Ribitol and flavinogenesis in Eremothecium ashbyii

1. Supplementation of cultures of Eremothecium ashbyii with ribitol leads to a twofold increase in riboflavin formation compared with unsupplemented cultures or those supplemented with ribose or ribulose phosphate. Addition of unlabelled ribitol decreases the incorporation of [1-(14)C]ribose into riboflavin, indicating that free ribitol is preferred to ribose for incorporation into riboflavin. 2. The enzymes ribitol kinase, d-ribose reductase, d-ribose 5'-phosphatase and GMP nucleosidase were demonstrated in the cell-free extracts. Ribitol induces the formation of ribitol kinase. The enzyme is activated in vitro by the flavinogenic purines, guanine and xanthine. d-Ribose reductase shows a specific requirement for NADPH and forms free ribitol from ribose. 3. The activities of ribitol kinase, ribose 5'-phosphatase and GMP nucleosidase reach their maximal values before riboflavin formation reaches a maximum. 4. [U-(14)C]GMP is taken up intact by the culture of E. ashbyii and is incorporated into riboflavin as well as into a blue fluorescent compound. The radioactivity from this compound is incorporated into riboflavin by the cell-free extract of E. ashbyii.     

Influencia de diversos factores sobre Eremothecium ashbyii

Resumen y conclusiones Se observa la diferencia entre las variables productoras y no productoras de riboflavina, establecidas por sus caracteres morfológicos y microscópicos, y se estudia la influencia de ciertos factores y la inmunidad de otros sobre las mismas.Observaciones microscópicas muestran a las variables flavinogénicas de fácil y rápida esporulación.Factores físicos como la luz, la temperatura, el calor, la radiación ultravioleta, no determinan alteraciones digunas de mención. Las cepas flavinogénicas pueden perder esa propiedad, por cercanía de contaminantes, contacto con substancias químicas, etc.Antibióticos y quimioterápicos no ejercen acción sobre ninguna de las formas deEremothecium. Las sulfas actuan levemente sobre la forma blanca menos que sobre la anaranjada.Factores nutricios como el extracto de papa, el almidón y la peptona estimulan la flavinogénesis, por lo que se considera que agregadas en las proporciones establecidas al Sabouraud común, se mejorará el medio de cultivo y se obtendrán cepas de mayor rendimiento.Cepas altamente flavinogénicas se obtienen por desarrollo en medios conteniendo extracto de tierra; por exposición de la cepa a la radiación ultravioleta en estado de hibernación; por pasaje por caldos peptonados y por desarrollo en medios conteniendo riboflavina.Se observa que efectuando los transplantes cada cuatro dias disminuye el porcentaje de variables blancas. Por crecimiento en medios conteniendo riboflavina, se induce a las formas blancas a la producción de vitamina.Finalmente aconsejamos como medio de mantenimiento el uso de Sabouraud al que se agrega riboflavina en niveles no superiores 1 mg/ml y efectuar los repiques cada cuatro dias.

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Summary The property ofEremothecium ashbyii to produce riboflavin is exploited for the industrial production of the vitamin. However, the fungus presents natural variability giving rise to several variants such as the orange one with a high level of riboflavin production, the yellow one with less productive capacity and the white one without flavinogenic properties. This problem is interesting from a biological and industrial point of view.The purpose of the present work was to detect possible differences between the two extreme variants ofEremothecium ashbyii (orange or strong producer of riboflavin and white or non producer form) so as to stimulate riboflavin production in the white strain and on the other hand, to stabilize the flavinic ones. To achieve this, the influence of different factors on the two selected variants was tested with the following results:Macroscopic observations showed morphological differences in the aspect of both variants and in the production of riboflavin.Microscopic observations showed that the flavinic strain sporulated rapidly whereas the white strain did so more slowly. Moreover, a relation was shown to exist between sporulation and riboflavin production.Physical factors such as light, temperature, heat and ultraviolet radiation did not determine appreciable alterations in the two forms studied. Antibiotics and chemotherapics did not excercise any action on either of the variants. The sulfamides had a slightly greater affect on the orange form than on the white one. Nutrient factors such as potato extract, starch and peptone stimulate flavinogenesis, and for that reason, it was considered that if added, in the established proportions to the Sabouraud medium they would improve the culture medium.Highly flavinogenic strains were obtained by development in a medium with earth extract, by exposure of the strain to ultraviolet radiation when it was in the hibernation state, by passage through peptoned broth and by development in a culture medium to which riboflavin had been added. The flavinic strains may lose its producing property due to the presence of contaminants, through contact with chemical substances etc.It was observed that by making transplants every four days and not weekly, the percentage of the white forms decreased. When the white form was developed in a culture medium with riboflavin, vitamin production was induced.Finally, it is suggested that Sabouraud's medium with the addition of 1 mg/ml riboflavin would be an adequate maintenance medium and that making transplants every four days, it would be possible to obtain a greater percentage of riboflavin producing strains.

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Trabajo realizado en el Instituto de Industrias Bioquímico Farmacéutico, Facultad de Farmacia y Bioquímica de la Universidad de Buenos Aires.     

Cytologische und physiologische Untersuchungen an Eremothecium ashbyii Guill.

Ohne ZusammenfassungTeilergebnisse aus einer Inauguraldissertation der philosophisch-naturwissenschaftlichen Fakultät der Universität Bern zur Erlangung der Doktorwürde 1961.     

Eremothecium ashbyii mutants resistant to 2,6-diaminopurine]

3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.     

8-azaguanine and flavinogenesis in Eremothecium ashbyii.

8-Azaguanine (10- minus 4 M) supplementation in synthetic medium inhibited flavinogenesis in Eremothecium ashbyii to far greater extent (68per cent) than the growth (25 per cent). That enzymes comprising the biosynthetic pathway of riboflavin are synthesized during early growth phase of the organism is supported by the data presented. 8-Azaguanine mediated inhibition in flavinogenesis was closely related with decreased levels of ribose-5'-phosphatase, ribose reductase and ribitol kinase, the enzymes involved in supplying ribitol for flavinogenesis. Addition of guanine and not ribitol during early growth phase to 8-azaguanine-added cultures released the inhibition of riboflavin synthesis and restored the enzyme levels in the presence of the antimetabolite.     

The kinetics of riboflavin secretion by Eremothecium ashbyii nrrl 1363

For riboflavin production a highly flavinogenic strain Eremothecium ashbyii is used. Vitamins are classified as chemical substances that control and effect the physiological processes; Riboflavin is one among them. The deficiency of riboflavin in human beings results in the cracking of lips and corners of mouth (cheilosis); nerve tissues are affected. For riboflavin production a highly flavinogenic strain Eremothecium ashbyii NRRL 1363 was used. Investigations were conducted in shake flask using inexpensive and abundantly available raw materials. Among the stimulants, a combination of hog casings and beef extract stimulated the highest and promoted the maximum riboflavin yield followed by the combination of fish meal and beef extract. The fermented broth (an enriched, riboflavin concentrate) can be directly used as a feed grade riboflavin. To upgrade it to pharmaceutical grade further investigations are required.     

Eremothecium ashbyii: revision y actualizacion de conceptos sobre la especie

Summary A general review of knowledge complemented with personal observations ofEremothecium ashbyii, a high level fungus producer of riboflavin (vitamin B₂), is made.Following a short history of the species, all the macro- and microscopic aspects of the cultures are investigated: hyphae, nuclei protoplasmatic inclusions, formation of sporangia (ascus) and spores germination of spores and appearance of riboflavin in the mycelial filaments. Bulbar forms, not previously reported in the literature, are described in this species for the first time. Finally, in view of the unusual morphology of the fungus, its systematic placing and phylogenetic relations are discussed.

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Trabajo realizado en el Instituto de Industrias Bioquímico Farmacéuticas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires.     

Application of agro-industrial by-products for riboflavin production by Eremothecium ashbyii NRRL 1363

Riboflavin production is significantly determined by the type and initial concentration of the carbon and nitrogen sources and also by other flavinogenic stimulants. Using an optimum carbon and nitrogen concentration, an industrial fermentation medium has been designed with molasses as the carbon source and peanut seed cake as the nitrogen source. In addition the stimulatory effect of some of the low-cost agro-industrial by-products on riboflavin yield was investigated. Received: 10 March 1996 / Received revision: 25 June 1996 / Accepted: 14 July 1996     

Eremothecium ashbyii T_(30)发酵产脂肪酶特性的研究

研究了一株核黄素高产突变株E .ashbyiiT3 0 产脂肪酶的条件 ,确定了比较合适的脂肪酶发酵培养基 :豆饼粉 30g/L ,玉米浆 30ml/L ,豆油 5ml/L ,KH2 PO4 5g/L ,MgSO4 ·7H2 O 0 15g/L ,pH消前7 5。T3 0 在此培养基上振荡培养 2 8h ,既定条件下测脂肪酶活力可达 2 32酶活单位 /ml(较原株提高 36 5 % ) ,从一个侧面证实其突变株特性。另外 ,对T3 0 所产脂肪酶的诱导酶属性及脂肪酶在T3 0 核黄素发酵过程中的作用也进行了初步探讨     

Statistical optimization of medium components for improved synthesis of riboflavin by Eremothecium ashbyii

Statistical designs were used to determine optimum levels of medium nutrients for riboflavin production in shake flask fermentation. The medium components considered include molasses, sesame seed cake (SSC), yeast extract, KH₂PO₄, MgSO₄·7H₂O, NaCl and Tween-80. Information about the effects of different medium components on riboflavin production were investigated by using the Plackett-Burman experimental design. MgSO₄·7H₂O and NaCl were found to significantly influence the riboflavin production (confidence levels above 70%). For obtaining the mutual interaction between the variables required, for achieving the optimal concentrations, a 2²-factorial central composite design was employed. The optimal concentrations for the enhanced production of riboflavin were (g/l): molasses =50.0 (glucose equivalents); SSC =50.0; yeast extract =2.0; KH₂PO₄=2.0; MgSO₄·7H₂O=0.117 and NaCl =1.13.