Involvement of a flavoprotein, acetohydroxyacid synthase, in growth and riboflavin production in riboflavin-overproducing Ashbya gossypii mutant.

BACKGROUND: Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria. RESULTS: In the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain. CONCLUSION: The significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii.

Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii.

The blockage of the de novo pyrimidine biosynthetic pathway at the orotidine-5'-phosphate decarboxylase level was previously demonstrated to affect riboflavin production in the industrial producer fungus Ashbya gossypii. However, the molecular basis for the unusual sensitivity to uracil displayed by the pyrimidine auxotroph A. gossypii Agura3 was unknown. Here, uridine was shown to be the only intermediate of the pyrimidine salvage pathway able to fully restore this mutant's growth. Conversely, uracil, which is routinely used to rescue pyrimidine auxotrophs, had a dose-dependent growth-inhibitory effect. Uracil phosphoribosyltransferase (UPRT) is the pyrimidine salvage pathway enzyme responsible for converting uracil to uridine monophosphate in the presence of phosphoribosyl pyrophosphate (PRPP). Characterization of the A. gossypii UPRT, as produced and purified from Escherichia coli, revealed that uracil concentrations above 1 mM negatively affected its activity, thus explaining the hypersensitivity of the Agura3 mutant to uracil. Accordingly, overexpression of the AgUPRT encoding-gene in A. gossypii Agura3 led to similar growth on rich medium containing 5 mM uracil or uridine. Decreased UPRT activity ultimately favors the preservation of PRPP, which otherwise may be directed to other pathways. In A. gossypii, increased PRPP availability promotes overproduction of riboflavin. Thus, this UPRT modulation mechanism reveals a putative means of saving precursors essential for riboflavin overproduction by this fungus. A similar uracil-mediated regulation mechanism of the UPRT activity is reported only in two protozoan parasites, whose survival depends on the availability of PRPP. Physiological evidence here discussed indicate that it may be extended to other distantly related flavinogenic fungi.

An Arf-GAP promotes endocytosis and hyphal growth of Ashbya gossypii.

The ADP-ribosylation factor (ARF) family of GTPases are highly conserved from yeast to human and regulate vesicle budding. Sec7 domain containing proteins stimulate the guanine nucleotide exchange on Arf proteins, while ARF-GTPase activating proteins stimulate the hydrolysis of GTP. Since vesicle trafficking is important for hyphal growth, we studied the Ashbya gossypii homolog of Saccharomyces cerevisiae ARF3 along with its putative GEF and GTPase-activating protein (GAP) encoded by YEL1 and GTS1, respectively. Deletion of YEL1 had no discernible phenotype and deletion of ARF3 had only a minor defect in vacuolar fusion. In contrast, deletion of GTS1 severely impaired hyphal growth, and mutants showed defects in the maintenance of polarity and the localization of cortical actin patches. The uptake of the lipophilic dye FM4-64 was delayed in gts1 hyphae, indicating a defect in endocytosis. Gts1 has several protein domains, of which the Arf-GAP domain is required for complementation of the gts1 mutant phenotype. GFP-tagged GTS1 under control of its endogenous promoter localized to the plasma membrane but was enriched at hyphal tips and septal sites corresponding to a role in polarized vesicle trafficking. Our results indicate that this ARF-GTPase module plays an important role for filamentous hyphal growth.

Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes.

The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 Å resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes. In contrast to the suggestions by earlier in vivo studies of mitochondrial processing, we found that these enzymes are genuine Xaa-Pro aminopeptidases, which hydrolyze peptides with proline at the second position (P1'). The mitochondrial processing activity involving cleavage of peptides lacking P1' proline was also detected in the purified enzymes. A wide proline pocket as well as molecular complementarity and capping at the S1 substrate site of XPNPEP3 provide the necessary structural features for processing the mitochondrial substrates. However, this activity was found to be significantly lower as compared with Xaa-Pro aminopeptidase activity. Because of similar activity profiles of Icp55 and XPNPEP3, we propose that XPNPEP3 plays the same mitochondrial role in humans as Icp55 does in yeast. Both Xaa-Pro aminopeptidase and mitochondrial processing activities of XPNPEP3 have implications toward mitochondrial fitness and cystic kidney disease. Furthermore, the presence of both these activities in Icp55 elucidates the unexplained processing of the mitochondrial cysteine desulfurase Nfs1 in yeast. The enzymatic and structural analyses reported here provide a valuable molecular framework for understanding the diverse cellular roles of XPNPEP3.

Increased riboflavin production by manipulation of inosine 5'-monophosphate dehydrogenase in Ashbya gossypii.

Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Herein, we report the functional and structural characterization of the IMPDH enzyme from the industrial fungus Ashbya gossypii. Our data show that the overexpression of the IMPDH gene increases the metabolic flux through the guanine pathway and ultimately enhances 40 % riboflavin production with respect to the wild type. Also, IMPDH disruption results in a 100-fold increase of inosine excretion to the culture media. Our results contribute to the developing metabolic engineering toolbox aiming at improving the production of metabolites with biotechnological interest in A. gossypii.

Developmental Growth Control Exerted via the Protein A Kinase Tpk2 in Ashbya gossypii.

Sporulation in Ashbya gossypii is induced by nutrient-limited conditions and leads to the formation of haploid spores. Using RNA-seq, we have determined a gene set induced upon sporulation, which bears considerable overlap with that of Saccharomyces cerevisiae but also contains A. gossypii-specific genes. Addition of cyclic AMP (cAMP) to nutrient-limited media blocks sporulation and represses the induction of sporulation specific genes. Deletion of the protein kinase A (PKA) catalytic subunits encoded by TPK1 and TPK2 showed reduced growth in tpk1 but enhanced growth in the tpk2 strain; however, both mutants sporulated well. Sporulation can be blocked by cAMP in tpk1 but not in tpk2 strains. Similarly, TPK2 acts at a second developmental switch promoting the break in spore dormancy. In S. cerevisiae, PKA phosphorylates and inhibits Msn2/4. The transcript profiles of the tpk1 and msn2/4 mutants were very similar to that of the wild type under sporulation conditions. However, deletion of the single A. gossypii MSN2/4 homolog generated a specific sporulation defect. We identified a set of genes involved in spore wall assembly that was downregulated in the msn2/4 mutant, particularly DIT2, suggesting that poor spore viability may be due to lysis of spores. Our results reveal specific functional differences between the two catalytic PKA subunits in A. gossypii and identified Tpk2 as the key A kinase that transduces developmental decisions of growth. Our data also suggest that Msn2/4 is involved only at a late step of sporulation in A. gossypii and is not a major regulator of IME1.

Increased production of inosine and guanosine by means of metabolic engineering of the purine pathway in Ashbya gossypii.

BACKGROUND: Inosine and guanosine monophosphate nucleotides are convenient sources of the umami flavor, with attributed beneficial health effects that have renewed commercial interest in nucleotide fermentations. Accordingly, several bacterial strains that excrete high levels of inosine and guanosine nucleosides are currently used in the food industry for this purpose. RESULTS: In the present study, we show that the filamentous fungus Ashbya gossypii, a natural riboflavin overproducer, excretes high amounts of inosine and guanosine nucleosides to the culture medium. Following a rational metabolic engineering approach of the de novo purine nucleotide biosynthetic pathway, we increased the excreted levels of inosine up to 27-fold. CONCLUSIONS: We generated Ashbya gossypii strains with improved production titers of inosine and guanosine. Our results point to Ashbya gossypii as the first eukaryotic microorganism representing a promising candidate, susceptible to further manipulation, for industrial nucleoside fermentation.

Genome-wide metabolic re-annotation of Ashbya gossypii: new insights into its metabolism through a comparative analysis with Saccharomyces cerevisiae and Kluyveromyces lactis.

BACKGROUND: Ashbya gossypii is an industrially relevant microorganism traditionally used for riboflavin production. Despite the high gene homology and gene order conservation comparatively with Saccharomyces cerevisiae, it presents a lower level of genomic complexity. Its type of growth, placing it among filamentous fungi, questions how close it really is from the budding yeast, namely in terms of metabolism, therefore raising the need for an extensive and thorough study of its entire metabolism. This work reports the first manual enzymatic genome-wide re-annotation of A. gossypii as well as the first annotation of membrane transport proteins. RESULTS: After applying a developed enzymatic re-annotation pipeline, 847 genes were assigned with metabolic functions. Comparatively to KEGG's annotation, these data corrected the function for 14% of the common genes and increased the information for 52 genes, either completing existing partial EC numbers or adding new ones. Furthermore, 22 unreported enzymatic functions were found, corresponding to a significant increase in the knowledge of the metabolism of this organism. The information retrieved from the metabolic re-annotation and transport annotation was used for a comprehensive analysis of A. gossypii's metabolism in comparison to the one of S. cerevisiae (post-WGD - whole genome duplication) and Kluyveromyces lactis (pre-WGD), suggesting some relevant differences in several parts of their metabolism, with the majority being found for the metabolism of purines, pyrimidines, nitrogen and lipids. A considerable number of enzymes were found exclusively in A. gossypii comparatively with K. lactis (90) and S. cerevisiae (13). In a similar way, 176 and 123 enzymatic functions were absent on A. gossypii comparatively to K. lactis and S. cerevisiae, respectively, confirming some of the well-known phenotypes of this organism. CONCLUSIONS: This high quality metabolic re-annotation, together with the first membrane transporters annotation and the metabolic comparative analysis, represents a new important tool for the study and better understanding of A. gossypii's metabolism.

Tuning single-cell oil production in Ashbya gossypii by engineering the elongation and desaturation systems.

Microbial oils represent a sustainable alternative to vegetable oils and animal fats as feedstock for both the chemical and biofuel industries. The applications of microbial oils depend on their fatty acid composition, which is defined by the relative amount of each fatty acid, also considering the length and unsaturations of the acyl chain. These two properties are determined by elongases and desaturases. In the present study, we characterized the elongase and desaturase systems in the filamentous fungus Ashbya gossypii, which is able to accumulate high amounts of lipids. Additionally, both the elongation and desaturation systems were engineered in order to broaden the potential applications of A. gossypii oils. Finally, the properties of the strains engineered for biodiesel production were analyzed, with the observation that A. gossypii is a good candidate for the microbial production of renewable biofuels.

Molecular and functional characterization of an invertase secreted by Ashbya gossypii.

The repertoire of hydrolytic enzymes natively secreted by the filamentous fungus Ashbya (Eremothecium) gossypii has been poorly explored. Here, an invertase secreted by this flavinogenic fungus was for the first time molecularly and functionally characterized. Invertase activity was detected in A. gossypii culture supernatants and cell-associated fractions. Extracellular invertase migrated in a native polyacrylamide gel as diffuse protein bands, indicating the occurrence of at least two invertase isoforms. Hydrolytic activity toward sucrose was approximately 10 times higher than toward raffinose. Inulin and levan were not hydrolyzed. Production of invertase by A. gossypii was repressed by the presence of glucose in the culture medium. The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is highly homologous to the Saccharomyces cerevisiae SUC2 (ScSUC2) gene. Agsuc2 null mutants were unable to hydrolyze sucrose, proving that invertase is encoded by a single gene in A. gossypii. This mutation was functionally complemented by the ScSUC2 and AgSUC2 genes, when expressed from a 2-µm-plasmid. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii.

Functional analysis of cis-aconitate decarboxylase and trans-aconitate metabolism in riboflavin-producing filamentous Ashbya gossypii.

In Ashbya gossypii, isocitrate lyase (ICL1) is a very crucial enzyme for riboflavin production. Itaconate, the inhibitor of ICL1, has been used as an antimetabolite for mutagenic studies in A. gossypii. It has been reported that itaconate is produced from cis-aconitate by cis-aconitate decarboxylase (CAD1) in Aspergillus terreus. In this study, identification of CAD1 gene and determination of the presence of itaconate in the riboflavin biosynthetic pathway in A. gossypii were carried out to confirm itaconate metabolism. Although no CAD1 candidate gene was found and no itaconate production was observed, cis- and trans-aconitate were detected in the riboflavin production phase. It is known that trans-aconitate inhibits aconitase (ACO1) in the tricarboxylic acid cycle. In A. gossypii, the transcription level of AGR110Wp, the homolog of trans-aconitate 3-methyltransferase (TMT1), was enhanced by almost threefold during riboflavin production than that during its growth phase. TMT1 catalyzes the methylation reaction of trans-aconitate in Saccharomyces cerevisiae. Thus, these results suggest that the enhancement of the transcription level of this TMT1 homolog decreases the trans-aconitate level, which may mitigate the inhibition of ACO1 by oxidative stress in the riboflavin biosynthetic pathway in A. gossypii. This is a novel finding in A. gossypii, which may open new metabolic engineering ideas for improving riboflavin productivity.

Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria.

The discovery of diverse codon reassignment events has demonstrated that the canonical genetic code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted tRNA(Ala)UAG is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that tRNA(Ala)UAG is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase (AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNA(Ala); a G3A change in Ashbya tRNA(Ala)UAG abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae tRNA(Thr)UAG confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.

Development of fluorescent reporter tagged RIB gene cassettes for replicative transformation, early expression, and enhanced riboflavin production in Eremothecium ashbyi.

Eremothecium ashbyi is a riboflavin overproducing filamentous fungus in which the metabolic pathways have not been genetically characterized. Two genes of the riboflavin biosynthetic (RIB) pathway, RIB1 and RIB3, which encode GTP-cyclohydrolase II (GCH II) and 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase respectively, were selected for the present study. The two RIB genes under their native promoters were obtained from Ashbya gossypii genomic library. Yeast enhanced green fluorescent protein (yEGFP) and mCherry genes were tagged to the C-terminal ends of RIB1 and RIB3 genes to analyse the functionality of the RIB transgenes in E. ashbyi. Shuttle vectors with the reporter tagged RIB genes contained the Escherichia coli kan(R) gene and Saccharomyces cerevisiae ARS element. On transformation with these plasmids, the ARS element was found to be functional in E. ashbyi. The E. ashbyi transcription factors could recognize the Ashbya RIB gene promoters and express the reporter tagged RIB genes as cytoplasmic proteins, in early cell development. Replicative transformants carrying RIB1-mCherry plasmids showed 2.95 times more GCH II activity and 2.44 times more riboflavin production when compared to untransformed. This is the first report of genetic transformation of E. ashbyi and is of significance as the first step towards genetic engineering of this genus.

The Ashbya gossypii EF-1α promoter of the ubiquitously used MX cassettes is toxic to Saccharomyces cerevisiae.

Protein overexpression based on introduction of multiple gene copies is well established. To improve purification or quantification, proteins are typically fused to peptide tags. In Saccharomyces cerevisiae, this has been hampered by multicopy toxicity of the TAP and GFP cassettes used in the global strain collections. Here, we show that this effect is due to the EF-1α promoter in the HIS3MX marker cassette rather than the tags per se. This promoter is frequently used in heterologous marker cassettes, including HIS3MX, KanMX, NatMX, PatMX and HphMX. Toxicity could be eliminated by promoter replacement or exclusion of the marker cassette. To our knowledge, this is the first report of toxicity caused by introduction of a heterologous promoter alone.

Sequence analysis and structural characterization of a glyceraldehyde-3-phosphate dehydrogenase gene from the phytopathogenic fungus Eremothecium ashbyi.

Eremothecium ashbyi is a phytopathogenic fungus infesting cotton, soybeans and several other plants. This highly flavinogenic fungus has been phylogenetically characterized, but the genetic aspects of its central metabolic and riboflavin biosynthetic pathways are unknown. An ORF of 996 bp was obtained from E. ashbyi by using degenerate primers for glyceraldehyde-3-phosphate dehydrogenase (GPD) through reverse transcriptase polymerase chain reaction (RT-PCR) and 5'-3' rapid amplification of cDNA ends (RACE-PCR). This nucleotide sequence had a high similarity of 88% with GPD sequence of Ashbya gossypii. The putative GPD peptide of 331-aa had a high similarity of 85% with the GPD sequence from other ascomycetes. The ORF had an unusually strong codon bias with 5 amino acids showing strict preference of a single codon. The theoretical molecular weight for the putative peptide was 35.58 kDa with an estimated pI of 5.7. A neighbor-joining tree showed that the putative peptide from E. ashbyi displayed the highest similarity to GPD of A. gossypii. The gene sequence is available at the GenBank, accession number EU717696. Homology modeling done with Kluyveromyces marxianus GPD (PDB: 2I5P) as template indicated high structural similarity.

Molecular and structural characterization of GTP-cyclohydrolase II in Eremothecium ashbyi NRRL Y-1363: cDNA cloning, comparative sequence analysis and molecular modeling.

GTP-cyclohydrolase II (GCH II) encoded by RIB1 gene catalyzes the first committed step in the riboflavin biosynthetic pathway. We report here the cloning and characterization of the entire RIB1 ORF (EaRIB1) of 942bp by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE-PCR) in Eremothecium ashbyi where it was found to be present as a single-copy gene. EaRIB1 sequence is available at the GenBank Accession Number EF565374. The putative peptide of 313-aa has a high similarity of 60-70% with GCH II sequences from other ascomycete fungi. Gene expression and alignment studies confirmed the functional annotation of this gene. Homology model was developed with Escherichia coli (PDB 2BZ1) as template to identify the catalytic domains and to explore its functional architecture. We report here the first three-dimensional model of any fungal GCH II which due to its absence in humans assumes significance for anti-fungal drug targeting.

Importance of malate synthase in the glyoxylate cycle of Ashbya gossypii for the efficient production of riboflavin.

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.

Regulation of distinct septin rings in a single cell by Elm1p and Gin4p kinases.

Septins are conserved, GTP-binding proteins that assemble into higher order structures, including filaments and rings with varied cellular functions. Using four-dimensional quantitative fluorescence microscopy of Ashbya gossypii fungal cells, we show that septins can assemble into morphologically distinct classes of rings that vary in dimensions, intensities, and positions within a single cell. Notably, these different classes coexist and persist for extended times, similar in appearance and behavior to septins in mammalian neurons and cultured cells. We demonstrate that new septin proteins can add through time to assembled rings, indicating that septins may continue to polymerize during ring maturation. Different classes of rings do not arise from the presence or absence of specific septin subunits and ring maintenance does not require the actin and microtubule cytoskeletons. Instead, morphological and behavioral differences in the rings require the Elm1p and Gin4p kinases. This work demonstrates that distinct higher order septin structures form within one cell because of the action of specific kinases.

An Ashbya gossypii cts2 mutant deficient in a sporulation-specific chitinase can be complemented by Candida albicans CHT4.

The Candida albicans genome encodes four chitinases, CHT1, CHT2, CHT3 and CHT4. All four C. albicans chitinase-encoding genes are non-essential. The corresponding proteins belong to two groups in which Cht1, Cht2 and Cht3 are more similar to Saccharomyces cerevisiae Cts1, while Cht4 is more similar to ScCts2. In the filamentous fungus Ashbya gossypii, a CTS2 homolog (ACL166w) was identified as the sole chitinase gene. The AgCts2 is 490 aa in Length and shows 42.3% overall identity to ScCts2 (511 aa) and 33.2% identity to CaCht4 (388 aa). The A. gossypii cts2 deletion mutant showed no growth retardation or vegetative morphogenetic defects. However, upon sporulation Agcts2 mutants revealed a defect in spore formation. Expression of AgCts2 using a lacZ reporter gene was only found in the centre of a mycelium corresponding to the sporogenous part of a colony. The mutant spore phenotype of Agcts2 could be complemented by either AgCTS2, the S. cerevisiae CTS2, or the C. albicans CHT4 gene when expressed by either the AgCTS2 or the AgTEF1 promoter.