Maria A. Santos

Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii

BackgroundPhosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability.ResultsHere we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C). We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Δagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains.ConclusionIt is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

Physiological Consequence of Disruption of the VMA1Gene in the Riboflavin Overproducer Ashbya gossypii

The vacuolar ATPase subunit A structural geneVMA1 of the biotechnologically important riboflavin overproducer Ashbya gossypii was cloned and disrupted to prevent riboflavin retention in the vacuolar compartment and to redirect the riboflavin flux into the medium. Cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequences of the Vma1 proteins from yeast and filamentous fungi. The deduced polypeptide comprises 617 amino acids with a calculated molecular mass of 67.8 kDa. The deduced amino acid sequence is highly similar to that of the catalytic subunits ofSaccharomyces cerevisiae (67 kDa), Candida tropicalis (67 kDa), and Neurospora crassa (67 kDa) with 89, 87, and 60% identity, respectively, and shows about 25% identity to the β-subunit of the FoF1-ATPase of S. cerevisiae and Schizosaccharomyces pombe. In contrast to S. cerevisiae, however, where disruption of the VMA1 gene was conditionally lethal, and to N. crassa, where viable disruptants could not be isolated, disruption of the VMA1 gene in A. gossypii did not cause a lethal phenotype. Disruption of the AgVMA1 gene led to complete excretion of riboflavin into the medium instead of retention in the vacuolar compartment, as observed in the wild type.

The Saccharomyces cerevisiae RIB4 Gene Codes for 6,7-Dimethyl- 8-ribityllumazine Synthase Involved in Riboflavin Biosynthesis MOLECULAR CHARACTERIZATION OF THE GENE AND PURIFICATION OF THE ENCODED PROTEIN

6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic precursor of riboflavin, is synthesized by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate. The gene coding for 6,7-dimethyl-8-ribityllumazine synthase in Saccharomyces cerevisiae (RIB4) has been cloned by functional complementation of a mutant accumulating 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which can grow on riboflavin- or diacetyl- but not on 3,4-dihydroxy-2-butanone-supplemented media. Gene disruption of the chromosomal copy of RIB4 led to riboflavin auxotrophy and loss of enzyme activity. Nucleotide sequencing revealed a 169-base pair open reading frame encoding a 18.6-kDa protein. Hybridization analysis indicated that RIB4 is a single copy gene located on the left arm of chromosome XV. Overexpression of the RIB4 coding sequence in yeast cells under the control of the strong TEF1 promoter allowed ready purification of 6,7-dimethyl-8-ribityllumazine synthase to apparent homogeneity by a simple procedure. Initial structural characterization of 6,7-dimethyl-8-ribityllumazine synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a pentamer of identical 16.8-kDa subunits. The derived amino acid sequence of RIB4 shows extensive homology to the sequences of the β subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes.

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

Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM 2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzymes specific activity revealed a reduction from 3 m-units/mg of protein to 0.5 m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM 1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM 2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from (13)C-labelling experiments. When 500 microM 99% [1-(13)C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM 1 had no detectable effect on serine labelling, but disruption of SHM 2 led to a decrease in serine (2-5%) and an increase in glycine (59-67%) labelling, indicating a changed carbon flux.

Strain design of Ashbya gossypii for single-cell oil production

ABSTRACT Single-cell oil (SCO) represents a sustainable alternative for the oil industry. Accordingly, the identification of microorganisms with either higher lipidogenic ability or novel capacities for the transformation of raw materials constitutes a major challenge for the field of oil biotechnology. With this in mind, here, we were prompted to address the lipidogenic profile of the filamentous hemiascomycete Ashbya gossypii, which is currently used for the microbial production of vitamins. We found that A. gossypii mostly accumulates unsaturated fatty acids (FAs), with more than 50% of the total FA content corresponding to oleic acid. In addition, we engineered A. gossypii strains both lacking the beta-oxidation pathway and also providing ATP-citrate lyase (ACL) activity to block the degradation of FA and to increase the cytosolic acetyl-coenzyme A (CoA) content, respectively. The lipidogenic profile of the newly developed strains demonstrates that the mere elimination of the beta-oxidation pathway in A. gossypii triggers a significant increase in lipid accumulation that can reach 70% of cell dry weight. The use of A. gossypii as a novel and robust tool for the production of added-value oils is further discussed.

Insertional Mutagenesis in the Vitamin B 2 Producer Fungus Ashbya gossypii

The ascomycete Ashbya gossypii, a filamentous fungus, is a natural overproducer of vitamin B2 and is currently exploited for the industrial production of this vitamin. Classical mutagenesis and selection of mutants showing improved production capacities have been routinely applied with success to the development of industrial strains of A. gossypii that overproduce vitamin B2. However, this approach does not allow the subsequent isolation and identification of affected genes in organisms such as A. gossypii that lack a sexual life cycle, thus hampering further improvements of the strains on the basis of rational strategies derived from knowledge of the function of the genes involved in the process. Here, we describe an efficient in vitro Himar1based transposition reaction of minitransposon elements on chromosomal A. gossypii DNA, which is subsequently reintroduced into A. gossypii by electrotransformation. Sequencing of the minitransposon insertion point in numerous transposition mutants and a limited screening of the insertional mutants demonstrate that this method can lead to the exhaustive mutagenesis of A. gossypii, allowing—for the first time—a genomic-scale mutational analysis of this biotechnologically important fungus.

Mapping of the RIB5 gene in Saccharomyces cerevisiae using UV light as an enhancer of rad52-mediated chromosome loss

SummaryRibs mutants of S. cerevisiae are blocked at the end of the riboflavin biosynthetic pathway. Using UV light to increase rad52-mediated chromosome loss, we have assigned the rib5 mutation to chromosome II. Tetrad analysis of crosses between rib5 and other markers on chromosome II shows that the RIB5 gene is located on the right arm of this chromosome, closely linked to HIS7.

Microbial production of vitamins

Abstract: This chapter is a short review of the production of vitamins with the main focus on the microbial fermentation processes that are currently being employed in the production of vitamins. The state of the art of the industry of vitamins is extensively analysed with a description of both chemical and biotechnological systems that have been developed for the production of each vitamin. Finally, future trends in the microbial production of vitamins are analysed and the recent advances in strain design for whole-cell production of vitamins are discussed.

Disruption of six unknown open reading frames from Saccharomyces cerevisiae reveals two genes involved in vacuolar morphogenesis and one gene required for sporulation

In this report we describe the construction and basic phenotypic analysis of deletion mutants in six open reading frames (ORFs) of unknown function from the yeast Saccharomyces cerevisiae. Using the dominant kanMX marker and polymerase chain reaction (PCR) methods, deletion cassettes were constructed for five ORFs (YNL099c, YNL100w, YNL101w, YNL106c and YNL242w ) located on chromosome XIV and one ORF (YOR109w ) located on chomosome XV. The recovery of viable haploid deletant strains among the meiotic products of heterozygous deletants for each ORF demonstrated that none of the analysed ORFs was essential. With the exception of YNL242w, no alterations in growth characteristics or mating and sporulation efficiencies associated with deletion of the ORFs were observed. Homozygous diploid ynl242wΔ cells obtained in three different genetic backgrounds were unable to sporulate, indicating that the product of this ORF is required for sporulation. Complementation of the sporulation defect by the cognate gene clone confirmed this observation. YNL106c and YOR109w are very similar and show strong sequence homology with a mammalian phosphatidylinositol‐phosphate 5‐phosphatase, synaptojanin, known to be involved in synaptic vesicle cycling. Strains bearing single and double deletions of YNL106c and YOR109w were seen to display abnormal vacuolar morphologies of varying degrees. Complementation tests indicated that YNL106c and YOR109w are redundant genes. Copyright