| 1. |
- Anasontzis, George E, 1980, et al.
(författare)
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Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris
- 2014
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 30:3, s. 728-735
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Tidskriftsartikel (refereegranskat)abstract
- Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. (c) 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728-735, 2014
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| 2. |
- Andersen, M. R., et al.
(författare)
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Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88
- 2011
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 21:6, s. 885-897
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Tidskriftsartikel (refereegranskat)abstract
- The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook wholegenome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi
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| 3. |
- Papini, Marta, 1981, et al.
(författare)
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Systems Biology of Industrial Microorganisms
- 2010
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Ingår i: Advances in Biochemical Engineering/Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145 .- 1616-8542. - 9783642142307 ; 120, s. 51-99
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Tidskriftsartikel (refereegranskat)abstract
- The field of industrial biotechnology is expanding rapidly as the chemical industry is looking towards more sustainable production of chemicals that can be used as fuels or building blocks for production of solvents and materials. In connection with the development of sustainable bioprocesses, it is a major challenge to design and develop efficient cell factories that can ensure cost efficient conversion of the raw material into the chemical of interest. This is achieved through metabolic engineering, where the metabolism of the cell factory is engineered such that there is an efficient conversion of sugars, the typical raw materials in the fermentation industry, into the desired product. However, engineering of cellular metabolism is often challenging due to the complex regulation that has evolved in connection with adaptation of the different microorganisms to their ecological niches. In order to map these regulatory structures and further de-regulate them, as well as identify ingenious metabolic engineering strategies that full-fill mass balance constraints, tools from systems biology can be applied. This involves both high-throughput analysis tools like transcriptome, proteome and metabolome analysis, as well as the use of mathematical modeling to simulate the phenotypes resulting from the different metabolic engineering strategies. It is in fact expected that systems biology may substantially improve the process of cell factory development, and we therefore propose the term Industrial Systems Biology for how systems biology will enhance the development of industrial biotechnology for sustainable chemical production.
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| 4. |
- Salazar Pena, Margarita, 1979, et al.
(författare)
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Recombinant galactose oxidase production in fed batch fermentations with Pichia pastoris
- 2010
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Ingår i: WWSC Second International Conference, 23-25 November 2010, Stockholm, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- This study concentrates on the optimization of the cultivation conditions, in order to achieve high galactose oxidase productivity from a recombinant strain of the methylotrophic yeast Pichia pastoris.
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| 5. |
- Salazar Pena, Margarita, 1979
(författare)
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Systems Biology of Glucose Sensing and Repression in Aspergillus niger: Lessons from Genomics and Transcriptomics
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aspergillus comprises a genus of multicellular eukaryotic microorganisms containing close to 200 different species. Among them, the filamentous fungus Aspergillus niger exhibits great diversity in its phenotype and it is found, both in marine and terrestrial habitats. A. niger produces many metabolites that are valuable commodities, e.g., organic acids, but is also able to secrete a wide range of hydrolytic enzymes, while some strains exhibit pathogenicity. Although A. niger is a widely used industrial species used for metabolite production, relatively little is known about the regulation of its metabolism. We therefore undertook a functional genomics approach to produce transcriptome data of high quality trying to uncover novel regulatory processes and the transcription factors associated to them. Throughout this thesis we worked with several wild type and gene deletion A. niger strains, i.e., the ancestor of an industrial glucoamylase producer strain, A. niger BO1; the acidogenic wild type strain ATCC 1015 and the industrial enzyme-producing strain CBS 513.88, both strains already sequenced. High variation within these two later strains was confirmed with the genomics and transcriptomics approaches used in combination with exo-metabolite profiling. Genotypic differences were observed to accumulate in metabolic pathways, e.g., essential to production of organic acids for A. niger ATCC 1015 and of protein synthesis for CBS 513.88. Overall, the studies reported in this thesis illustrate the complexity of the regulatory circuits regulating cellular processes. For example, through comparative genomics approaches, we exposed the reasons why glycerol metabolism and maltose metabolism are different in closely related species. We demonstrated that the two industrially relevant Aspergillus species, A. niger and A. oryzae do not possess the same maltose uptake, metabolism and regulatory mechanisms. While, the two later studies reported in this thesis were not as focused as the previous ones, but more exploratory in terms of looking for novel regulatory roles played by the transcription factors studied: AdrA, FacB, CreA and AreB; we think that our results could help to improve understanding of the metabolic regulation in A. niger by several transcriptional regulators that will lead to better tuning and improvement of production processes and wider exploitation of A. niger as a cell factory.
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| 6. |
- Salazar Pena, Margarita, 1979, et al.
(författare)
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Uncovering transcriptional regulation of glycerol metabolism in Aspergilli through genome-wide gene expression data analysis
- 2009
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Ingår i: Molecular Genetics and Genomics. - : Springer Science and Business Media LLC. - 1617-4615 .- 1617-4623. ; 282:6, s. 571-586
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Tidskriftsartikel (refereegranskat)abstract
- Glycerol is catabolized by a wide range of microorganisms including Aspergillus species. To identify the transcriptional regulation of glycerol metabolism in Aspergillus, we analyzed data from triplicate batch fermentations of three different Aspergilli (Aspergillus nidulans, Aspergillus oryzae and Aspergillus niger) with glucose and glycerol as carbon sources. Protein comparisons and cross-analysis with gene expression data of all three species resulted in the identification of 88 genes having a conserved response across the three Aspergilli. A promoter analysis of the up-regulated genes led to the identification of a conserved binding site for a putative regulator to be 5'-TGCGGGGA-3', a binding site that is similar to the binding site for Adr1 in yeast and humans. We show that this Adr1 consensus binding sequence was over-represented on promoter regions of several genes in A. nidulans, A. oryzae and A. niger. Our transcriptome analysis indicated that genes involved in ethanol, glycerol, fatty acid, amino acids and formate utilization are putatively regulated by Adr1 in Aspergilli as in Saccharomyces cerevisiae and this transcription factor therefore is likely to be cross-species conserved among Saccharomyces and distant Ascomycetes. Transcriptome data were further used to evaluate the high osmolarity glycerol pathway. All the components of this pathway present in yeast have orthologues in the three Aspergilli studied and its gene expression response suggested that this pathway functions as in S. cerevisiae. Our study clearly demonstrates that cross-species evolutionary comparisons among filamentous fungi, using comparative genomics and transcriptomics, are a powerful tool for uncovering regulatory systems.
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| 7. |
- Udatha, Gupta, 1984, et al.
(författare)
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Deciphering the signaling mechanisms of the plant cell wall degradation machinery in Aspergillus oryzae
- 2015
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Ingår i: BMC Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 9:1, s. 20-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The gene expression and secretion of fungal lignocellulolytic enzymes are tightly controlled at the transcription level using independent mechanisms to respond to distinct inducers from plant biomass. An advanced systems-level understanding of transcriptional regulatory networks is required to rationally engineer filamentous fungi for more efficient bioconversion of different types of biomass. Results: In this study we focused on ten chemically defined inducers to drive expression of cellulases, hemicellulases and accessory enzymes in the model filamentous fungus Aspergillus oryzae and shed light on the complex network of transcriptional activators required. The chemical diversity analysis of the inducers, based on 186 chemical descriptors calculated from the structure, resulted into three clusters, however, the global, metabolic and extracellular protein transcription of the A. oryzae genome were only partially explained by the chemical similarity of the enzyme inducers. Genes encoding enzymes that have attracted considerable interest such as cellobiose dehydrogenases and copper-dependent polysaccharide mono-oxygenases presented a substrate-specific induction. Several homology-model structures were derived using ab-initio multiple threading alignment in our effort to elucidate the interplay of transcription factors involved in regulating plant-deconstructing enzymes and metabolites. Systematic investigation of metabolite-protein interactions, using the 814 unique reactants involved in 2360 reactions in the genome scale metabolic network of A. oryzae, was performed through a two-step molecular docking against the binding pockets of the transcription factors AoXlnR and AoAmyR. A total of six metabolites viz., sulfite (H2SO3), sulfate (SLF), uroporphyrinogen III (UPGIII), ethanolamine phosphate (PETHM), D-glyceraldehyde 3-phosphate (T3P1) and taurine (TAUR) were found as strong binders, whereas the genes involved in the metabolic reactions that these metabolites appear were found to be significantly differentially expressed when comparing the inducers with glucose. Conclusions: Based on our observations, we believe that specific binding of sulfite to the regulator of the cellulase gene expression, AoXlnR, may be the molecular basis for the connection of sulfur metabolism and cellulase gene expression in filamentous fungi. Further characterization and manipulation of the regulatory network components identified in this study, will enable rational engineering of industrial strains for improved production of the sophisticated set of enzymes necessary to break-down chemically divergent plant biomass.
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| 8. |
- Vongsangnak, Wanwipa, 1982, et al.
(författare)
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Analysis of Genome-Wide Coexpression and Coevolution of Aspergillus oryzae and Aspergillus niger
- 2010
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Ingår i: OMICS A Journal of Integrative Biology. - : Mary Ann Liebert Inc. - 1536-2310 .- 1557-8100. ; 14:2, s. 165-175
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of coexpressed genes in response to different perturbations at the genome-level can provide new insight into global regulatory structures. Here we performed integrated data analysis for a crossspecies comparative investigation by exploring genomes and transcriptional coexpression profiles in Aspergillus oryzae and Aspergillus niger. Based on our analysis of conserved coexpressed genes, fatty acid catabolism via beta-oxidation, fatty acid transport, the glyoxylate bypass, and peroxisomal biogenesis were identified as core coevolved pathways between the two species. The occurrence of coexpression patterns, allowed for identification of DNA regulatory motifs and putative corresponding transcription factors, and we hereby show that comparative transcriptome analysis between two closely related fungi allows for identification of how genes involved in the utilization of fatty acids, peroxisomal biogenesis, and the glyoxylate bypass are regulated. Interestingly, "CCTCGG'' was identified as a core binding site for the putative FarA and FarB transcription factors that govern the underlined biological processes. Phylogeny and domain architecture analysis of amino acid sequences of FarA and FarB in eight species of aspergilli, clearly indicate that these proteins are evolutionarily conserved across Aspergillus species as well as they are conserved in other fungi.
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| 9. |
- Vongsangnak, Wanwipa, 1982, et al.
(författare)
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Genome-wide analysis of maltose utilization and regulation in aspergilli
- 2009
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Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 155:12, s. 3893-3902
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Tidskriftsartikel (refereegranskat)abstract
- Maltose utilization and regulation in aspergilli is of great importancefor cellular physiology and industrial fermentation processes.In Aspergillus oryzae, maltose utilization requires a functionalMAL locus, each composed of three genes: MALR encoding a regulatoryprotein, MALT encoding maltose permease and MALS encodingmaltase. Through a comparative genome and transcriptomeanalysis we show that the MAL regulon system is active in A.oryzae while it is not present in Aspergillus niger. In order to utilizemaltose, A. niger requires a different regulatory system thatinvolves the AmyR regulator for glucoamylase (glaA) induction.Analysis of reporter metabolites and subnetworks illustrate themajor route of maltose transport and metabolism in A. oryzae. Thisdemonstrates that overall metabolic responses of A. oryzae occur interms of genes, enzymes, and metabolites when altering the carbonsource. Although the amount of knowledge on maltose transportand metabolism is far from being complete in Aspergillus spp., ourstudy not only helps to understand the sugar preference in industrialfermentation processes, but also indicates how maltose affectsgene expression and overall metabolism.
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