavin/g glucose. To our knowledge, this is the first report of engineered E. coli strains that can produce more than 10g riboflavin/l in fed-batch cultivation, indicating that E. coli has potential for riboflavin production.

Trace heavy metal ions promoted extracellular electron transfer and power generation by Shewanella in microbial fuel cells

Although microbial fuel cells (MFCs) is considered as one of the most promising technology for renewable energy harvesting, low power output still accounts one of the bottlenecks and limits its further development. In this work, it is found that Cu2+ (0.1μgL−1–0.1mgL−1) or Cd2+ (0.1μgL−1–1mgL−1) significantly improve the electricity generation in MFCs. The maximum power output achieved with trace level of Cu2+ (∼6nM) or Cd2+ (∼5nM) is 1.3 times and 1.6 times higher than that of the control, respectively. Further analysis verifies that addition of Cu2+ or Cd2+ effectively improves riboflavin production and bacteria attachment on the electrode, which enhances bacterial extracellular electron transfer (EET) in MFCs. These results unveil the mechanism for power output enhancement by Cu2+ or Cd2+ addition, and suggest that metal ion addition should be a promising strategy to enhance EET as well as power generation of MFCs.

Effect of indigenous lactic acid bacteria isolated from goat milk and cheeses on folate and riboflavin content of fermented goat milk

The aim of the present study was to isolate riboflavin- and folate-producing lactic acid bacteria (LAB) from raw goat milk and cheeses, identify them and evaluate their capability to increase the content of these vitamins in fermented goat milk, envisaging potential application for development of novel bioenriched goat milk products. From 179 LAB isolates obtained, 151 (84%) were capable to produce at least one of these vitamins. The average production of total folate and riboflavin in vitamin-free media was 138 ng/ml and 364 ng/ml, respectively. Based on RAPD-PCR and 16S rDNA sequencing, 19 different genetic profiles were obtained and 7 species were identified, with predominance of Streptococcus thermophilus (7), Weissella paramensenteroides (6), and Lactococcus lactis (4). Seven isolates that produced folate and riboflavin above the average were tested for vitamins production in UHT goat milk. Five isolates were capable to increase four to six fold the original amount of folate in the milk in 24 h. Folate content in milk fermented with Lc. lactis FP368 for 24 h was 313 ng/ml that could provide 19% of the recommended daily intake of this vitamin. In addition, St. thermophilus FP268 increased the folate concentration in the milk almost four fold in only 6 h.

Screening of Riboflavin-Producing Lactobacilli by a Polymerase-Chain-Reaction-Based Approach and Microbiological Assay.

Riboflavin has an important role in various cellular metabolic activities through its participation in oxidation-reduction reactions. In this study, as many as 60 lactobacilli were screened for the presence or absence of riboflavin biosynthesis genes and riboflavin production. Of these, only 14 strains were able to grow in a commercial riboflavin-free medium. We observed that the presence of riboflavin biosynthesis genes is strain-specific across different species of lactobacilli. The microbiological assay was found to be appreciably reproducible, sensitive, rapid, and inexpensive and, hence, can be employed for screening the riboflavin-producing strains. The study thus represents a convenient and efficient method for selection of novel riboflavin producers. These riboflavin(+) strains thus identified and characterized could be explored as potent candidates for the development of a wide range of dairy- and cereal-based foods for the delivery of in situ riboflavin to consumers.

Biotechnology of riboflavin.

Riboflavin (vitamin B2) production has shifted from chemical synthesis to exclusive biotechnological synthesis in less than 15 years. The underlying extraordinary achievement in metabolic engineering and bioprocess engineering is reviewed in this article with regard to the two most important industrial producers Bacillus subtilis and Ashbya gossypii. The respective biosynthetic routes and modifications are discussed, and also the regulation of riboflavin synthesis. As the terminal biosynthesis of riboflavin starts from the two precursors, ribulose 5-phosphate and guanosine triphosphate (GTP), both strains have been optimized for an improved flux through the pentose phosphate pathway as well as the purine biosynthetic pathway. Specific targets for improvement of A. gossypii were the increase of the glycine pool and the increase of carbon flow through the glyoxylic shunt. In B. subtilis, research interest, amongst others, has focused on gluconeogenesis and overexpression of the rib operon. In addition, insight into large-scale production of vitamin B2 is given, as well as future prospects and possible developments.

Lactic acid bacteria as a cell factory for riboflavin production

SummaryConsumers are increasingly becoming aware of their health and nutritional requirements, and in this context, vitamins produced in situ by microbes may suit their needs and expectations. B groups vitamins are essential components of cellular metabolism and among them riboflavin is one of the vital vitamins required by bacteria, plants, animals and humans. Here, we focus on the importance of microbial production of riboflavin over chemical synthesis. In addition, genetic abilities for riboflavin biosynthesis by lactic acid bacteria are discussed. Genetically modified strains by employing genetic engineering and chemical analogues have been developed to enhance riboflavin production. The present review attempts to collect the currently available information on riboflavin production by microbes in general, while placing greater emphasis on food grade lactic acid bacteria and human gut commensals. For designing riboflavin‐enriched functional foods, proper selection and exploitation of riboflavin‐producing lactic acid bacteria is essential. Moreover, eliminating the in situ vitamin fortification step will decrease the cost of food production.

Isolation and Purification of Lipase from the Riboflavin Overproducing Fungus Eremothecium Ashbyii

An extracellular lipase was observed in the hemiascomycete riboflavin overproducing fungus Eremotheciumashbyii . It was isolated and purified by salting out, dialysis, ion exchange chromatography on DEAE Sephadex and FPLC to 75-fold purity. The molecular weight of the purified enzyme was estimated to be 66 KDa and it showed a preference for unsaturated fatty acids. Maximum lipase was induced on lipid substrates containing a large percentage of unsaturated fatty acids. Thus olive oil substrate induced maximum lipase activity. It had an optimum pH of 7.0, an optimum temperature of 30°C, was stimulated by Zn 2+ , Mg 2+ , Fe 3+ , Ca 2+ and inhibited byCu 2+ , Co 2+ . An inhibition of activity upon incubation with PMSF and guanidine chloride, suggested the involvement of Serine and Arginine respectively in catalysis, whereas absence of inhibition by mercapto ethanol ruled out involvement of thiol groups in catalysis. This is the first report on the isolation and characterization of the lipase from the riboflavin over producer E. ashbyii. The possibility of lipase providing precursors for riboflavin over production can be exploited further to improve riboflavin production.

Metabolic engineering of riboflavin production in Ashbya gossypii through pathway optimization.

BackgroundThe industrial production of riboflavin mostly relies on the microbial fermentation of flavinogenic microorganisms and Ashbya gossypii is the main industrial producer of the vitamin. Accordingly, bioengineering strategies aimed at increasing riboflavin production in A. gossypii are highly valuable for industry.Results We analyze the contribution of all the RIB genes to the production of riboflavin in A. gossypii. Two important metabolic rate-limiting steps that limit the overproduction of riboflavin have been found: first, low mRNA levels of the RIB genes hindered the overproduction of riboflavin; second, the competition of the AMP branch for purinogenic precursors also represents a limitation for riboflavin overproduction. Thus, overexpression of the RIB genes resulted in a significant increase in riboflavin yield. Moreover, both the inactivation and the underexpression of the ADE12 gene, which controls the first step of the AMP branch, also proved to have a positive effect on riboflavin production. Accordingly, a strain that combines both the overexpression of the RIB genes and the underexpression of the ADE12 gene was engineered. This strain produced 523 mg/L of riboflavin (5.4-fold higher than the wild-type), which is the highest titer of riboflavin obtained by metabolic engineering in A. gossypii so far.ConclusionsRiboflavin production in A. gossypii is limited by a low transcription activity of the RIB genes. Flux limitation towards AMP provides committed substrate GTP for riboflavin overproduction without detrimental effects on biomass formation. A multiple-engineered Ashbya strain that produces up to 523 mg/L of riboflavin was generated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0354-x) contains supplementary material, which is available to authorized users.

Improvement of the riboflavin production by engineering the precursor biosynthesis pathways in Escherichia coli

3,4-Dihydroxy-2-butanone 4-phosphate (DHBP) and GTP are the precursors for riboflavin biosynthesis. In this research, improving the precursor supply for riboflavin production was attempted by overexpressing ribB and engineering purine pathway in a riboflavin-producing Escherichia coli strain. Initially, ribB gene was overexpressed to increase the flux from ribulose 5-phosphate (Ru-5-P) to DHBP. Then ndk and gmk genes were overexpressed to enhance GTP supply. Subsequently, a R419L mutation was introduced into purA to reduce the flux from IMP to AMP. Finally, co-overexpression of mutant purF and prs genes further increased riboflavin production. The final strain RF18S produced 387.6mgriboflavin·L−1 with a yield of 44.8mg riboflavin per gram glucose in shake-flask fermentations. The final titer and yield were 72.2% and 55.6% higher than those of RF01S, respectively. It was concluded that simultaneously engineering the DHBP synthase and GTP biosynthetic pathway by rational metabolic engineering can efficiently boost riboflavin production in E. coli.

Improvement of medium components for high riboflavin production by Aspergillus terreus using response surface methodology

The current study was designed to improve riboflavin production utilizing the filamentous fungus Aspergillus terreus. The effect of different nutritional and environmental factors was carried out by applying one-factor-at-a-time method. The selected high producer fungal strain was identified on the bases of the sequences of the gene encoding 18S rRNA and designated as A. terreus ASU1 (KP793450). In order to investigate the quantitative, optimal levels and the reciprocal interactions between five main medium components, response surface methodology (RSM) was subsequently employed. The results proved that optimal concentrations for enhancement of riboflavin production were: corn starch 30 (g/l); tryptone 0.5 (g/l) and 1 (g/l) for each glycine, KH2PO4 and MgSO4. The maximum riboflavin production of 344.60 mg/l (predicted 342.86 mg/l) was determined under the optimal conditions and the regression analysis (R 2) of RSM showed 97.7 %. In comparison to the production of original level (63 mg/l), 5.4-fold increase had been obtained.

Increased riboflavin production by manipulation of inosine 5'-monophosphate dehydrogenase in Ashbya gossypii.

Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Herein, we report the functional and structural characterization of the IMPDH enzyme from the industrial fungus Ashbya gossypii. Our data show that the overexpression of the IMPDH gene increases the metabolic flux through the guanine pathway and ultimately enhances 40 % riboflavin production with respect to the wild type. Also, IMPDH disruption results in a 100-fold increase of inosine excretion to the culture media. Our results contribute to the developing metabolic engineering toolbox aiming at improving the production of metabolites with biotechnological interest in A. gossypii.

Exploring Indigenous Lactobacillus Species from Diverse Niches for Riboflavin Production

Background: Riboflavin has received rather less attention in the past years, but interest is increasing because it is recognized as an essential component of cellular biochemistry, it is one of the vital vitamins required by human, bacteria, animals and plants. Today, bio production of riboflavin is competing with chemically synthesized one. Lactobacilli are well reckoned for their technological and probiotic significance and their exercise in dairy products for trade and industry. Materials and Methods: The present study was carried out to isolate and characterize riboflavin lactobacilli from various dairy and non-dairy sources of Indian origin. The isolates were subjected to phenotypic and genotypic characterizations followed by partial 16S rDNA sequencing. Results: As many as 40 isolates were identified as Lactobacilli and among them 14 were identified as L.plantarum , 5 as L. fermentum , 5 as L. delbrueckii subsp . bulgaricus , 4 L. brevis , 2 L. pentosus and 1 isolate as L. mucosae . Further isolates were screened for riboflavin production by chemical assay method and among all the tested isolates, 2 isolates viz. KTLF1 (L.fermentum) and KTLP13 (L.planatrum) have been observed to produce appreciable amount of riboflavin in MRS and RAM. Conclusion: These riboflavin producing lactobacilli can be used as a model for the development of strains that have the potential to produce an essential vitamin in situ which would contribute significantly to the functional value of certain fermented foods. Key words: Health benefits, Lactobacilli, Nutrition, Probiotic, Riboflavin, Vitamin.

Engineering Ashbya gossypii for efficient biolipid production

Ashbya gossypii is a filamentous fungus that naturally overproduces riboflavin. Indeed, engineered strains are currently used for the industrial production of riboflavin, replacing the chemical synthesis processes formerly used. The utilization of A. gossypii for biotechnological applications affords significant advantages that involve low-cost media use and cheap downstream processing for some applications. Although A. gossypii cannot be considered a bona fide oleaginous microorganism, the accumulation of lipid droplets within hyphae has been described. In view of the genomic and molecular tools available for its manipulation, the metabolism of A. gossypii was engineered aiming to increase total lipid accumulation. Blocking the β-oxidation pathway through the knock-out of the AgPOX1 gene was sufficient to obtain strains with high lipid yields, comparable to those of the best oleaginous microorganisms. Thus, the poxΔ strain of A. gossypii constitutes a novel promising tool for the production of microbial oils in forthcoming modified A. gossypii strains.

Investigation of protein secretion and secretion stress in Ashbya gossypii.

BackgroundAshbya gossypii is a filamentous Saccharomycete used for the industrial production of riboflavin that has been recently explored as a host system for recombinant protein production. To gain insight into the protein secretory pathway of this biotechnologically relevant fungus, we undertook genome-wide analyses to explore its secretome and its transcriptional responses to protein secretion stress.ResultsA computational pipeline was used to predict the inventory of proteins putatively secreted by A. gossypii via the general secretory pathway. The proteins actually secreted by this fungus into the supernatants of submerged cultures in minimal and rich medium were mapped by two-dimensional gel electrophoresis, revealing that most of the A. gossypii secreted proteins have an isoelectric point between 4 and 6, and a molecular mass above 25 kDa. These analyses together indicated that 1-4% of A. gossypii proteins are likely to be secreted, of which less than 33% are putative hydrolases. Furthermore, transcriptomic analyses carried out in A. gossypii cells under recombinant protein secretion conditions and dithiothreitol-induced secretion stress unexpectedly revealed that a conventional unfolded protein response (UPR) was not activated in any of the conditions, as the expression levels of several well-known UPR target genes (e.g. IRE1, KAR2, HAC1 and PDI1 homologs) remained unaffected. However, several other genes involved in protein unfolding, endoplasmatic reticulum-associated degradation, proteolysis, vesicle trafficking, vacuolar protein sorting, secretion and mRNA degradation were up-regulated by dithiothreitol-induced secretion stress. Conversely, the transcription of several genes encoding secretory proteins, such as components of the glycosylation pathway, was severely repressed by dithiothreitolConclusionsThis study provides the first insights into the secretion stress response of A. gossypii, as well as a basic understanding of its protein secretion potential, which is more similar to that of yeast than to that of other filamentous fungi. Contrary to what has been widely described for yeast and fungi, a conventional UPR was not observed in A. gossypii, but alternative protein quality control mechanisms enabled it to cope with secretion stress. These data will help provide strategies for improving heterologous protein secretion in A. gossypii.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1137) contains supplementary material, which is available to authorized users.

Blockage of the pyrimidine biosynthetic pathway affects riboflavin production in Ashbya gossypii

The Ashbya gossypii riboflavin biosynthetic pathway and its connection with the purine pathway have been well studied. However, the outcome of genetic alterations in the pyrimidine pathway on riboflavin production by A. gossypii had not yet been assessed. Here, we report that the blockage of the de novo pyrimidine biosynthetic pathway in the recently generated A. gossypii Agura3 uridine/uracil auxotrophic strain led to improved riboflavin production on standard agar-solidified complex medium. When extra uridine/uracil was supplied, the production of riboflavin by this auxotroph was repressed. High concentrations of uracil hampered this (and the parent) strain growth, whereas excess uridine favored the A. gossypii Agura3 growth. Considering that the riboflavin and the pyrimidine pathways share the same precursors and that riboflavin overproduction may be triggered by nutritional stress, we suggest that overproduction of riboflavin by the A. gossypii Agura3 may occur as an outcome of a nutritional stress response and/or of an increased availability in precursors for riboflavin biosynthesis, due to their reduced consumption by the pyrimidine pathway.