Ribitol and flavinogenesis in Eremothecium ashbyii

Abstract

1. Supplementation of cultures of Erenwthecium 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 [1-14C]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-_4C]GMP is taken up intact by the culture ofE. 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.Micro-organisms have been used extensively to study the biosynthesis of riboflavin, since some of them produce this vitamin in large excess. Although considerable information is available on the biosynthesis of riboflavin, the mode of insertion of the ribitol moiety into the vitamin molecule still remains obscure (Bacher & Lingens, 1970;Baugh & Krumdieck, 1969;Goodwin, 1963; Goodwin & Jones, 1956;Harvey & Plaut, 1966;Maley & Plaut, 1959;McNutt, 1954;Plaut, 1961). The present investigation was undertaken to study the effect of availability of ribitol and guanine on enzymic constitution and flavinogenesis in Eremothecium ashbyii. Materials and Methods OrganismA flavinogenic strain of Eremothecium ashbyii (no. 0944), obtained from the Type Culture Collection, Tokyo, Japan, was maintained on Sabourauds' agar (Difco Laboratories, Detroit, Mich., U.S.A.) slant cultures. The yeast was cultivated in the liquid fermentation medium as described by Goodwin & Pendlington (1954), supplemented with 0.5% vitamin-free Casamino acids (Difco Laboratories). The inoculated flasks were incubated at 28 ±20C in a dark incubator; unless otherwise specified, the cultures were incubated for 4 days. The surface cultures grown in the above liquid medium ('normal medium') are termed 'normal cultures'; when ribose (5 or lOmg/ 50ml) or ribitol (5 or lOmg/50ml) or guanine (3 mg/50ml) was included in this medium, the culVol. 130 tures are referred to as 'ribose-supplemented', 'ribitol-supplemented' or 'guanine-supplemented' respectively. Radioactivity experimentsTo study the fate of [1-14C]ribose during growth and flavinogenesis, the organisms were grown for 48h in the non-radioactive normal medium. Cells were collected by centrifugation and resuspended in the fermented broth to give a concentration ofapprox. 0.2mg dry wt./ml of suspension. Samples (5ml) from this suspension were distributed in lOOm...

Inhibition of purified isocitrate lyase identified itaconate and oxalate as potential antimetabolites for the riboflavin overproducer Ashbya gossypii

“…A possible bottleneck role of the ICL in supplying the riboflavin biosynthesis with precursors should become evident by this approach. Yield-enhancing effects of glycine (Hanson, 1967) and ribitol (Mehta et al ., 1972) support the idea that such limitations in precursors are responsible for suboptimal riboflavin yields.…”

Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii

“…In its metabolism the availability of the educts GTP and ribulose 5-phosphate is of great importance for increasing riboflavin overproduction, since riboflavin formation was found to be enhanced by supplementation of the culture with precursors of the riboflavin molecule, e.g. ribitol [4], purines [5] and glycine [3,5,6]. Supplementation of the medium with glycine, a precursor of purine biosynthesis, increases riboflavin overproduction in A. gossypii in the range of 30 % [6] to 650 % [5], depending on the culture conditions.…”

Effects of various metabolites (sugars, carboxylic acids and alcohols) on riboflavin formation in non-growing cells of Ashbya gossypii.

“…Accordingly, the biosynthetic pathway of the pyrimi dine and pyrazine moieties in the isoalloxazine ring of riboflavin has been fairly well elucidated. However, the metabolic pathways of the o-xylene ring and the ribityl side chain on the riboflavin molecule are still unclear, in spite of many works with labelled (11)(12)(13)(14)(15)(16)(17)(18) or nonlabelled compounds (19)(20)(21).…”