| 1. |
- Grijseels, S., et al.
(författare)
-
Identification of the decumbenone biosynthetic gene cluster in penicillium decumbens and the importance for production of calbistrin
- 2018
-
Ingår i: Fungal Biology and Biotechnology. - : Springer Science and Business Media LLC. - 2054-3085. ; 5:1, s. 1-17
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicy-clic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor-related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol. Results: A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (calA to calM). Implementation of the CRISPR/ Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase (calA), a major facilitator pump (calB) and a binuclear zinc cluster transcription factor (calC). Detailed metabolic profiling, using UHPLC-MS, of the ∆calA (PKS) and ∆calC ( TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B. Conclusion: Here we report the discovery of a gene cluster (calA-M) involved in the biosynthesis of the polyketide calbistrin in P. decumbens. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.
|
|
| 2. |
- Grijseels, S., et al.
(författare)
-
Physiological characterization of secondary metabolite producing Penicillium cell factories
- 2017
-
Ingår i: Fungal Biology and Biotechnology. - : Springer Science and Business Media LLC. - 2054-3085. ; 4, s. 8-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Penicillium species are important producers of bioactive secondary metabolites. However, the immense diversity of the fungal kingdom is only scarcely represented in industrial bioprocesses and the upscaling of compound production remains a costly and labor intensive challenge. In order to facilitate the development of novel secondary metabolite producing processes, two routes are typically explored: optimization of the native producer or transferring the enzymatic pathway into a heterologous host. Recent genome sequencing of ten Penicillium species showed the vast amount of secondary metabolite gene clusters present in their genomes, and makes them accessible for rational strain improvement. In this study, we aimed to characterize the potential of these ten Penicillium species as native producing cell factories by testing their growth performance and secondary metabolite production in submerged cultivations.Results: Cultivation of the fungal species in controlled submerged bioreactors showed that the ten wild type Penicillium species had promising, highly reproducible growth characteristics in two different media. Analysis of the secondary metabolite production using liquid chromatography coupled with high resolution mass spectrometry proved that the species produced a broad range of secondary metabolites, at different stages of the fermentations. Metabolite profiling for identification of the known compounds resulted in identification of 34 metabolites; which included several with bioactive properties such as antibacterial, antifungal and anticancer activities. Additionally, several novel species metabolite relationships were found.Conclusions: This study demonstrates that the fermentation characteristics and the highly reproducible performance in bioreactors of ten recently genome sequenced Penicillium species should be considered as very encouraging for the application of native hosts for production via submerged fermentation. The results are particularly promising for the potential development of the ten analysed Penicillium species for production of novel bioactive compounds via submerged fermentations
|
|
| 3. |
- Zhao, Meng, et al.
(författare)
-
Pathway engineering in yeast for synthesizing the complex polyketide bikaverin
- 2020
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast.
|
|
| 4. |
- Caspeta-Guadarrama, Luis, 1974, et al.
(författare)
-
Genome-scale metabolic reconstructions of Pichia stipitis and Pichia pastoris and in-silico evaluation of their potentials
- 2012
-
Ingår i: BMC Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 6:24
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundPichia stipitis and Pichia pastoris have long been investigated due to their native abilities to metabolize every sugar from lignocellulose and to modulate methanol consumption, respectively. The latter has been driving the production of several recombinant proteins. As a result, significant advances in their biochemical knowledge, as well as in genetic engineering and fermentation methods have been generated. The release of their genome sequences has allowed systems level research. ResultsIn this work, genome-scale metabolic models (GEMs) of P. stipitis (iSS884) and P. pastoris (iLC915) were reconstructed. iSS884 includes 1332 reactions, 922 metabolites, and 4 compartments. iLC915 contains 1423 reactions, 899 metabolites, and 7 compartments. Compared with the previous GEMs of P. pastoris, PpaMBEL1254 and iPP668, iLC915 contains more genes and metabolic functions, as well as improved predictive capabilities. Simulations of physiological responses for the growth of both yeasts on selected carbon sources using iSS884 and iLC915 closely reproduced the experimental data. Additionally, the iSS884 model was used to predict ethanol production from xylose at different oxygen uptake rates. Simulations with iLC915 closely reproduced the effect of oxygen uptake rate on physiological states of P. pastoris expressing a recombinant protein. The potential of P. stipitis for the conversion of xylose and glucose into ethanol using reactors in series, and of P. pastoris to produce recombinant proteins using mixtures of methanol and glycerol or sorbitol are also discussed. ConclusionsIn conclusion the first GEM of P. stipitis (iSS884) was reconstructed and validated. The expanded version of the P. pastoris GEM, iLC915, is more complete and has improved capabilities over the existing models. Both GEMs are useful frameworks to explore the versatility of these yeasts and to capitalize on their biotechnological potentials.
|
|
| 5. |
- Cvijovic, Marija, 1977, et al.
(författare)
-
BioMet Toolbox: genome-wide analysis of metabolism
- 2010
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:SUPPL. 2, s. W144-W149
-
Tidskriftsartikel (refereegranskat)abstract
- The rapid progress of molecular biology tools for directed genetic modifications, accurate quantitative experimental approaches, high-throughput measurements, together with development of genome sequencing has made the foundation for a new area of metabolic engineering that is driven by metabolic models. Systematic analysis of biological processes by means of modelling and simulations has made the identification of metabolic networks and prediction of metabolic capabilities under different conditions possible. For facilitating such systemic analysis, we have developed the BioMet Toolbox, a web-based resource for stoichiometric analysis and for integration of transcriptome and interactome data, thereby exploiting the capabilities of genome-scale metabolic models. The BioMet Toolbox provides an effective user-friendly way to perform linear programming simulations towards maximized or minimized growth rates, substrate uptake rates and metabolic production rates by detecting relevant fluxes, simulate single and double gene deletions or detect metabolites around which major transcriptional changes are concentrated. These tools can be used for high-throughput in silico screening and allows fully standardized simulations. Model files for various model organisms (fungi and bacteria) are included. Overall, the BioMet Toolbox serves as a valuable resource for exploring the capabilities of these metabolic networks. BioMet Toolbox is freely available at www.sysbio.se/BioMet/.
|
|
| 6. |
- Liu, Liming, 1976, et al.
(författare)
-
Use of genome-scale metabolic models for understanding microbial physiology
- 2010
-
Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 584:12, s. 2556-2564
-
Tidskriftsartikel (refereegranskat)abstract
- The exploitation of microorganisms in industrial, medical, food and environmental biotechnology requires a comprehensive understanding of their physiology. The availability of genome sequences and accumulation of high-throughput data allows gaining understanding of microbial physiology at the systems level, and genome-scale metabolic models represent a valuable framework for integrative analysis of metabolism of microorganisms. Genome-scale metabolic models are reconstructed based on a combination of genome sequence information and detailed biochemical information, and these reconstructed models can be used for analyzing and simulating the operation of metabolism in response to different stimuli. Here we discuss the requirement for having detailed physiological insight in order to exploit microorganisms for production of fuels, chemicals and pharmaceuticals. We further describe the reconstruction process of genome-scale metabolic models and different algorithms that can be used to apply these models to gain improved insight into microbial physiology. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
|
|
| 7. |
- Mardinoglu, Adil, 1982, et al.
(författare)
-
Integration of clinical data with a genome-scale metabolic model of the human adipocyte
- 2013
-
Ingår i: Molecular Systems Biology. - : EMBO. - 1744-4292 .- 1744-4292. ; 9, s. 649-
-
Tidskriftsartikel (refereegranskat)abstract
- We evaluated the presence/absence of proteins encoded by 14 077 genes in adipocytes obtained from different tissue samples using immunohistochemistry. By combining this with previously published adipocyte-specific proteome data, we identified proteins associated with 7340 genes in human adipocytes. This information was used to reconstruct a comprehensive and functional genome-scale metabolic model of adipocyte metabolism. The resulting metabolic model, iAdipocytes1809, enables mechanistic insights into adipocyte metabolism on a genome-wide level, and can serve as a scaffold for integration of omics data to understand the genotype-phenotype relationship in obese subjects. By integrating human transcriptome and fluxome data, we found an increase in the metabolic activity around androsterone, ganglioside GM2 and degradation products of heparan sulfate and keratan sulfate, and a decrease in mitochondrial metabolic activities in obese subjects compared with lean subjects. Our study hereby shows a path to identify new therapeutic targets for treating obesity through combination of high throughput patient data and metabolic modeling.
|
|
| 8. |
- Pabinger, S., et al.
(författare)
-
MEMOSys: Bioinformatics platform for genome-scale metabolic models
- 2011
-
Ingår i: BMC Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Recent advances in genomic sequencing have enabled the use of genome sequencing in standard biological and biotechnological research projects. The challenge is how to integrate the large amount of data in order to gain novel biological insights. One way to leverage sequence data is to use genome-scale metabolic models. We have therefore designed and implemented a bioinformatics platform which supports the development of such metabolic models. Results: MEMOSys (MEtabolic MOdel research and development System) is a versatile platform for the management, storage, and development of genome-scale metabolic models. It supports the development of new models by providing a built-in version control system which offers access to the complete developmental history. Moreover, the integrated web board, the authorization system, and the definition of user roles allow collaborations across departments and institutions. Research on existing models is facilitated by a search system, references to external databases, and a feature-rich comparison mechanism. MEMOSys provides customizable data exchange mechanisms using the SBML format to enable analysis in external tools. The web application is based on the Java EE framework and offers an intuitive user interface. It currently contains six annotated microbial metabolic models. Conclusions: We have developed a web-based system designed to provide researchers a novel application facilitating the management and development of metabolic models. The system is freely available at http://www.icbi.at/MEMOSys.
|
|
| 9. |
- Thiele, I., et al.
(författare)
-
A community-driven global reconstruction of human metabolism
- 2013
-
Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 31:5, s. 419-
-
Tidskriftsartikel (refereegranskat)abstract
- Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus 'metabolic reconstruction', which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including similar to 2x more reactions and similar to 1.7x more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type-specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/.
|
|
| 10. |
- Tymoshenko, S., et al.
(författare)
-
Metabolic Needs and Capabilities of Toxoplasma gondii through Combined Computational and Experimental Analysis
- 2015
-
Ingår i: PLoS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358. ; 11:5, s. Art. no. e1004261-
-
Tidskriftsartikel (refereegranskat)abstract
- Toxoplasma gondii is a human pathogen prevalent worldwide that poses a challenging and unmet need for novel treatment of toxoplasmosis. Using a semi-automated reconstruction algorithm, we reconstructed a genome-scale metabolic model, ToxoNet1. The reconstruction process and flux-balance analysis of the model offer a systematic overview of the metabolic capabilities of this parasite. Using ToxoNet1 we have identified significant gaps in the current knowledge of Toxoplasma metabolic pathways and have clarified its minimal nutritional requirements for replication. By probing the model via metabolic tasks, we have further defined sets of alternative precursors necessary for parasite growth. Within a human host cell environment, ToxoNet1 predicts a minimal set of 53 enzyme-coding genes and 76 reactions to be essential for parasite replication. Double-gene-essentiality analysis identified 20 pairs of genes for which simultaneous deletion is deleterious. To validate several predictions of ToxoNet1 we have performed experimental analyses of cytosolic acetyl-CoA biosynthesis. ATP-citrate lyase and acetyl-CoA synthase were localised and their corresponding genes disrupted, establishing that each of these enzymes is dispensable for the growth of T. gondii, however together they make a synthetic lethal pair.
|
|
