Riboflavin Biosynthesis by Candida robusta

During the study on the oxidative sugar metabolism of yeasts, it was found that six strains of Candida robusta, isolated from fruits, produced large amounts of yellow pigment in shaking culture and this pigment was identified as riboflavin. Riboflavin production by C. robusta has never been reported.

Some notable characteristics of C. robusta in riboflavin production were found. As nitrogen sources, ammonium salts and urea were favorable, but nitrate and organic nitrogen sources such as glycine, asparagine and peptone were not utilized for riboflavin production. Riboflavin was not produced in still culture; a highly aerobic condition, as may be obtained by shaking culture, wa, essential. The addition of excess CaCO₃ was also necessary. Acetic acid, added as the Ca salt in its production as a sole carbon source, was more effective than sugars and optimum concentration of this acid was 7%. Riboflavin were obtained in yields as high as 32 to 34 mg % from the acetate medium after 8 days.     

Integrated control of tobacco black shank by combined use of riboflavin and <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain Tpb55

We investigated the effect of riboflavin on the biocontrol activity of Bacillus subtilis Tpb55 against Phytophthora nicotianae (Pn), which causes tobacco black shank. Riboflavin (0.2 mg ml^?1) significantly improved the biocontrol activity of Tpb55 (2.0 × 10⁸ cfu ml^?1). Riboflavin (0.02–0.5 mg ml^?1) alone could not significantly inhibit Pn growth. However, it enhanced the B. subtilis population, both in vitro and in tobacco roots and significantly increased the activity of defense enzymes, peroxidase, catalase, superoxide dismutase, and β-1,3-glucanase, in the roots of B. subtilis-treated tobacco seedlings. Our results indicate that riboflavin can stimulate the growth of B. subtilis Tpb55 and induce resistance to Pn in tobacco plants. These findings should boost the prospects for practical application of B. subtilis Tpb55 as a biocontrol agent against black shank of tobacco.     

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii‐specific serum antibodies might play an important role in protection. Immuno‐proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA‐like protein of B. holmesii. An aBH vaccine prepared from a BipA‐like protein‐deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA‐like protein plays an important role in the protective efficacy of aBH vaccine.     

Riboflavin, overproduced during sporulation of Ashbya gossypii, protects its hyaline spores against ultraviolet light

Riboflavin (vitamin B₂), essential in tiny amounts as a precursor for oxidoreductase coenzymes, is a yellow pigment. Although it causes cytotoxicity via photoinduced damage of macromolecules, several microorganisms are striking overproducers. A question, unanswered for decades, is whether riboflavin overproducers can benefit from this property. Here, we report an ultraviolet (UV) protective effect of riboflavin. The spores of Ashbya gossypii , a riboflavin-overproducing fungus, are more sensitive to UV than those of Aspergillus nidulans . The addition of riboflavin to suspensions improves the UV resistance of both spore types. Interestingly, we show that regulation of sporulation and riboflavin overproduction in A. gossypii are linked. In batch culture, both were elevated when growth ceased. At constant growth rates, obtained in a chemostat culture, neither was elevated. Supplementation of cultures by cAMP, a known stress signal, negatively affected sporulation as well as riboflavin overproduction, establishing a second, independent argument for the linkage.     

Bioproduction of riboflavin: a bright yellow history

Riboflavin (vitamin B₂) is an essential nutrient for humans and animals that must be obtained from the diet. To ensure an optimal supply, riboflavin is used on a large scale as additive in the food and feed industries. Here, we describe a historical overview of the industrial process of riboflavin production starting from its discovery and the need to produce the vitamin in bulk at prices that would allow for their use in human and animal nutrition. Riboflavin was produced industrially by chemical synthesis for many decades. At present, the development of economical and eco-efficient fermentation processes, which are mainly based on Bacillus subtilis and Ashbya gossypii strains, has replaced the synthetic process at industrial scale. A detailed account is given of the development of the riboflavin overproducer strains as well as future prospects for its improvement.     

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis . It was found that this homolog, named AfuA_Bpp, is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O‐antigen, a molecule that has been found to shield surface antigens on B. parapertussis , showed no influence on antibody recognition of AfuA_Bpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

     

Non‐invasive optical method for real‐time assessment of intracorneal riboflavin concentration and efficacy of corneal cross‐linking

Keratoconus is the primary cause of corneal transplantation in young adults worldwide. Riboflavin/UV‐A corneal cross‐linking may effectively halt the progression of keratoconus if an adequate amount of riboflavin enriches the corneal stroma and is photo‐oxidated by UV‐A light for generating additional cross‐linking bonds between stromal proteins and strengthening the biomechanics of the weakened cornea. Here we reported an UV‐A theranostic prototype device for performing corneal cross‐linking with the ability to assess corneal intrastromal concentration of riboflavin and to estimate treatment efficacy in real time. Seventeen human donor corneas were treated according to the conventional riboflavin/UV‐A corneal cross‐linking protocol. Ten of these tissues were probed with atomic force microscopy in order to correlate the intrastromal riboflavin concentration recorded during treatment with the increase in elastic modulus of the anterior corneal stroma. The intrastromal riboflavin concentration and its consumption during UV‐A irradiation of the cornea were highly significantly correlated (R = 0.79; P = .03) with the treatment‐induced stromal stiffening effect. The present study showed an ophthalmic device that provided an innovative, non‐invasive, real‐time monitoring solution for estimating corneal cross‐linking treatment efficacy on a personalized basis.      

Improving the <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> butanol fermentation by engineering the strain for co-production of riboflavin

Solvent-producing clostridia are well known for their capacity to use a wide variety of renewable biomass and agricultural waste materials for biobutanol production. To investigate the possibility of co-production of a high value chemical during biobutanol production, the Clostridium acetobutylicum riboflavin operon ribGBAH was over-expressed in C. acetobutylicum on Escherichia coli–Clostridium shuttle vector pJIR750. Constructs that either maintained the original C. acetobutylicum translational start codon or modified the start codons of ribG and ribB from TTG to ATG were designed. Riboflavin was successfully produced in both E. coli and C. acetobutylicum using these plasmids, and riboflavin could accumulate up to 27 mg/l in Clostridium culture. Furthermore, the C. acetobutylicum purine pathway was modified by over-expression of the Clostridium purF gene, which encodes the enzyme PRPP amidotransferase. The function of the plasmid pJaF bearing C. acetobutylicum purF was verified by its ability to complement an E. coli purF mutation. However, co-production of riboflavin with biobutanol by use of the purF over-expression plasmid was not improved under the experimental conditions examined. Further rational mutation of the purF gene was conducted by replacement of amino acid codons D302 V and K325Q to make it similar to the feedback-resistant enzymes of other species. However, the co-expression of ribGBAH and purFC in C. acetobutylicum also did not improve riboflavin production. By buffering the culture pH, C. acetobutylicum ATCC 824(pJpGN) could accumulate more than 70 mg/l riboflavin while producing 190 mM butanol in static cultures. Riboflavin production was shown to exert no effect on solvent production at these levels.     

On the Character of Riboflavin Producing Mutant of Zygosaccharomyces soja

Riboflavin producing mutant of Zygosaccharomyces soja* was obtained by a treatment with cycloheximide. This mutant actively utilized various sugars and excreted riboflavin to the culture medium in a concentration of 30 to 40 μg per ml. Aerobic condition was prefered to sustain the growth of mutant and glucose catabolism was altered from alcohol fermentation in case of mother strain to respiration in mutant. This paper presents data obtained from morphological and physiological investigations.     

Effect of light quality on the growth and proximal composition of <Emphasis Type="Italic">Amphora</Emphasis> sp.

We determined the effects of various light spectra (white, blue, green, yellow, and Grolux) on the growth rate, proximate composition, pigment content, and cell size of the marine benthic diatom Amphora sp. during two growth phases of batch cultures. The growth rate was higher under green light and lowest with Grolux and yellow light. Cell size differed significantly between growth phases but remained unaffected by light spectra; the smallest cells were observed on the initial day of culture, whereas larger cells developed in the stationary phase under Grolux treatment. The proximate composition was modified by the light spectra and growth phase. In the exponential growth phase, the protein content was higher with yellow and white light, and lipid content peaked with Grolux. The pigment content (chlorophyll a, carotenoids) was also higher under yellow light. In the stationary growth phase, we noted a higher carbohydrate content under Grolux and yellow light. Our results show that light spectra and growth phase influence the metabolism of Amphora sp., changing its proximate composition, pigment content, growth rate, and cell size.     

Protective effects of in vivo‐expressed autotransporters against Bordetella pertussis infection

Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re‐emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine.

     

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata

Riboflavin (vitamin B₂) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.     

Specific substrates for isolation and differentiation of Azotobacter vinelandii

Summary Resorcinol, ethylene glycol and glutaric acid have been found to be specific substrates for the enrichment of Azotobacter vinelandii in the presence of other Azotobacter species. The three compounds may also be used for species differentiation in the genus Azotobacter. In contrast to other reports strains of A. chroococcum and A. beijerinckii have been isolated which are able to use L-rhamnose for growth.Conditions for the production of a yellow pigment and green fluorescence by A. vinelandii from non-aromatic substrates on agar-plates have been tested.The production of water soluble yellow pigments in benzoate containing media cannot be used as a criterium for the presence of A. vinelandii. The pigment seems to be -hydroxy-muconic semialdehyde, the splitting product resulting from metacleavage of catechol. It may be formed by all Azotobacter species that can metabolize aromatic substrates.     

The roles of the shikimate pathway genes,aroA and aroB,in virulence,growth and UV tolerance of Burkholderia glumae strain 411gr‐6

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow–green and purple) pigment compounds in casein–peptone–glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment‐deficient mutant derivatives of the virulent and pigment‐proficient strain 411gr‐6, having mini‐Tn5gus insertions in aroA encoding 3‐phosphoshikimate 1‐carboxyvinyltransferase and aroB encoding 3‐dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.     

Identification of active constraints in dynamic flux balance analysis

This study deals with the calibration of dynamic metabolic flux models that are formulated as the maximization of an objective subject to constraints. Two approaches were applied for identifying the constraints from data. In the first approach a minimal active number of limiting constraints is found based on data that are assumed to be bounded within sets whereas, in the second approach, the limiting constraints are found based on parametric sensitivity analysis. The ability of these approaches to finding the active limiting constraints was verified through their application to two case studies: an in‐silico (simulated) data‐based study describing the growth of E. coli and an experimental data‐based study for Bordetella pertussis (B. pertussis). © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:26–36, 2017     

A red pigment produced by aerobic sporeforming bacteria

Summary The production of an extracellular, water-insoluble, red pigment by strains of Bacillus cereus, B. circulans, B. licheniformis, B. macerans, and B. subtilis was found to be dependent on the presence of iron in the medium. B. cereus (strain TS) produced approximately 13 mg of pigment per gram of cells (dry weight), the pigment being formed during growth and not accumulating when growth ceased. Pigment-less mutants were usually present in pigmented cultures, their frequency increasing as the cultures aged. The absorption spectra (UV and visible regions) of pigments from representatives of the above-mentioned species were identical, with a maximum at approximately 385 and one at 490 m. These spectra were remarkably similar to that of pulcherrimin, a red, iron-containing pigment produced by the yeast Candida pulcherrima.The pigment from B. cereus TS was purified by treatment with alkaline methanol, followed by precipitation with acid in the presence of excess FeCl₃. Chemical analysis of the purified pigment indicated that it consisted of a ferric complex of substituted pyrazine rings with isobutyl groups bound to positions 2 and 5. The structure of the bacterial pigment was found to be essentially identical with that of pulcherrimin, the degree of chelation in the former being less extensive than in the latter. The name pulcherrimin a is proposed for the pigment produced by B. cereus TS.     

Effect of riboflavin‐producing bacteria against chemically induced colitis in mice

Aim

To assess the anti‐inflammatory effect associated with individual probiotic suspensions of riboflavin‐producing lactic acid bacteria (LAB) in a colitis murine model.

Methods and Results

Mice intrarectally inoculated with trinitrobenzene sulfonic acid (TNBS) were orally administered with individual suspensions of riboflavin‐producing strains: Lactobacillus (Lact.) plantarum CRL2130, Lact. paracasei CRL76, Lact. bulgaricus CRL871 and Streptococcus thermophilus CRL803; and a nonriboflavin‐producing strain or commercial riboflavin. The extent of colonic damage and inflammation and microbial translocation to liver were evaluated. iNOs enzyme was analysed in the intestinal tissues and cytokine concentrations in the intestinal fluids. Animals given either one of the four riboflavin‐producing strains showed lower macroscopic and histologic damage scores, lower microbial translocation to liver, significant decreases of iNOs+ cells in their large intestines and decreased proinflammatory cytokines, compared with mice without treatment. The administration of pure riboflavin showed similar benefits. Lact. paracasei CRL76 accompanied its anti‐inflammatory effect with increased IL‐10 levels demonstrating other beneficial properties in addition to the vitamin production.

Conclusion

Administration of riboflavin‐producing strains prevented the intestinal damage induced by TNBS in mice.

Significance and Impact of the Study

Riboflavin‐producing phenotype in LAB represents a potent tool to select them for preventing/treating IBD.     

Unusual toxicity of riboflavin and flavin mononucleotide forCardiobacterium hominis

The chemical composition of a completely defined culture medium for the growth of strains ofCardiobacterium hominis is described.Riboflavin and flavin mononucleotide at low concentrations completely inhibit the growth response ofC. hominis. Only leucine, among a wide variety of substrates tested, prevents this toxicity over a range of riboflavin or flavin mononucleotide levels.     

Studies on the extracellular xylanase activity of some thermophilic actinomycetes

Summary An agar plate-clearing assay was used to screen 37 thermophilic actinomycete strains for extracellular xylanase production. The xylanase activity in culture supernatants of strains representing Saccharomonospora viridis and three Thermomonospora spp. was characterised by measurement of reducing sugar released from oat spelt xylan and analysis of degradation products by thin-layer chromatography. In all four species, xylanase activity was optimal within the temperature range 60–75°C and between pH 5 and pH 8. While culture supernatants of Thermomonospora strains incubated at 70°C for 60 min retained >80% of their activity, that of S. viridis was almost, totally inactivated.All of the culture supernatants initially hydrolysed xylan to a mixture of oligomeric products, indicating that the main activity was of the endoxylanase type. Prolonged incubation for 24h resulted in the hydrolysis of xylan to d-xylose by T curvata and T. fusca preparations, indicating the additional presence of exoxylanase or -xylosidase activity. Xylanase production was induced by growth on xylan although low levels of activity were also detected in glucose-grown cultures. Thermomonospora curvata MT815 culture supernatant was the most active and produced d-xylose from milled wheat straw in yields approximately 10% of those from oat spelt xylan.