The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid 310 , lysine 333 , and alanine 417 ) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter).

Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing epsilon-(gamma-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIalpha) at the expense of centers located mainly in stroma thylakoids (PSIIbeta). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.

Ashbya gossypii is a natural riboflavin overproducer used in the industrial production of the vitamin. We have isolated an insertional mutant exhibiting higher levels of riboflavin production than the wild type. DNA analysis of the targeted locus in the mutant strain revealed that a syntenic homolog of the Saccharomyces cerevisiae BAS1 gene, a member of the Myb family of transcription factors, was inactivated. Directed gene disruption of AgBAS1 confirmed the phenotype observed for the insertional mutant, and the ⌬bas1 mutant also showed auxotrophy for adenine and several growth defects, such as a delay in the germination of the spores and an abnormally prolonged trophic phase. Additionally, we demonstrate that the DNA-binding domain of AgBas1p is able to bind to the Bas1-binding motifs in the AgADE4 promoter; we also show a clear nuclear localization of a green fluorescent protein-Bas1 fusion protein. Real-time quantitative PCR analyses comparing the wild type and the ⌬bas1 mutant revealed that AgBAS1 was responsible for the adenine-mediated regulation of the purine and glycine pathways, since the transcription of the ADE4 and SHM2 genes was virtually abolished in the ⌬bas1 mutant. Furthermore, the transcription of ADE4 and SHM2 in the ⌬bas1 mutant did not diminish during the transition from the trophic to the productive phase did not diminish, in contrast to what occurred in the wild-type strain. A C-terminal deletion in the AgBAS1 gene, comprising a hypothetical regulatory domain, caused constitutive activation of the purine and glycine pathways, enhanced riboflavin overproduction, and prolonged the trophic phase. Taking these results together, we propose that in A. gossypii, AgBAS1 is an important transcription factor that is involved in the regulation of different physiological processes, such as purine and glycine biosynthesis, riboflavin overproduction, and growth.

Riboflavin (vitamin B 2 ) serves as a precursor of the flavinnucleotide cofactors riboflavin monophosphate (FMN) 1 and flavin adenine dinucleotide (FAD). The formation of FAD depends on the sequential utilization of two molecules of ATP in reactions that first involve the riboflavin (flavokinase, EC 2.7.1.26) kinase-phosphorylation of riboflavin to form FMN and then FAD (EC 2.7.7.2) synthetase-catalyzed adenylylation of the latter to form FAD. The enzymes responsible for catalyzing the two steps have been purified from several sources (1-9). In Corynebacterium ammoniagenes and Bacillus subtilis, flavokinase and FAD synthetase co-purify and are present in a single, bifunctional flavokinase/FAD synthetase enzyme (7, 10). To date, a large number of homologs to the bifunctional flavokinase/FAD synthetase ribC gene of B. subtilis have been identified in archaea and eubacteria, indicating that this type of gene organization is common in prokaryotes. In contrast, both enzymatic activities have been purified separately in eukaryotic organisms (1,6,8). In mammalian tissues, the smaller flavokinase (1) is readily separable from the larger FAD synthetase (3). In the yeast Saccharomyces cerevisiae, the gene encoding a monofunctional FAD synthetase (FAD1) has recently been cloned as an extragenic suppressor of a respiratory deficient pet mutant (11). However, the gene encoding flavokinase in yeast as well as in the rest of eukaryotic organisms remains to be identified.The formation of holo-flavoproteins, by binding of the coenzyme to the apo-protein, depends on the availability of the cognate flavocoenzyme. It is known that most cell flavoproteins are located in the mitochondria but little is known about the subcellular distribution of the enzymes involved in flavin metabolism. It has been described that flavokinase is a cytosolic enzyme in plant and mammalian tissues (4, 6) and the synthesis of FMN and FAD occurs in hepatocyte cytosol (12). However, the synthesis of both flavocoenzymes from externally added riboflavin by isolated mitochondria from rat liver has been also reported (13). Whereas it has been determined that the synthesis of riboflavin occurs in the cytosol of S. cerevisiae cells (14, 15), the exact location of the synthesis of FMN and FAD and their compartmentation is controversial in this organism. It has been reported that yeast mitochondria do not contain FAD synthetase and that a specific mitochondrial carrier protein, Flx1p, is involved in flavin transport across the mitochondrial membrane (16). However, it has also been described that FMN and FAD synthesis occurs in isolated yeast mitochondria (17) and that yeast cells overexpressing the FAD1 gene show mitochondrial FAD synthetase activity (11).Homeostasis of flavin coenzymes is achieved by an intricate interplay of the different enzymes in the pathway responding to regulators (18), by organelle compartmentation (11,19), and by altered susceptibility of holo-and apo-flavoproteins to proteolytic digestion (20). Since yeast and other eukaryotes share the...