The Experts below are selected from a list of 924 Experts worldwide ranked by ideXlab platform
Domingues Lucília - One of the best experts on this subject based on the ideXlab platform.
-
Bioinformatic analysis of promoters from Ashbya Gossypii
Universidade do Minho, 2021Co-Authors: Silva, Pedro Moreira Montenegro Baptista, Domingues Lucília, Aguiar, Tatiana QuintaAbstract:Ashbya Gossypii is a filamentous fungus that produces large quantities of riboflavin, a living factory that generates nearly half of this vitamins world supply. The fungus can grow on industrial wastes and can produce other value-added compounds including recombinant proteins, single cell oil, nucleosides, folic acid and organoleptic compounds. At the genome level, 95% of the genes have homologues in Saccharomyces cerevisiae (90% in syntenic positions). Many molecular tools used in yeast are functional in both organisms, including the autonomously replicating sequences. Fine-tuning heterologous metabolic pathways requires the availability of promoters with a wide range of activity. Endogenous promoters provide the main regulatory elements for gene expression control, however, there is a limited range of well characterised promoters available for metabolic engineering of A. Gossypii. Repeated copies of promoters for genome editing purposes increases the probability of homologous recombination and causes strain instability, what is disadvantageous when extensive pathway engineering is performed. The identification of transcription factor (TF) binding sites (TFBSs) in promoters is important for rational design of regulatory crosstalk in metabolic engineering strategies. TFs usually bind to degenerated sequences that can be identified using bioinformatics tools. The MEME algorithm can be used to mine DNA motifs from upstream sequences of co-expressed or co-regulated genes. The obtained motifs can further be compared to TFBSs in databases for S. cerevisiae (i.e. JASPAR, TRANSFAC, YEASTRACT). This procedure allowed the identification of 4 DNA motifs, 8 matching putative TFs, and TATA-box as important elements for high level gene expression. Thus, the bioinformatic approach used in this study, in intergenic regions from A. Gossypii, can be also performed in sequences of other organisms with the aim of identifying potential candidate motifs for subsequent experimental characterization and allowing future construction of hybrid semi-synthetic promoters.info:eu-repo/semantics/publishedVersio
-
New genome-editing tools for Ashbya Gossypii
2021Co-Authors: Silva, Pedro Moreira Montenegro Baptista, Domingues Lucília, Aguiar, Tatiana QuintaAbstract:Ashbya Gossypii is a splendid riboflavin biofactory that suppresses nearly half of the worldwide demand for vitamin B2. The availability of its genome sequence allowed a better understanding of this fungus metabolism and the development of targeted genome editing tools. Such progresses allowed increasing the vitamin B2 natural production levels, and to expand the potential of this fungus towards the production of nucleosides, folic acid, single cell oil and fragrances [1,2]. Now, with a broadening range of potential genetic targets, modular and flexible pathway assembly tools are more convenient than ever. There is also an urgent need for a larger set of well characterized promoters for metabolic flux optimization towards improved production of target products. As such, this work aimed at developing a modular, flexible and fast pathway assembly platform for targeted chromosomal integration in A. Gossypii that offers a wide range of expression strengths (promoters). The developed pathway assembly platform is in an early-stage, designed to easily allow fast cloning and exchange of selection markers, promoters, genes and terminators. As endogenous promoters provide the required regulatory elements for gene expression, several transcriptomic datasets were scanned for medium to high strength promoters. A set of uni- and bi-directional promoters was selected, and each promoter cloned into promoter-probes for characterization in terms of expression strength and regulation, a task that is under way using multiple reporter systems. Preliminary results revealed that the bi-directional promoter AgCCW12/HOG1p drives moderate gene expression through the HOG1p side and strong expression through the CCW12p side. The datasets were also used for transcription factor binding motif mining, in order to find important regulation elements that confer strong gene expression values. Five transcription factor binding motifs were identified as strong targets for promoter engineering. This information can hereafter enable the rational design of synthetic/semi-synthetic minimal promoters. With the current genetic engineering turnaround time to genetically modify A. Gossypii being a limiting factor to establish this fungus as a state-of-the-art microbial factory, this pathway assembly platform has great relevance. With a growing number of promoters characterized, it will become increasingly easier to genetically channel the A. Gossypii metabolism towards the production of value-added chemicals.Study supported by FCT, Compete2020 and Portugal2020 through the strategic funding of UIDB/04469/2020 and Project EcoBioInks4SmartTextiles (PTDC/CTM-TEX/30298/2017–POCI-01-0145-FEDER-030298)info:eu-repo/semantics/publishedVersio
-
Metabolic engineering of Ashbya Gossypii for deciphering the de novo biosynthesis of γ-lactones
Springer Nature, 2019Co-Authors: Silva Rui, Aguiar, Tatiana Quinta, Revuelta, José Luis, Jiménez Alberto, Coelho Eduardo, Domingues LucíliaAbstract:Background Lactones are highly valuable cyclic esters of hydroxy fatty acids that find application as pure fragrances or as building blocks of speciality chemicals. While chemical synthesis often leads to undesired racemic mixtures, microbial production allows obtaining optically pure lactones. The production of a specific lactone by biotransformation depends on the supply of the corresponding hydroxy fatty acid, which has economic and industrial value similar to γ-lactones. Hence, the identification and exploration of microorganisms with the rare natural ability for de novo biosynthesis of lactones will contribute to the long-term sustainability of microbial production. In this study, the innate ability of Ashbya Gossypii for de novo production of γ-lactones from glucose was evaluated and improved. Results Characterization of the volatile organic compounds produced by nine strains of this industrial filamentous fungus in glucose-based medium revealed the noteworthy presence of seven chemically different γ-lactones. To decipher and understand the de novo biosynthesis of γ-lactones from glucose, we developed metabolic engineering strategies focused on the fatty acid biosynthesis and the β-oxidation pathways. Overexpression of AgDES589, encoding a desaturase for the conversion of oleic acid (C18:1) into linoleic acid (C18:2), and deletion of AgELO624, which encodes an elongase that catalyses the formation of C20:0 and C22:0 fatty acids, greatly increased the production of γ-lactones (up to 6.4-fold; (7.6 ± 0.8) × 103 µg/gCell Dry Weight). Further substitution of AgPOX1, encoding the exclusive acyl-CoA oxidase in A. Gossypii, by a codon-optimized POX2 gene from Yarrowia lipolytica, which encodes a specific long chain acyl-CoA oxidase, fine-tuned the biosynthesis of γ-decalactone to a relative production of more than 99%. Conclusions This study demonstrates the potential of A. Gossypii as a model and future platform for de novo biosynthesis of γ-lactones. By means of metabolic engineering, key enzymatic steps involved in their production were elucidated. Moreover, the combinatorial metabolic engineering strategies developed resulted in improved de novo biosynthesis of γ-decalactone. In sum, these proof-of-concept data revealed yet unknown metabolic and genetic determinants important for the future exploration of the de novo production of γ-lactones as an alternative to biotransformation processes.This study was supported by: the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) (Post-Doc grant to T. Q. Aguiar and Ph.D. grant to E. Coelho), Ph.D. Grant PD/BD/113812/2015 to R. Silva (Doctoral Program in Applied and Environmental Microbiology), and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte; the Spanish Ministerio de Economía y Competitividad (BIO2014-56930-P and BIO2017-88435-R) and Junta de Castilla y León (SA016P17) to A. Jiménez and J.L. Revuelta.info:eu-repo/semantics/publishedVersio
-
Microbial lipids from industrial wastes using xylose-utilizing Ashbya Gossypii strains
'Elsevier BV', 2019Co-Authors: Díaz Fernández David, Aguiar, Tatiana Quinta, Martín, Victoria Isabel, Romani, Aloia Perez, Silva Rui, Domingues Lucília, Revuelta, José Luis, Jiménez AlbertoAbstract:Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122054.This work presents the exploitation of waste industrial by-products as raw materials for the production of microbial lipids in engineered strains of the filamentous fungus Ashbya Gossypii. A lipogenic xylose-utilizing strain was used to apply a metabolic engineering approach aiming at relieving regulatory mechanisms to further increase the biosynthesis of lipids. Three genomic manipulations were applied: the overexpression of a feedback resistant form of the acetyl-CoA carboxylase enzyme; the expression of a truncated form of Mga2, a regulator of the main 9 desaturase gene; and the overexpression of an additional copy of DGA1 that codes for diacylglycerol acyltransferase. The performance of the engineered strain was evaluated in culture media containing mixed formulations of corn-cob hydrolysates, sugarcane molasses or crude glycerol. Our results demonstrate the efficiency of the engineered strains, which were able to accumulate about 40% of cell dry weight (CDW) in lipid content using organic industrial wastes as feedstocks.This work was supported by: the Spanish Ministerio de Economía y Competitividad (grant numbers BIO2014-56930-PandBIO2017-88435R) and the Junta de Castilla y León (grant number SA016P17); the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019, PhD grant PD/ BD/113812/2015 to R. Silva (Doctoral Program in Applied and Environmental Microbiology), the MultiBiorefinery project (POCI-010145-FEDER-016403) and the BioTecNorte operation (NORTE-010145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. DDF was recipient of USAL predoctoral fellowship from the University of Salamanca.info:eu-repo/semantics/publishedVersio
-
Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya Gossypii riboflavin-overproducing strains
'Oxford University Press (OUP)', 2019Co-Authors: Silva Rui, Aguiar, Tatiana Quinta, Rui Pedro Soares De ,oliveira, Domingues LucíliaAbstract:The overproduction of riboflavin (vitamin B2) by Ashbya Gossypii, one of the most distinctive traits of this filamentous hemiascomycete, has been proposed to act as an ecological defense mechanism, since it is triggered by environmental stress. The interaction of endogenous riboflavin with light generates reactive oxygen species (ROS) and induces oxidative DNA damage in mammalian cells, but exogenous riboflavin was shown to protect A. Gossypii spores against ultraviolet light. Envisioning a better understanding of this biotechnologically relevant trait, here we investigated the putative genotoxic effects associated with the overproduction of riboflavin by A. Gossypii. For assessing that we developed the Ashbya Comet Assay, which was able to reproducibly measure oxidative (H2O2/menadione-mediated) and non-oxidative (camptothecin-mediated) DNA damage in A. Gossypii. Using this protocol, we determined that exposure to sunlight-mimicking light during growth significantly increased the DNA damage accumulation in riboflavin-overproducing cells, but not in non-overproducing ones. The exposure of overproducing cells to light induced the intracellular accumulation of ROS and increased the production of riboflavin 1.5-fold. These results show that riboflavin-overproducing strains are highly susceptible to photo-induced oxidative DNA damage and draw attention for the importance of controlling the exposure to light of biotechnological riboflavin production processes with A. Gossypii.This study was supported by the Portuguese Foundation for Science and Technology (FCT) through the strategic funding of UID/BIO/04469/2013 (Post-Doc grant to TQA) and UID/ AGR/04033/2013 units, supported by COMPETE 2020 (POCI-01- 0145-FEDER-006684 and POCI-01-0145-FEDER-006958), through the PhD grant PD/BD/113812/2015 to RS (Doctoral Program in Applied and Environmental Microbiology) and through the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio
José L. Revuelta - One of the best experts on this subject based on the ideXlab platform.
-
one vector crispr cas9 genome engineering of the industrial fungus Ashbya Gossypii
Microbial Biotechnology, 2019Co-Authors: Alberto Jiménez, Ruben M Buey, Rodrigo Ledesmaamaro, Gloria Munozfernandez, José L. RevueltaAbstract:The filamentous fungus Ashbya Gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. Gossypii to grow using low‐cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. Gossypii of other high‐added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. Gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one‐vector CRISPR/Cas9 system for genomic engineering of A. Gossypii. We demonstrate the efficiency of the system as a marker‐less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. Gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker‐less manner.
-
Microbial lipids from industrial wastes using xylose-utilizing Ashbya Gossypii strains.
Bioresource technology, 2019Co-Authors: David Díaz-fernández, Tatiana Quinta Aguiar, Rui Silva, Lucília Domingues, Victoria Isabel Martín, Aloia Romaní, José L. Revuelta, Alberto JiménezAbstract:Abstract This work presents the exploitation of waste industrial by-products as raw materials for the production of microbial lipids in engineered strains of the filamentous fungus Ashbya Gossypii. A lipogenic xylose-utilizing strain was used to apply a metabolic engineering approach aiming at relieving regulatory mechanisms to further increase the biosynthesis of lipids. Three genomic manipulations were applied: the overexpression of a feedback resistant form of the acetyl-CoA carboxylase enzyme; the expression of a truncated form of Mga2, a regulator of the main Δ9 desaturase gene; and the overexpression of an additional copy of DGA1 that codes for diacylglycerol acyltransferase. The performance of the engineered strain was evaluated in culture media containing mixed formulations of corn-cob hydrolysates, sugarcane molasses or crude glycerol. Our results demonstrate the efficiency of the engineered strains, which were able to accumulate about 40% of cell dry weight (CDW) in lipid content using organic industrial wastes as feedstocks.
-
utilization of xylose by engineered strains of Ashbya Gossypii for the production of microbial oils
Biotechnology for Biofuels, 2017Co-Authors: David Diazfernandez, Ruben M Buey, José L. Revuelta, Patricia Lozanomartinez, Alberto JiménezAbstract:Ashbya Gossypii is a filamentous fungus that is currently exploited for the industrial production of riboflavin. The utilization of A. Gossypii as a microbial biocatalyst is further supported by its ability to grow in low-cost feedstocks, inexpensive downstream processing and the availability of an ease to use molecular toolbox for genetic and genomic modifications. Consequently, A. Gossypii has been also introduced as an ideal biotechnological chassis for the production of inosine, folic acid, and microbial oils. However, A. Gossypii cannot use xylose, the most common pentose in hydrolysates of plant biomass. In this work, we aimed at designing A. Gossypii strains able to utilize xylose as the carbon source for the production of biolipids. An endogenous xylose utilization pathway was identified and overexpressed, resulting in an A. Gossypii xylose-metabolizing strain showing prominent conversion rates of xylose to xylitol (up to 97% after 48 h). In addition, metabolic flux channeling from xylulose-5-phosphate to acetyl-CoA, using aheterologous phosphoketolase pathway, increased the lipid content in the xylose-metabolizing strain a 54% over the parental strain growing in glucose-based media. This increase raised to 69% when lipid accumulation was further boosted by blocking the beta-oxidation pathway. Ashbya Gossypii has been engineered for the utilization of xylose. We present here a proof-of-concept study for the production of microbial oils from xylose in A. Gossypii, thus introducing a novel biocatalyst with very promising properties in developing consolidated bioprocessing to produce fine chemicals and biofuels from xylose-rich hydrolysates of plant biomass.
-
increased riboflavin production by manipulation of inosine 5 monophosphate dehydrogenase in Ashbya Gossypii
Applied Microbiology and Biotechnology, 2015Co-Authors: Ruben M Buey, Rodrigo Ledesmaamaro, Monica Balsera, Jose M De Pereda, José L. RevueltaAbstract: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.
-
strain design of Ashbya Gossypii for single cell oil production
Applied and Environmental Microbiology, 2014Co-Authors: Rodrigo Ledesmaamaro, Alberto Jiménez, Maria A Santos, José L. RevueltaAbstract: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.
Ruben M Buey - One of the best experts on this subject based on the ideXlab platform.
-
one vector crispr cas9 genome engineering of the industrial fungus Ashbya Gossypii
Microbial Biotechnology, 2019Co-Authors: Alberto Jiménez, Ruben M Buey, Rodrigo Ledesmaamaro, Gloria Munozfernandez, José L. RevueltaAbstract:The filamentous fungus Ashbya Gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. Gossypii to grow using low‐cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. Gossypii of other high‐added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. Gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one‐vector CRISPR/Cas9 system for genomic engineering of A. Gossypii. We demonstrate the efficiency of the system as a marker‐less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. Gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker‐less manner.
-
utilization of xylose by engineered strains of Ashbya Gossypii for the production of microbial oils
Biotechnology for Biofuels, 2017Co-Authors: David Diazfernandez, Ruben M Buey, José L. Revuelta, Patricia Lozanomartinez, Alberto JiménezAbstract:Ashbya Gossypii is a filamentous fungus that is currently exploited for the industrial production of riboflavin. The utilization of A. Gossypii as a microbial biocatalyst is further supported by its ability to grow in low-cost feedstocks, inexpensive downstream processing and the availability of an ease to use molecular toolbox for genetic and genomic modifications. Consequently, A. Gossypii has been also introduced as an ideal biotechnological chassis for the production of inosine, folic acid, and microbial oils. However, A. Gossypii cannot use xylose, the most common pentose in hydrolysates of plant biomass. In this work, we aimed at designing A. Gossypii strains able to utilize xylose as the carbon source for the production of biolipids. An endogenous xylose utilization pathway was identified and overexpressed, resulting in an A. Gossypii xylose-metabolizing strain showing prominent conversion rates of xylose to xylitol (up to 97% after 48 h). In addition, metabolic flux channeling from xylulose-5-phosphate to acetyl-CoA, using aheterologous phosphoketolase pathway, increased the lipid content in the xylose-metabolizing strain a 54% over the parental strain growing in glucose-based media. This increase raised to 69% when lipid accumulation was further boosted by blocking the beta-oxidation pathway. Ashbya Gossypii has been engineered for the utilization of xylose. We present here a proof-of-concept study for the production of microbial oils from xylose in A. Gossypii, thus introducing a novel biocatalyst with very promising properties in developing consolidated bioprocessing to produce fine chemicals and biofuels from xylose-rich hydrolysates of plant biomass.
-
Engineering Ashbya Gossypii strains for de novo lipid production using industrial by-products
Microbial Biotechnology, 2017Co-Authors: Patricia Lozano-martinez, Ruben M Buey, Alberto Jiménez, Rodrigo Ledesma-amaro, Jose Luis RevueltaAbstract:Ashbya Gossypii is a filamentous fungus that naturally overproduces riboflavin, and it is currently exploited for the industrial production of this vitamin. The utilization of A. Gossypii for biotechnological applications presents important advantages such as the utilization of low-cost culture media, inexpensive downstream processing and a wide range of molecular tools for genetic manipulation, thus making A. Gossypii a valuable biotechnological chassis for metabolic engineering. A. Gossypii has been shown to accumulate high levels of lipids in oil-based culture media; however, the lipid biosynthesis capacity is rather limited when grown in sugar-based culture media. In this study, by altering the fatty acyl-CoA pool and manipulating the regulation of the main.9 desaturase gene, we have obtained A. Gossypii strains with significantly increased (up to fourfold) de novo lipid biosynthesis using glucose as the only carbon source in the fermentation broth. Moreover, these strains were efficient biocatalysts for the conversion of carbohydrates from sugarcane molasses to biolipids, able to accumulate lipids up to 25% of its cell dry weight. Our results represent a proof of principle showing the promising potential of A. Gossypii as a competitive microorganism for industrial biolipid production using cost-effective feed stocks.
-
increased riboflavin production by manipulation of inosine 5 monophosphate dehydrogenase in Ashbya Gossypii
Applied Microbiology and Biotechnology, 2015Co-Authors: Ruben M Buey, Rodrigo Ledesmaamaro, Monica Balsera, Jose M De Pereda, José L. RevueltaAbstract: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.
-
increased production of inosine and guanosine by means of metabolic engineering of the purine pathway in Ashbya Gossypii
Microbial Cell Factories, 2015Co-Authors: Rodrigo Ledesmaamaro, Ruben M Buey, Jose Luis RevueltaAbstract: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. 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. 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.
Peter Philippsen - One of the best experts on this subject based on the ideXlab platform.
-
regulation of exit from mitosis in multinucleate Ashbya Gossypii cells relies on a minimal network of genes
Molecular Biology of the Cell, 2011Co-Authors: Mark R Finlayson, Katrin A Helferhungerbuhler, Peter PhilippsenAbstract:In Saccharomyces cerevisiae, mitosis is coupled to cell division by the action of the Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN) regulatory networks, which mediate exit from mitosis by activation of the phosphatase Cdc14. The closely related filamentous ascomycete Ashbya Gossypii provides a unique cellular setting to study the evolution of these networks. Within its multinucleate hyphae, nuclei are free to divide without the spatial and temporal constraints described for budding yeast. To investigate how this highly conserved system has adapted to these circumstances, we constructed a series of mutants lacking homologues of core components of MEN and FEAR and monitored phenomena such as progression through mitosis and Cdc14 activation. MEN homologues in A. Gossypii were shown to have diverged from their anticipated role in Cdc14 release and exit from mitosis. We observed defects in septation, as well as a partial metaphase arrest, in Agtem1Δ, Agcdc15Δ, Agdbf2/dbf20Δ, and Agmob1Δ. A. Gossypii homologues of the FEAR network, on the other hand, have a conserved and more pronounced role in regulation of the M/G1 transition. Agcdc55Δ mutants are unable to sequester AgCdc14 throughout interphase. We propose a reduced model of the networks described in yeast, with a low degree of functional redundancy, convenient for further investigations into these networks.
-
Maximal polar growth potential depends on the polarisome component AgSpa2 in the filamentous fungus Ashbya Gossypii
Molecular biology of the cell, 2003Co-Authors: Philipp Knechtle, Fred S. Dietrich, Peter PhilippsenAbstract:We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya Gossypii. The development starts with an initial isotropic growth phase followed by the emergence of germ tubes. The initial tip growth speed of 6-10 microm/h increases during early stages of development. This increase is transiently interrupted in response to the establishment of lateral branches or septa. The hyphal tip growth speed finally reaches a maximum of up to 200 micro/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae. A search for A. Gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain. AgSpa2 colocalizes with sites of polarized actin. Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips. Polarization at sites of septation is transient. During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided. To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion. The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern. We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process.
-
pcr based gene targeting in the filamentous fungus Ashbya Gossypii
Gene, 2000Co-Authors: Jurgen Wendland, Y Ayaddurieux, Philipp Knechtle, C Rebischung, Peter PhilippsenAbstract:Abstract We have investigated a PCR-based approach for one-step gene targeting in the filamentous fungus Ashbya Gossypii. Short guide sequences with 40–46 bp of homology to two sequences of a targeted gene, provided by PCR, were sufficient to mediate homologous recombination. The PCR products used for transformation were generated from the newly constructed chimeric selection marker GEN3. This consists of the open reading frame of the Escherichia coli kanR gene under the control of promoter and terminator sequences of the Saccharomyces cerevisiae TEF2 gene and allows selection of G418/geneticin-resistant transformants. Verification of gene targeting was performed either by PCR or by DNA hybridization analyses, and in all 18 cases tested, correct targeting was confirmed. This approach was used for the complete deletion of the open reading frame of the A. Gossypii RHO4 gene for which a double-strand sequence was available as information source for the design of PCR primers. We also demonstrated successful partial deletion of four other ORFs using single-read sequences (SRS) as sole information for the design of targeting primers. A Gossypii is the first filamentous fungus in which a PCR-based gene disruption technique has been established. Since short target guide sequences are sufficient to direct homologous integration into the A. Gossypii genome it is not necessary to obtain and sequence large DNA fragments from a target locus to provide the long flanking homology regions usually required for efficient targeting of cloned disruption cassettes in filamentous fungi. Thus functional analysis of A. Gossypii genes is already possible, based on single-pass sequence information.
-
sequence and promoter analysis of the highly expressed tef gene of the filamentous fungus Ashbya Gossypii
Molecular Genetics and Genomics, 1994Co-Authors: Sabine Steiner, Peter PhilippsenAbstract:Ashbya Gossypii carries only a single gene (TEF) coding for the abundant translation elongation factor 1α. Cloning and sequencing of this gene and deletion analysis of the promoter region revealed an extremely high degree of similarity with the well studied TEF genes of the yeast Saccharomyces cerevisiae including promoter upstream activation sequence (UAS) elements. The open reading frames in both species are 458 codons long and show 88.6% identity at the DNA level and 93.7% identity at the protein level. A short DNA segment in the promoter, between nucleotides -268 and -213 upstream of the ATG start codon, is essential for high-level expression of the A. Gossypii TEF gene. It carries two sequences, GCCCATACAT and ATCCATACAT, with high homology to the UASrpg sequence of S. cerevisiae, which is an essential promoter element in genes coding for highly expressed components of the translational apparatus. UASrpg sequences are binding sites for the S. cerevisiae protein TUF, also called RAP1 or GRF1. In gel retardation with A. Gossypii protein extracts we demonstrated specific protein binding to the short TEF promoter segment carrying the UASrpg homologous sequences.
Lucília Domingues - One of the best experts on this subject based on the ideXlab platform.
-
Microbial lipids from industrial wastes using xylose-utilizing Ashbya Gossypii strains.
Bioresource technology, 2019Co-Authors: David Díaz-fernández, Tatiana Quinta Aguiar, Rui Silva, Lucília Domingues, Victoria Isabel Martín, Aloia Romaní, José L. Revuelta, Alberto JiménezAbstract:Abstract This work presents the exploitation of waste industrial by-products as raw materials for the production of microbial lipids in engineered strains of the filamentous fungus Ashbya Gossypii. A lipogenic xylose-utilizing strain was used to apply a metabolic engineering approach aiming at relieving regulatory mechanisms to further increase the biosynthesis of lipids. Three genomic manipulations were applied: the overexpression of a feedback resistant form of the acetyl-CoA carboxylase enzyme; the expression of a truncated form of Mga2, a regulator of the main Δ9 desaturase gene; and the overexpression of an additional copy of DGA1 that codes for diacylglycerol acyltransferase. The performance of the engineered strain was evaluated in culture media containing mixed formulations of corn-cob hydrolysates, sugarcane molasses or crude glycerol. Our results demonstrate the efficiency of the engineered strains, which were able to accumulate about 40% of cell dry weight (CDW) in lipid content using organic industrial wastes as feedstocks.
-
physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya Gossypii
New Biotechnology, 2019Co-Authors: Rui Silva, Tatiana Quinta Aguiar, Carla Cristina Marques De ,oliveira, Lucília DominguesAbstract:Abstract 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.
-
Metabolic engineering of Ashbya Gossypii for deciphering the de novo biosynthesis of γ-lactones
BMC, 2019Co-Authors: Rui Silva, Jose Luis Revuelta, Alberto Jiménez, Tatiana Q. Aguiar, Eduardo Coelho, Lucília DominguesAbstract:Abstract Background Lactones are highly valuable cyclic esters of hydroxy fatty acids that find application as pure fragrances or as building blocks of speciality chemicals. While chemical synthesis often leads to undesired racemic mixtures, microbial production allows obtaining optically pure lactones. The production of a specific lactone by biotransformation depends on the supply of the corresponding hydroxy fatty acid, which has economic and industrial value similar to γ-lactones. Hence, the identification and exploration of microorganisms with the rare natural ability for de novo biosynthesis of lactones will contribute to the long-term sustainability of microbial production. In this study, the innate ability of Ashbya Gossypii for de novo production of γ-lactones from glucose was evaluated and improved. Results Characterization of the volatile organic compounds produced by nine strains of this industrial filamentous fungus in glucose-based medium revealed the noteworthy presence of seven chemically different γ-lactones. To decipher and understand the de novo biosynthesis of γ-lactones from glucose, we developed metabolic engineering strategies focused on the fatty acid biosynthesis and the β-oxidation pathways. Overexpression of AgDES589, encoding a desaturase for the conversion of oleic acid (C18:1) into linoleic acid (C18:2), and deletion of AgELO624, which encodes an elongase that catalyses the formation of C20:0 and C22:0 fatty acids, greatly increased the production of γ-lactones (up to 6.4-fold; (7.6 ± 0.8) × 103 µg/gCell Dry Weight). Further substitution of AgPOX1, encoding the exclusive acyl-CoA oxidase in A. Gossypii, by a codon-optimized POX2 gene from Yarrowia lipolytica, which encodes a specific long chain acyl-CoA oxidase, fine-tuned the biosynthesis of γ-decalactone to a relative production of more than 99%. Conclusions This study demonstrates the potential of A. Gossypii as a model and future platform for de novo biosynthesis of γ-lactones. By means of metabolic engineering, key enzymatic steps involved in their production were elucidated. Moreover, the combinatorial metabolic engineering strategies developed resulted in improved de novo biosynthesis of γ-decalactone. In sum, these proof-of-concept data revealed yet unknown metabolic and genetic determinants important for the future exploration of the de novo production of γ-lactones as an alternative to biotransformation processes
-
deciphering the uracil growth sensitivity of Ashbya Gossypii pyrimidine auxotrophs
2017Co-Authors: Rui Silva, Tatiana Quinta Aguiar, Lucília DominguesAbstract:The authors thank the financial support from FCT, Portugal: strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) (Post-Doc fellowship to TQ Aguiar), BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by FEDER under the scope of Norte2020 - Programa Operacional Regional do Norte, and PhD grant to R Silva (PD/BD/113812/2015) through the FCT PhD Programme in Applied and Environmental Microbiology.
-
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya Gossypii
2014Co-Authors: Orquidea Ribeiro, Lucília Domingues, Marja Ilmen, Marilyn Wiebe, Merja PenttilaAbstract:Abstract To explore the potential of Ashbya Gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohy-drolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. Gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. Gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-D-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. Gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae
