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.

Glycine metabolism in Candida albicans: characterization of the serine hydroxymethyltransferase (SHM1, SHM2) and threonine aldolase (GLY1) genes.

Genes encoding the mitochondrial (SHM1) and cytosolic (SHM2) serine hydroxymethyltransferases, and the L-threonine aldolase gene (GLY1) from Candida albicans were cloned and sequenced. All three genes are involved in glycine metabolism. The C. albicans Shm1 protein is 82% identical to that from Saccharomyces cerevisiae and 56% identical to that from Homo sapiens. The corresponding identities for the Shm2 proteins are 68% and 53%. The Gly1 protein shares significant identity with the S. cerevisiae L-threonine aldolase (55%) and also with threonine aldolases from Aeromonas jandiae (36%) and Escherichia coli (36%). Genetic ablation experiments show that GLY1 is a non-essential gene in C. albicans and that L-threonine aldolase plays a lesser role in glycine metabolism than it does in S. cerevisiae. GenBank Accession Nos of the C. albicans SHM1 and SHM2 are AF009965 and AF009966, respectively. Accession No. for C. albicans GLY1 is AF009967.

Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii.

Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg-1 was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.

Identification of Saccharomyces cerevisiae GLY1 as a threonine aldolase: a key enzyme in glycine biosynthesis.

Determination of enzyme-specific activities revealed that GLY1 encodes a threonine aldolase (TA) in Saccharomyces cerevisiae. A knock-out mutant auxotrophic for glycine lacked detectable activity. After transformation with YEp24GLY1 glycine prototrophy was restored and TA-specific activity was 16-fold higher than in the wild type. Growth experiments using glucose as the sole carbon source showed that GLY1 is more important for glycine biosynthesis than SHM1 and SHM2 encoding alternative serine hydroxymethyltransferases. On ethanol as carbon source simultaneous disruption of GLY1, SHM1 and SHM2 did not lead to glycine auxotrophy because glycine biosynthesis proceeds via alanine glyoxylate aminotransferase.

Structural properties of native and sonicated cinerean, a beta- (1-->3) (1-->6)-D-glucan produced by Botrytis cinerea.

Cinerean, the extracellular beta-(1-->3) (1-->6)-D-glucan of the fungus Botrytis cinerea was studied. Electron micrographs of the native polysaccharide revealed quasi-endless fibrils with an estimated diameter of ca. 1.5 nm. A particle mass of 10(9)-10(10) daltons was determined from dilute solutions by low-angle laser light scattering. Sonication of increasing duration led to fragmentation of the native polymer with an approximately exponential decrease of mass in the range of average molecular masses between 250,000 and 50,000 daltons. Shadowed by platinum, cinerean fibril fragments with a weight-average molecular mass of 172,000 +/- 3000 daltons could be characterized from electron micrographs as a distribution of rods of most probable length of 45 nm and an average length of 72 nm. Small-angle X-ray scattering confirmed the fibrillar structure of the native cinerean and the rodlike structure of sonicated cinerean. A rod diameter of 1.9 +/- 0.2 nm and a mass per unit length of 2250 +/- 490 daltons/nm were found. The latter is in agreement with the value of 1830 daltons/nm calculated from the length distribution determined from the electron micrographs. These data-especially the mass per unit length-suggest a quaternary structure for the polysaccharide. Such a structure would explain the rigidity of the rods which, in turn, is responsible for the characteristic phase separation behaviour in aqueous solutions observed by nephelometry and viscometry.