Functional characterization of a xylose transporter in Aspergillus nidulans

BACKGROUND: The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization. RESULTS: In this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene. CONCLUSIONS: This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.We would like to thank the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) for providing financial support. We also thank Dr Eckardt Boles for providing the EBY.VW4000 yeast strain, Dr Ronald Hector for providing the plasmids pRH274 and pRH195, Dr Michel Flipphi for providing the Delta creA4 strain, and the two anonymous reviewers for their comments and suggestions. We also acknowledge the Program project grant GM068087 (PI Jay Dunlap) for providing the deletion cassettes

Aspergillus nidulans protein kinase A plays an important role in cellulase production

Abstract\ud \ud Background\ud The production of bioethanol from lignocellulosic feedstocks is dependent on lignocellulosic biomass degradation by hydrolytic enzymes. The main component of lignocellulose is cellulose and different types of organisms are able to secrete cellulases. The filamentous fungus Aspergillus nidulans serves as a model organism to study cellulase production and the available tools allow exploring more in depth the mechanisms governing cellulase production and carbon catabolite repression.\ud \ud \ud Results\ud In A. nidulans, microarray data identified the cAMP-dependent protein kinase A (PkaA) as being involved in the transcriptional modulation and the production of lignocellulolytic enzymes in the presence of cellulose. Deletion of pkaA resulted in increased hydrolytic enzyme secretion, but reduced growth in the presence of lignocellulosic components and various other carbon sources. Furthermore, genes involved in fungal development were increased in the ΔpkaA strain, probably leading to the increased hyphal branching as was observed in this strain. This would allow the secretion of higher amounts of proteins. In addition, the expression of SynA, encoding a V-SNARE synaptobrevin protein involved in secretion, was increased in the ΔpkaA mutant. Deletion of pkaA also resulted in the reduced nuclear localization of the carbon catabolite repressor CreA in the presence of glucose and in partial de-repression when grown on cellulose. PkaA is involved in the glucose signaling pathway as the absence of this protein resulted in reduced glucose uptake and lower hexokinase/glucokinase activity, directing the cell to starvation conditions. Genome-wide transcriptomics showed that the expression of genes encoding proteins involved in fatty acid metabolism, mitochondrial function and in the use of cell storages was increased.\ud \ud \ud Conclusions\ud This study shows that PkaA is involved in hydrolytic enzyme production in A. nidulans. It appears that this protein kinase blocks the glucose pathway, hence forcing the cell to change to starvation conditions, increasing hydrolytic enzyme secretion and inducing the usage of cellular storages. This work uncovered new regulatory avenues governing the tight interplay between the metabolic states of the cell, which are important for the production of hydrolytic enzymes targeting lignocellulosic biomass. Deletion of pkaA resulted in a strain with increased hydrolytic enzyme secretion and reduced biomass formation.We would like to thank the Fundação de Amparo a Pesquisa do Estado de\ud São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e\ud Tecnológico (CNPq), Brazil, for providing financial support. We also thank the\ud two reviewers and the editor for their suggestions and comments

Mitogen activated kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation

We would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for providing financial support. LW and CM would like to acknowledge support from the University of Aberdeen and the MRC Centre for Medical Mycology (MR/N006364/1). All authors declare no financial conflict of interest.Peer reviewedPostprin

The low affinity glucose transporter HxtB is also involved in glucose signalling and metabolism in Aspergillus nidulans

One of the drawbacks during second-generation biofuel production from plant lignocellulosic biomass is the accumulation of glucose, the preferred carbon source of microorganisms, which causes the repression of hydrolytic enzyme secretion by industrially relevant filamentous fungi. Glucose sensing, subsequent transport and cellular signalling pathways have been barely elucidated in these organisms. This study therefore characterized the transcriptional response of the filamentous fungus Aspergillus nidulans to the presence of high and low glucose concentrations under continuous chemostat cultivation with the aim to identify novel factors involved in glucose sensing and signalling. Several transcription factor- and transporter-encoding genes were identified as being differentially regulated, including the previously characterized glucose and xylose transporter HxtB. HxtB was confirmed to be a low affinity glucose transporter, localizing to the plasma membrane under low- and high-glucose conditions. Furthermore, HxtB was shown to be involved in conidiation-related processes and may play a role in downstream glucose signalling. A gene predicted to encode the protein kinase PskA was also identified as being important for glucose metabolism. This study identified several proteins with predicted roles in glucose metabolic processes and provides a foundation for further investigation into the response of biotechnologically important filamentous fungi to glucose

The influence of genetic stability on Aspergillus fumigatus virulence and azole resistance

Genetic stability is extremely important for the survival of every living organism, and a very complex set of genes has evolved to cope with DNA repair upon DNA damage. Here, we investigated the Aspergillus fumigatus AtmA (Ataxia-telangiectasia mutated, ATM) and AtrA kinases, and how they impact virulence and the evolution of azole resistance. We demonstrated that A. fumigatus atmA and atrA null mutants are haploid and have a discrete chromosomal polymorphism. The ?atmA and ?atrA strains are sensitive to several DNA-damaging agents, but surprisingly both strains were more resistant than the wild-type strain to paraquat, menadione, and hydrogen peroxide. The atmA and atrA genes showed synthetic lethality emphasizing the cooperation between both enzymes and their consequent redundancy. The lack of atmA and atrA does not cause any significant virulence reduction in A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonela. Wild-type, ?atmA, and ?atrA populations that were previously transferred 10 times in minimal medium (MM) in the absence of voriconazole have not shown any significant changes in drug resistance acquisition. In contrast, ?atmA and ?atrA populations that similarly evolved in the presence of a subinhibitory concentration of voriconazole showed an ~5-10-fold increase when compared to the original minimal inhibitory concentration (MIC) values. There are discrete alterations in the voriconazole target Cyp51A/Erg11A or cyp51/erg11 and/or Cdr1B efflux transporter overexpression that do not seem to be the main mechanisms to explain voriconazole resistance in these evolved populations. Taken together, these results suggest that genetic instability caused by ?atmA and ?atrA mutations can confer an adaptive advantage, mainly in the intensity of voriconazole resistance acquisition.We thank the Fundação de Amparo a Pesquisa do Estado de São Paulo and the Conselho Nacional de Desenvolvimento Cientí fi co e Tecnológico, Brazil, for fi nancial support. F.R. and B.H.F. were supported by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (NORTE-01-0145-FEDER-000013)info:eu-repo/semantics/publishedVersio

Novel homologous lactate transporter improves l‑lactic acid production from glycerol in recombinant strains of Pichia pastoris

Background: Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous l-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for l-lactic acid production. Results: Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. Conclusions: We showed that P. pastoris has a great potential as a fermentative organism for producing l-lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half

Fundamentals of Brazilian Honey Analysis: An Overview

Brazilian honey possesses large floral sources with various colors and flavors due to botanical and geographical differences and the large extension of the country. The absence of antibiotics and pesticides contamination positively differentiates Brazilian honey in the international market. Thus, the present chapter presents an overview of regulatory aspects for identity and quality evaluation of honey produced and commercialized in Brazil and international markets, as well as, it compares the production and consumption of honey with other countries. In addition, the chapter presents physicochemical and microbiological analysis commonly used in honey, as fundamentals of the technics and literature results with different kinds of honey obtained in Brazil. Physicochemical quality control and microbiological analysis of honey samples is of fundamental importance for assessing their quality, possible adulteration and storage conditions. In the literature, several methodologies exist to be used in the performance of honey quality control and each one complements the results in order to have an idea about the quality of the product, the absence of adulteration, deterioration, and environmental pollution and geographical area. Finally, we will present the market scenario nowadays with future perspectives and some recognition obtained for Brazilian bee products in international events

Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5′-CACAGCCAC-3′ and 5′-CCCTGCCCC-3′ sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis

Identification of glucose transporters in Aspergillus nidulans

o characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.The authors would like to thank the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript