Riboflavin formation by mould fungi cultivated on hydrocarbon-containing media

The potentiality of some mould fungi, isolated from petroleum sludge to produce riboflavin when grown on hydrocarbon-containing media was tested. Aspergillus terreus was found to be distinguished by its capacity to produce riboflavin when cultivated on the different culture media tested. It was able to grow more luxuriantly and produce good riboflavin output on solar-containing medium. A solar concentration of 5% v/v favoured high riboflavin productivity. The maximal vitamin B2 yields were achieved after 20 days incubation at 30 degrees C. An initial pH value of 5.5-6.0 was found to be the optimum for growth of A. terreus and for riboflavin production.     

Growth and metabolism of Penicillium lilacinum Thom. with reference to the effects of riboflavin and nicotinic acid

Summary Growth and metabolism of Penicillium lilacinum were followed over a period of incubation of 18 days on a high sugar-salts medium favourable for fat formation with or without the addition of riboflavin or nicotinic acid to the growth medium. The high sugar content in the culture media helped rapid uptake and vigorous growth. Nicotinic acid and to a less extent riboflavin, enhanced sugar and nitrogen absorption and the rate of building up of cellular material in consequence. Nitrogenous compounds have been released from the mycelial cells into the external media before growth started to decline; the release being earlier and more rapid in the presence of nicotinic acid. It is suggested that the release of nitrogenous compounds in this case is not purely due to autolysis, and that nicotinic acid affected this process by increasing cell permeability. Both riboflavin and nicotinic acid accelerated the accumulation of carbohydrates and fat in the mycelium. Fat formation became active only when the nitrogen content of the culture media dropped to a very low value and the building of nitrogenous compounds almost stopped. The inverse relationship between synthesis of fat and of complicated nitrogenous compounds was quite clear under the present experimental conditions and was not affected by either riboflavin or nicotinic acid.     

Dietary ascorbic acid,pyridoxine and riboflavin reduce the light sensitivity of larvae and pupae of Drosophila melanogaster

Drosophilia melanogaster is light sensitive. On low yeast media, light induces high mortality during the development from egg to adulthood and increases development time. This effect of light is strongly dependent on the yeast-concentration. Addition of 8 vitamins, normally present in yeast, protects Drosophila against light under laboratory conditions. In this study we have analyzed the significance of the individual vitamins for both survival and development at high light intensities. Two D. melanogaster strains were utilized: a control strain C and a strain P. The latter had been adapted to a palmitic acid supplemented medium. In addition, we investigated the effect of vitamin C, a vitamin typically found in fruit, but not in yeast. It appears that both pyridoxine and riboflavin are essential for the survival of the control strain C under high light intensities, and they act synergistically. The other 6 tested vitamins can be omitted in these survival experiments. Moreover survival under high light conditions also improved strongly on media supplemented with vitamin C. The other strain (P), which was for many generations kept on a different food-medium, also was protected on yeast media by riboflavin and pyridoxine, and by vitamin C, although the survival at high light intensities on media with riboflavin and pyridoxine was less than the survival of the control strain.     

Production of B-group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Azotobacter vinelandii strain ATCC 12837 and A. chroococcum strain H23 (CECT 4435) were able to grow on N-free or NH4Cl-amended chemically-defined (Burk's) media, with protocatechuic acid (1-2 mmol 1(-1)) or sodium p-hydroxybenzoate (1-10 mmol 1(-1)) as sole carbon (C) sources. At a concentration of 2 mmol 1(-1), both substrates supported nitrogen fixation (acetylene reduction assay) at similar or higher rates than bacteria grown in control media amended with 2 mmol 1(-1) sodium succinate as C source. The two strains produced the B-group vitamins niacin, pantothenic acid, thiamine, riboflavin and biotin after 72 h of growth in chemically-defined media with 2 mmol 1(-1) protocatechuic acid, sodium phydroxybenzoate or sodium succinate as sole C source, either in N-free media or in media amended with 0.1% NH4Cl. Quantitative production of all vitamins was affected by the use of the different C and N substrates.     

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B₂) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-l-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3–3.5 times higher as compared to the parental strain. It produced 50–70 times more riboflavin in iron sufficient synthetic media relative to the parental wild-type strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.     

Importance of malate synthase in the glyoxylate cycle of <Emphasis Type="Italic">Ashbya gossypii</Emphasis> for the efficient production of riboflavin

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.     

Analgos of riboflavin, lumiflavin and alloxazine derivatives. II. Effect of roseoflavin on 6,7-dimethyl-8-ribityllumazine and riboflavin synthetase synthesis and growth of Bacillus subtilis]

The replacement of 8-CH3 group in the riboflavin molecule results in the formation of specific antimetabolites. They are rozeoflavin, 7-desmethylrozeoflavin, 8-amino (nor) riboflavin, 8-ribitylamino (nor) riboflavin. Effect of rozeoflavin and other riboflavin analogues on the growth and regulatory characteristics of Bacillus subtilis strains with different genetic state of riboflavin operon is studied. Roseoflavin at a concentration of 0.05 mkg/ml inhibits DRL synthesis in rib-b110 strain. An analogue inhibits the growth of auxotrophic and prototrophic strains at concentrations of 0.5 mkg/ml and 50 mkg/ml respectively. Riboflavin (1 mkg/ml) recovers the growth of bacteria. The curve of rozeoflavin regulation of DRL and riboflavin synthetase synthesis is shifted in 100 times in the direction of lesser concentrations as compared with riboflavin and 8 amino (nor) riboflavin. 180 mutants resistant to 100 mkg/ml of rozeoflavin were selected. 150 mutants over-synthetize riboflavin.     

Regulation of Riboflavin Biosynthesis in Bacillus subtilis Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by ribC

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.     

Cloning of structural genes involved in riboflavin synthesis of the yeast Candida famata

The riboflavin overproducing mutants of the flavinogenic yeast Candida famata isolated by conventional selection methods are used for the industrial production of vitamin B2. Recently, a transformation system was developed for C. famata using the leu2 mutant as a recipient strain and Saccharomyces cerevislae LEU2 gene as a selective marker. In this paper the cloning of C. famata genes for riboflavin synthesis on the basis of developed transformation system for this yeast species is described. Riboflavin autotrophic mutants were isolated from a previously selected C. famata leu2 strain. C. famata genomic DNA library was constructed and used for cloning of the corresponding structural genes for riboflavin synthesis by complementation of the growth defects on a medium without leucine and riboflavin. As a result, the DNA fragments harboring genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding GTP cyclohydrolase, reductase, dimethylribityllumazine synthase, dihydroxybutanone phosphate synthase and riboflavin synthase, were isolated and subsequently subcloned to the smallest possible fragments. The plasmids with these genes successfully complemented riboflavin auxotrophies of the corresponding mutants of another flavinogenic yeast Pichia guilliermondii. This suggested that C. famata structural genes for riboflavin synthesis and not some of the supressor genes were cloned.     

Isolation of an oxalate-resistant Ashbya gossypii strain and its improved riboflavin production

An oxalate-resistant strain of Ashbya gossypii was naturally isolated from spores grown on an oxalate-containing medium, and its medium was optimized to improve riboflavin production. Riboflavin production by the resistant strain was three-fold higher than that by the wild-type organism when grown in flask cultures. Medium optimization increased the riboflavin production by the resistant strain to 5 g l⁻¹, which was five-fold higher than that obtained by the wild-type strain. The productivity was reproduced in a 3-l bioreactor. During the early growth phase, the specific activity of isocitrate lyase in the oxalate-resistant strain was slightly higher than that in the wild-type strain. Proteomic analysis of the oxalate-resistant strain revealed that the expression of aldose reductase and cobalamin-independent methionine synthase decreased significantly. This is the first report that describes the natural isolation of a riboflavin producer using an antimetabolite-containing medium to enhance the riboflavin production level. This method should also be useful for improving the productivity of other bioproducts since it does not require any mutations or genetic modifications of the microorganism.     

Redirection electron flow to high coupling efficiency of terminal oxidase to enhance riboflavin biosynthesis

The metabolic impact of redirection electron flow to high coupling efficiency of terminal oxidases on riboflavin biosynthetic ability was quantitatively assessed during batch culture in this paper. While disruption of the low coupling bd oxidase of the riboflavin overproducing B. subtilis PK, the apparent phenotype with more rapid specific growth rate and higher biomass yield was achieved. Compared to by-products formation, a discernible shift to less acetate and more acetoin in cyd mutant was observed. As the overflow metabolism was decreased in B. subtilis PK cyd, more carbon source was directed to biomass and riboflavin biosynthetic pathway, which resulted in higher biomass and about 30% improvement of riboflavin biosynthetic ability. The higher product-corrected biomass yield in mutant showed that the efficient energy generation is an important factor for exponential growth of riboflavin overproducing B. subtilis strain in batch culture.     

Mutants of Pichia guilliermondii yeasts with multiple sensitivity to antibiotics and antimetabolites. I. The selection and properties of the mutants

Riboflavin deficient mutant Pichia guilliermondii MS1 which requires approximately 1000-fold lower concentration of exogenous vitamin B2 for growth when compared with a non-adapted riboflavin deficient mutants of this species was isolated by means of of UV-irradiation. The growth of the mutant was strongly inhibited by actinomycin D and L-canavanine. The revertant MS8 and MS14 which synthesized riboflavin were selected from the strain MS1. These revertants posses a multiple sensitivity to actinomycin D, rifamycin, euflavine, mitomycin C, antimycin A, 8-azaadenine, 8-azaguanine, L-canavanine and 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine. The ability to utilized glycerol and ethanol as a sole carbon source for growth was impaired in these mutants. The mutants which can utilize glycerol were isolated from the strain MS14. Such mutants were resistant to actonomycin D. Mutation (s) which determines a multiple sensitivity and inability to utilized glycerol was recessive.     

Isolation of Ashbya gossypii mutant for an improved riboflavin production targeting for biorefinery technology

AIMS: To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. METHODS AND RESULTS: Ashbya gossypii spores were mutagenized by exposure to UV light and mutant ZP4 strain, producing riboflavin threefold the riboflavin that of the wild-type strain, was isolated by the first and second screenings. Proteomic analysis of ZP4 strain showed the expression patterns of eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain and those enzyme activities were investigated. When activated bleaching earth (ABE) containing 75 g l(-1) rapeseed oil was added in the culture of the ZP4 strain with oxygen-enriched air supplied, riboflavin concentration increased to 8.7 g l(-1) at 5 days of culture. Riboflavin production yield was 0.17 g g(-1) of consumed oil, which was eightfold higher than that of the wild-type strain. CONCLUSIONS: The results show that the mutant ZP4 strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source. SIGNIFICANCE AND IMPACT OF STUDY: Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.     

Enhancement of riboflavin production by overexpression of acetolactate synthase in a pta mutant of Bacillus subtilis

Genetic alterations of carbon flux into the acetoin biosynthesis pathway as a possible means to reduce acid accumulation were investigated in the riboflavin-producing Bacillus subtilis during growth on glucose. The lower rates of cell growth and riboflavin production were found in the pta-disrupted mutant while the rate of acetate formation was reduced. In contrast, acid accumulation was significantly reduced, to one-fifth that of the parental strain RH33::[pRB63](n), and a 50% increase in the riboflavin yield was obtained when the expression of the gene encoding acetolactate synthase was increased in the pta-disrupted mutant. Metabolic analysis, together with enzyme activity assays, indicated that the tricarboxylic acid cycle fluxes are significantly increased in response to acetolactate synthase overexpression in pta-disrupted mutant. Moreover, the intracellular ATP-to-ADP ratio also increased 5.8-fold. The high concentration of ATP could explain the increased riboflavin production.     

Differential riboflavin deposition in white and variegated white mutants of Drosophila hydei

Riboflavin deposition in organs of Drosophila hydei was studied by means of a growth test using a riboflavin-deficient strain of the fungus Aspergillus nidulans. In wild-type animals, riboflavin is deposited in Malpighian tubules (MT) and testes but not in adult eyes. Certain white (w) mutants do not contain riboflavin, whereas intermediately colored w mutants contain minor amounts of the substance. Riboflavin-containing MT cells contain special globules that can be fixed and stained with the redox dye phenazine-methosulphate. The number and size of these granules is related to growth effect and point to a role of the w locus in the intracellular deposition of riboflavin in special organs. In white-mottled (wm) position-effect variegation mutants, a significant correlation was found between the extent of variegation (percentage of yellow cells) and riboflavin content (growth effect) of the MT. However, the individual variation of cell phenotype was extremely large and exaggerated types were observed indicating "overdominance" of the rearranged w+ gene. This contradicts an unsubstantiated dogma of position-effect variegation that assumes that the affected gene simply switches between the on and off state, as is discussed.     

Effect of rib83 mutation on riboflavin biosynthesis and iron assimilation in Pichia guilliermondii]

The monogenic rib83 mutation blocked riboflavin oversynthesis in the yeast Pichia guilliermondii and lowered iron acquisition by cells, their ferric reductase activity, and the growth rate in iron-deficient media. Mutants with the combined mutations of rib83 with rib80 and rib81 (the last two mutations impair the negative control of riboflavin synthesis and thus cause its oversynthesis) were unable to depress the enzymes of flavinogenesis (GTP cyclohydrolase and riboflavin synthase) and to overproduce riboflavin in both iron-deficient and iron-sufficient media. This suggests that the rib83 mutation is epistatic with respect to the rib80 and rib81 mutations. The RIB83 gene may positively control both riboflavin synthesis and iron acquisition in the yeast P. guilliermondii.     

Kinetics of riboflavin production by Brewers'' yeasts

The kinetics of riboflavin production by Saccharomyces cerevisiae and Saccharomyces carlsbergensis in synthetic media and wort were studied. The results indicated that riboflavin was produced by growing cells only. Riboflavin production rate was proportional to growth rate of the yeasts in the exponential phase. Riboflavin was depleted in the stationary phase. The depletion rate was expressed with a first-order kinetic expression in yeast concentration. The kinetics of substrate utilization and ethanol production were also given to describe better the associated phenomena and fermentation pattern.     

Characterization of a shiitake mutant strain producing ballshaped fruiting bodies

Growth characteristics of a spontaneous mutant of shiitake Lentinula edodes (Berk.) Pegler were studied. The mutant was first detected as a result of changes in the growth habit of the normal strain in the liquid medium. Abundant formation of aerial hyphae was distinctive. In sawdust logs the mutant strain produced abnormal basidiocarps, lacking stipe, gill and spore formation.
Growth rates of the normal and the mutant strain were compared in two liquid media: malt-yeast extract and Leatham's medium. The increase in dry weight of the mutant's mycelium was much higher than that of the wild type in both media, which indicated better adaptation to liquid culture. In the sawdust, however, growth of the mutant was slower than that of the normal strain. The mutant's intracellular protein content was lower than that of the normal strain. The pH of the liquid cultures differed: the wild type decreased the pH during growth, while the mutant increased the pH. Comparison of the protein and esterase isoenzyme profiles of the vegetative hyphae of both strains indicated profound differences. One protein (pI 6.5, 39 kDa), which in earlier studies has been found to be typical of L. edodes species, was absent from the mutant's profile. Differences in the esterase profile were also clear.