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1.
J Environ Manage ; 316: 115216, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35550960

RESUMO

In treating mine-impacted waters using sulfate-reducing bacteria (SRB), metal inhibition and substrate selection are important factors affecting the efficiency of the bioprocess. This work investigated the role of the substrate (i.e. lactate, formate, glycerol and glucose) on Ni inhibition to SRB with sulfate-reducing activity tests at initial pH 5, 7 and 9 and 100 mg/L of Ni. Results indicated that the type of substrate was a significant factor affecting Ni inhibition in SRB, which was the most negligible in the lactate system, followed by glycerol, glucose, and formate. Although less significant, Ni inhibition also varied with the pH, leading for instance, to a reduction of 77% in the sulfate reducing activity for the formate system, but only of 28% for lactate at pH 5. The added substrate also influenced the precipitation kinetics and the characteristics of the precipitates, reaching Ni precipitation extents above 95%, except for glucose (83.2%).


Assuntos
Desulfovibrio , Glicerol , Formiatos , Glucose , Lactatos , Sulfatos
2.
BMC Biotechnol ; 20(1): 12, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111201

RESUMO

BACKGROUND: Sugarcane bagasse is a major source of lignocellulosic biomass, yet its economic potential is not fully realised. To add value to bagasse, processing is needed to gain access to the embodied recalcitrant biomaterials. When bagasse is stored in piles in the open for long periods it is colonised by microbes originating from the sugarcane, the soil nearby or spores in the environment. For these microorganisms to proliferate they must digest the bagasse to access carbon for growth. The microbial community in bagasse piles is thus a potential resource for the discovery of useful and novel microbes and industrial enzymes. We used culturing and metabarcoding to understand the diversity of microorganisms found in a uniquely undisturbed bagasse storage pile and screened the cultured organisms for fibre-degrading enzymes. RESULTS: Samples collected from 60 to 80 cm deep in the bagasse pile showed hemicellulose and partial lignin degradation. One hundred and four microbes were cultured from different layers and included a high proportion of oleaginous yeast and biomass-degrading fungi. Overall, 70, 67, 70 and 57% of the microbes showed carboxy-methyl cellulase, xylanase, laccase and peroxidase activity, respectively. These percentages were higher in microbes selectively cultured from deep layers, with all four activities found for 44% of these organisms. Culturing and amplicon sequencing showed that there was less diversity and therefore more selection in the deeper layers, which were dominated by thermophiles and acid tolerant organisms, compared with the top of pile. Amplicon sequencing indicated that novel fungi were present in the pile. CONCLUSIONS: A combination of culture-dependent and independent methods was successful in exploring the diversity in the bagasse pile. The variety of species that was found and that are known for biomass degradation shows that the bagasse pile was a valuable selective environment for the identification of new microbes and enzymes with biotechnological potential. In particular, lignin-modifying activities have not been reported previously for many of the species that were identified, suggesting future studies are warranted.


Assuntos
Bactérias/crescimento & desenvolvimento , Celulose/química , Fungos/crescimento & desenvolvimento , Saccharum/metabolismo , Análise de Sequência de DNA/métodos , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Carbono/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/classificação , Fungos/isolamento & purificação , Fungos/metabolismo , Hidrólise , Técnicas Microbiológicas , Filogenia , Microbiologia do Solo
3.
J Ind Microbiol Biotechnol ; 47(12): 1059-1073, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33175241

RESUMO

Tetanus is a fatal disease caused by Clostridium tetani infections. To prevent infections, a toxoid vaccine, developed almost a century ago, is routinely used in humans and animals. The vaccine is listed in the World Health Organisation list of Essential Medicines and can be produced and administered very cheaply in the developing world for less than one US Dollar per dose. Recent developments in both analytical tools and frameworks for systems biology provide industry with an opportunity to gain a deeper understanding of the parameters that determine C. tetani virulence and physiological behaviour in bioreactors. Here, we compared a traditional fermentation process with a fermentation medium supplemented with five heavily consumed amino acids. The experiment demonstrated that amino acid catabolism plays a key role in the virulence of C. tetani. The addition of the five amino acids favoured growth, decreased toxin production and changed C. tetani morphology. Using time-course transcriptomics, we created a "fermentation map", which shows that the tetanus toxin transcriptional regulator BotR, P21 and the tetanus toxin gene was downregulated. Moreover, this in-depth analysis revealed potential genes that might be involved in C. tetani virulence regulation. We observed differential expression of genes related to cell separation, surface/cell adhesion, pyrimidine biosynthesis and salvage, flagellar motility, and prophage genes. Overall, the fermentation map shows that, mediated by free amino acid concentrations, virulence in C. tetani is regulated at the transcriptional level and affects a plethora of metabolic functions.


Assuntos
Aminoácidos , Clostridium tetani , Aminoácidos/metabolismo , Animais , Clostridium tetani/genética , Clostridium tetani/metabolismo , Clostridium tetani/patogenicidade , Humanos , Toxina Tetânica/biossíntese , Toxina Tetânica/genética , Transcriptoma
4.
Metab Eng ; 53: 14-23, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30641139

RESUMO

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.


Assuntos
Monóxido de Carbono/metabolismo , Clostridium , Hidrogênio/metabolismo , Hidroxibutiratos/metabolismo , Engenharia Metabólica , Poliésteres/metabolismo , Clostridium/genética , Clostridium/metabolismo , Metabolismo Energético/genética
5.
Appl Environ Microbiol ; 84(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29330186

RESUMO

Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs).IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.


Assuntos
Proteínas Fúngicas/genética , Pichia/genética , Proteínas Fúngicas/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Pichia/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Plant Biotechnol J ; 14(2): 567-80, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26015295

RESUMO

In planta production of the bioplastic polyhydroxybutyrate (PHB) is one important way in which plant biotechnology can address environmental problems and emerging issues related to peak oil. However, high biomass C4 plants such as maize, switch grass and sugarcane develop adverse phenotypes including stunting, chlorosis and reduced biomass as PHB levels in leaves increase. In this study, we explore limitations to PHB accumulation in sugarcane chloroplasts using a systems biology approach, coupled with a metabolic model of C4 photosynthesis. Decreased assimilation was evident in high PHB-producing sugarcane plants, which also showed a dramatic decrease in sucrose and starch content of leaves. A subtle decrease in the C/N ratio was found which was not associated with a decrease in total protein content. An increase in amino acids used for nitrogen recapture was also observed. Based on the accumulation of substrates of ATP-dependent reactions, we hypothesized ATP starvation in bundle sheath chloroplasts. This was supported by mRNA differential expression patterns. The disruption in ATP supply in bundle sheath cells appears to be linked to the physical presence of the PHB polymer which may disrupt photosynthesis by scattering photosynthetically active radiation and/or physically disrupting thylakoid membranes.


Assuntos
Carbono/metabolismo , Engenharia Metabólica/métodos , Modelos Biológicos , Folhas de Planta/metabolismo , Saccharum/metabolismo , Biologia de Sistemas/métodos , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Ritmo Circadiano , Regulação da Expressão Gênica de Plantas , Hidroxibutiratos/metabolismo , Metaboloma , Nitrogênio/metabolismo , Fotossíntese , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharum/genética
7.
Appl Environ Microbiol ; 81(10): 3316-25, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25746998

RESUMO

Monoterpenes are liquid hydrocarbons with applications ranging from flavor and fragrance to replacement jet fuel. Their toxicity, however, presents a major challenge for microbial synthesis. Here we evolved limonene-tolerant Saccharomyces cerevisiae strains and sequenced six strains across the 200-generation evolutionary time course. Mutations were found in the tricalbin proteins Tcb2p and Tcb3p. Genomic reconstruction in the parent strain showed that truncation of a single protein (tTcb3p(1-989)), but not its complete deletion, was sufficient to recover the evolved phenotype improving limonene fitness 9-fold. tTcb3p(1-989) increased tolerance toward two other monoterpenes (ß-pinene and myrcene) 11- and 8-fold, respectively, and tolerance toward the biojet fuel blend AMJ-700t (10% cymene, 50% limonene, 40% farnesene) 4-fold. tTcb3p(1-989) is the first example of successful engineering of phase tolerance and creates opportunities for production of the highly toxic C10 alkenes in yeast.


Assuntos
Evolução Biológica , Hidrocarbonetos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cicloexenos/metabolismo , Limoneno , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Engenharia Metabólica , Monoterpenos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo
8.
Microb Biotechnol ; 17(4): e14452, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38568755

RESUMO

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2-to-ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.


Assuntos
Dióxido de Carbono , Clostridium , Proteoma , Dióxido de Carbono/metabolismo , Monóxido de Carbono/metabolismo , Hidrogênio/metabolismo , Fermentação , Etanol/metabolismo , Metaboloma
9.
BMC Genomics ; 14: 699, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-24118942

RESUMO

BACKGROUND: Accurate bacterial genome annotations provide a framework to understanding cellular functions, behavior and pathogenicity and are essential for metabolic engineering. Annotations based only on in silico predictions are inaccurate, particularly for large, high G + C content genomes due to the lack of similarities in gene length and gene organization to model organisms. RESULTS: Here we describe a 2D systems biology driven re-annotation of the Saccharopolyspora erythraea genome using proteogenomics, a genome-scale metabolic reconstruction, RNA-sequencing and small-RNA-sequencing. We observed transcription of more than 300 intergenic regions, detected 59 peptides in intergenic regions, confirmed 164 open reading frames previously annotated as hypothetical proteins and reassigned function to open reading frames using the genome-scale metabolic reconstruction. Finally, we present a novel way of mapping ribosomal binding sites across the genome by sequencing small RNAs. CONCLUSIONS: The work presented here describes a novel framework for annotation of the Saccharopolyspora erythraea genome. Based on experimental observations, the 2D annotation framework greatly reduces errors that are commonly made when annotating large-high G + C content genomes using computational prediction algorithms.


Assuntos
Genoma Bacteriano/genética , Anotação de Sequência Molecular/métodos , Saccharopolyspora/genética , Biologia de Sistemas/métodos , Composição de Bases/genética , Sítios de Ligação/genética , DNA Intergênico/genética , Genes Bacterianos/genética , Fases de Leitura Aberta/genética , Proteômica , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Software
10.
BMC Genomics ; 14: 15, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23324121

RESUMO

BACKGROUND: Actinobacteria form a major bacterial phylum that includes numerous human pathogens. Actinobacteria are primary contributors to carbon cycling and also represent a primary source of industrial high value products such as antibiotics and biopesticides. Consistent with other members of the actinobacterial phylum, Saccharopolyspora erythraea undergo a transitional switch. This switch is characterized by numerous metabolic and morphological changes. RESULTS: We performed RNA sequencing to analyze the transcriptional changes that occur during growth of Saccharopolyspora erythraea in batch culture. By sequencing RNA across the fermentation time course, at a mean coverage of 4000X, we found the vast majority of genes to be prominently expressed, showing that we attained close to saturating sequencing coverage of the transcriptome. During the metabolic switch, global changes in gene expression influence the metabolic machinery of Saccharopolyspora erythraea, resetting an entirely novel gene expression program. After the switch, global changes include the broad repression of half the genes regulated by complex transcriptional mechanisms. Paralogous transposon clusters, delineate these transcriptional programs. The new transcriptional program is orchestrated by a bottleneck event during which mRNA levels are severely restricted by targeted mRNA degradation. CONCLUSIONS: Our results, which attained close to saturating sequencing coverage of the transcriptome, revealed unanticipated transcriptional complexity with almost one third of transcriptional content originating from un-annotated sequences. We showed that the metabolic switch is a sophisticated mechanism of transcriptional regulation capable of resetting and re-synchronizing gene expression programs at extraordinary speed and scale.


Assuntos
Genoma Bacteriano , Estabilidade de RNA/genética , Saccharopolyspora/genética , Transcrição Gênica , Eritromicina/biossíntese , Eritromicina/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes de Troca , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Redes e Vias Metabólicas/genética , Saccharopolyspora/patogenicidade
11.
Mol Omics ; 18(3): 226-236, 2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-34989730

RESUMO

The emergence of multidrug-resistant pathogenic bacteria creates a demand for novel antibiotics with distinct mechanisms of action. Advances in next-generation genome sequencing promised a paradigm shift in the quest to find new bioactive secondary metabolites. Genome mining has proven successful for predicting putative biosynthetic elements in secondary metabolite superproducers such as Streptomycetes. However, genome mining approaches do not inform whether biosynthetic gene clusters are dormant or active under given culture conditions. Here we show that using a multi-omics approach in combination with antiSMASH, it is possible to assess the secondary metabolic potential of a Streptomyces strain capable of producing mannopeptimycin, an important cyclic peptide effective against Gram-positive infections. The genome of Streptomyces hygroscopicus NRRL 30439 was first sequenced using PacBio RSII to obtain a closed genome. A chemically defined medium was then used to elicit a nutrient stress response in S. hygroscopicus NRRL 30439. Detailed extracellular metabolomics and intracellular proteomics were used to profile and segregate primary and secondary metabolism. Our results demonstrate that the combination of genomics, proteomics and metabolomics enables rapid evaluation of a strain's performance in bioreactors for industrial production of secondary metabolites.


Assuntos
Streptomyces , Genômica , Família Multigênica , Metabolismo Secundário/genética , Streptomyces/genética , Streptomyces/metabolismo
12.
BMC Genomics ; 12: 446, 2011 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-21906285

RESUMO

BACKGROUND: Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported. RESULTS: De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes. CONCLUSION: This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, DsIV genes expressed in the host and also improves our current understanding of this host-parasitoid interaction.


Assuntos
Interações Hospedeiro-Parasita/genética , Mariposas/genética , Mariposas/parasitologia , Polydnaviridae/genética , Transcriptoma , Vespas/fisiologia , Animais , Perfilação da Expressão Gênica , Genes de Insetos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Larva/genética , Larva/parasitologia , Larva/virologia , Mariposas/virologia , Análise de Sequência de DNA/métodos , Vespas/virologia
13.
BMC Genomics ; 12 Suppl 4: S5, 2011 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-22369158

RESUMO

BACKGROUND: Microalgae have the potential to deliver biofuels without the associated competition for land resources. In order to realise the rates and titres necessary for commercial production, however, system-level metabolic engineering will be required. Genome scale metabolic reconstructions have revolutionized microbial metabolic engineering and are used routinely for in silico analysis and design. While genome scale metabolic reconstructions have been developed for many prokaryotes and model eukaryotes, the application to less well characterized eukaryotes such as algae is challenging not at least due to a lack of compartmentalization data. RESULTS: We have developed a genome-scale metabolic network model (named AlgaGEM) covering the metabolism for a compartmentalized algae cell based on the Chlamydomonas reinhardtii genome. AlgaGEM is a comprehensive literature-based genome scale metabolic reconstruction that accounts for the functions of 866 unique ORFs, 1862 metabolites, 2249 gene-enzyme-reaction-association entries, and 1725 unique reactions. The reconstruction was compartmentalized into the cytoplasm, mitochondrion, plastid and microbody using available data for algae complemented with compartmentalisation data for Arabidopsis thaliana. AlgaGEM describes a functional primary metabolism of Chlamydomonas and significantly predicts distinct algal behaviours such as the catabolism or secretion rather than recycling of phosphoglycolate in photorespiration. AlgaGEM was validated through the simulation of growth and algae metabolic functions inferred from literature. Using efficient resource utilisation as the optimality criterion, AlgaGEM predicted observed metabolic effects under autotrophic, heterotrophic and mixotrophic conditions. AlgaGEM predicts increased hydrogen production when cyclic electron flow is disrupted as seen in a high producing mutant derived from mutational studies. The model also predicted the physiological pathway for H2 production and identified new targets to further improve H2 yield. CONCLUSIONS: AlgaGEM is a viable and comprehensive framework for in silico functional analysis and can be used to derive new, non-trivial hypotheses for exploring this metabolically versatile organism. Flux balance analysis can be used to identify bottlenecks and new targets to metabolically engineer microalgae for production of biofuels.


Assuntos
Chlamydomonas reinhardtii/genética , Genoma , Arabidopsis/genética , Biocombustíveis , Chlamydomonas reinhardtii/metabolismo , Bases de Dados Factuais , Hidrogênio/metabolismo , Redes e Vias Metabólicas/genética , Modelos Genéticos
14.
Plant Physiol ; 152(2): 579-89, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20044452

RESUMO

Genome-scale metabolic network models have been successfully used to describe metabolism in a variety of microbial organisms as well as specific mammalian cell types and organelles. This systems-based framework enables the exploration of global phenotypic effects of gene knockouts, gene insertion, and up-regulation of gene expression. We have developed a genome-scale metabolic network model (AraGEM) covering primary metabolism for a compartmentalized plant cell based on the Arabidopsis (Arabidopsis thaliana) genome. AraGEM is a comprehensive literature-based, genome-scale metabolic reconstruction that accounts for the functions of 1,419 unique open reading frames, 1,748 metabolites, 5,253 gene-enzyme reaction-association entries, and 1,567 unique reactions compartmentalized into the cytoplasm, mitochondrion, plastid, peroxisome, and vacuole. The curation process identified 75 essential reactions with respective enzyme associations not assigned to any particular gene in the Kyoto Encyclopedia of Genes and Genomes or AraCyc. With the addition of these reactions, AraGEM describes a functional primary metabolism of Arabidopsis. The reconstructed network was transformed into an in silico metabolic flux model of plant metabolism and validated through the simulation of plant metabolic functions inferred from the literature. Using efficient resource utilization as the optimality criterion, AraGEM predicted the classical photorespiratory cycle as well as known key differences between redox metabolism in photosynthetic and nonphotosynthetic plant cells. AraGEM is a viable framework for in silico functional analysis and can be used to derive new, nontrivial hypotheses for exploring plant metabolism.


Assuntos
Arabidopsis/metabolismo , Biologia Computacional/métodos , Redes e Vias Metabólicas , Modelos Genéticos , Arabidopsis/genética , Simulação por Computador , Genoma de Planta
15.
Plant Physiol ; 154(4): 1871-85, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20974891

RESUMO

Leaves of C(4) grasses (such as maize [Zea mays], sugarcane [Saccharum officinarum], and sorghum [Sorghum bicolor]) form a classical Kranz leaf anatomy. Unlike C(3) plants, where photosynthetic CO(2) fixation proceeds in the mesophyll (M), the fixation process in C(4) plants is distributed between two cell types, the M cell and the bundle sheath (BS) cell. Here, we develop a C(4) genome-scale model (C4GEM) for the investigation of flux distribution in M and BS cells during C(4) photosynthesis. C4GEM, to our knowledge, is the first large-scale metabolic model that encapsulates metabolic interactions between two different cell types. C4GEM is based on the Arabidopsis (Arabidopsis thaliana) model (AraGEM) but has been extended by adding reactions and transporters responsible to represent three different C(4) subtypes (NADP-ME [for malic enzyme], NAD-ME, and phosphoenolpyruvate carboxykinase). C4GEM has been validated for its ability to synthesize 47 biomass components and consists of 1,588 unique reactions, 1,755 metabolites, 83 interorganelle transporters, and 29 external transporters (including transport through plasmodesmata). Reactions in the common C(4) model have been associated with well-annotated C(4) species (NADP-ME subtypes): 3,557 genes in sorghum, 11,623 genes in maize, and 3,881 genes in sugarcane. The number of essential reactions not assigned to genes is 131, 135, and 156 in sorghum, maize, and sugarcane, respectively. Flux balance analysis was used to assess the metabolic activity in M and BS cells during C(4) photosynthesis. Our simulations were consistent with chloroplast proteomic studies, and C4GEM predicted the classical C(4) photosynthesis pathway and its major effect in organelle function in M and BS. The model also highlights differences in metabolic activities around photosystem I and photosystem II for three different C(4) subtypes. Effects of CO(2) leakage were also explored. C4GEM is a viable framework for in silico analysis of cell cooperation between M and BS cells during photosynthesis and can be used to explore C(4) plant metabolism.


Assuntos
Arabidopsis/genética , Genoma de Planta , Modelos Biológicos
16.
Front Genet ; 12: 610116, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995471

RESUMO

Spermatogenesis relies on complex molecular mechanisms, essential for the genesis and differentiation of the male gamete. Germ cell differentiation starts at the testicular parenchyma and finishes in the epididymis, which has three main regions: head, body, and tail. RNA-sequencing data of the testicular parenchyma (TP), head epididymis (HE), and tail epididymis (TE) from four bulls (three biopsies per bull: 12 samples) were subjected to differential expression analyses, functional enrichment analyses, and co-expression analyses. The aim was to investigate the co-expression and infer possible regulatory roles for transcripts involved in the spermatogenesis of Bos indicus bulls. Across the three pairwise comparisons, 3,826 differentially expressed (DE) transcripts were identified, of which 384 are small RNAs. Functional enrichment analysis pointed to gene ontology (GO) terms related to ion channel activity, detoxification of copper, neuroactive receptors, and spermatogenesis. Using the regulatory impact factor (RIF) algorithm, we detected 70 DE small RNAs likely to regulate the DE transcripts considering all pairwise comparisons among tissues. The pattern of small RNA co-expression suggested that these elements are involved in spermatogenesis regulation. The 3,826 DE transcripts (mRNAs and small RNAs) were further subjected to co-expression analyses using the partial correlation and information theory (PCIT) algorithm for network prediction. Significant correlations underpinned the co-expression network, which had 2,216 transcripts connected by 158,807 predicted interactions. The larger network cluster was enriched for male gamete generation and had 15 miRNAs with significant RIF. The miRNA bta-mir-2886 showed the highest number of connections (601) and was predicted to down-regulate ELOVL3, FEZF2, and HOXA13 (negative co-expression correlations and confirmed with TargetScan). In short, we suggest that bta-mir-2886 and other small RNAs might modulate gene expression in the testis and epididymis, in Bos indicus cattle.

17.
Genes (Basel) ; 11(10)2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32977700

RESUMO

Propionibacteria have been studied extensively since the early 1930s due to their relevance to industry and importance as human pathogens. Still, their unique metabolism is far from fully understood. This is partly due to their signature high GC content, which has previously hampered the acquisition of quality sequence data, the accurate annotation of the available genomes, and the functional characterization of genes. The recent completion of the genome sequences for several species has led researchers to reassess the taxonomical classification of the genus Propionibacterium, which has been divided into several new genres. Such data also enable a comparative genomic approach to annotation and provide a new opportunity to revisit our understanding of their metabolism. Using pan-genome analysis combined with the reconstruction of the first high-quality Propionibacterium genome-scale metabolic model and a pan-metabolic model of current and former members of the genus Propionibacterium, we demonstrate that despite sharing unique metabolic traits, these organisms have an unexpected diversity in central carbon metabolism and a hidden layer of metabolic complexity. This combined approach gave us new insights into the evolution of Propionibacterium metabolism and led us to propose a novel, putative ferredoxin-linked energy conservation strategy. The pan-genomic approach highlighted key differences in Propionibacterium metabolism that reflect adaptation to their environment. Results were mathematically captured in genome-scale metabolic reconstructions that can be used to further explore metabolism using metabolic modeling techniques. Overall, the data provide a platform to explore Propionibacterium metabolism and a tool for the rational design of strains.


Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Redes e Vias Metabólicas , Propionibacterium/metabolismo , Proteínas de Bactérias/genética , Composição de Bases , Mapeamento Cromossômico , DNA Bacteriano/análise , Humanos , Filogenia , Propionibacterium/classificação , Propionibacterium/genética , Propionibacterium/crescimento & desenvolvimento
18.
Front Microbiol ; 10: 2549, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31803150

RESUMO

Acetogens can fix carbon (CO or CO2) into acetyl-CoA via the Wood-Ljungdahl pathway (WLP) that also makes them attractive cell factories for the production of fuels and chemicals from waste feedstocks. Although most biochemical details of the WLP are well understood and systems-level characterization of acetogen metabolism has recently improved, key transcriptional features such as promoter motifs and transcriptional regulators are still unknown in acetogens. Here, we use differential RNA-sequencing to identify a previously undescribed promoter motif associated with essential genes for autotrophic growth of the model-acetogen Clostridium autoethanogenum. RNA polymerase was shown to bind to the new promoter motif using a DNA-binding protein assay and proteomics enabled the discovery of four candidates to potentially function directly in control of transcription of the WLP and other key genes of C1 fixation metabolism. Next, in vivo experiments showed that a TetR-family transcriptional regulator (CAETHG_0459) and the housekeeping sigma factor (σA) activate expression of a reporter protein (GFP) in-frame with the new promoter motif from a fusion vector in Escherichia coli. Lastly, a protein-protein interaction assay with the RNA polymerase (RNAP) shows that CAETHG_0459 directly binds to the RNAP. Together, the data presented here advance the fundamental understanding of transcriptional regulation of C1 fixation in acetogens and provide a strategy for improving the performance of gas-fermenting bacteria by genetic engineering.

19.
Biotechnol J ; 13(3): e1700231, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29316330

RESUMO

The development of next-generation sequencing technologies has opened new opportunities to better characterize complex eukaryotic cells. Chinese hamster ovary (CHO) cells play a primary role in therapeutic protein production, with currently five of the top ten blockbuster drugs produced in CHO. However, engineering superior CHO cells with improved production features has had limited success to date and cell lines are still developed through the generation and screening of large strain pools. Here, we applied RNA sequencing to contrast a high and a low monoclonal antibody producing cell line. Rigorous experimental design achieved high reproducibility between biological replicates, remarkably reducing variation to less than 10%. More than 14 000 gene-transcripts are identified and surprisingly 58% are classified as differentially expressed, including 2900 with a fold change higher than 1.5. The largest differences are found for gene-transcripts belonging to regulation of apoptosis, cell death, and protein intracellular transport GO term classifications, which are found to be significantly enriched in the high producing cell line. RNA sequencing is also performed on subclones, where down-regulation of genes encoding secreted glycoproteins is found to be the most significant change. The large number of significant differences even between subclones challenges the notion of identifying and manipulating a few key genes to generate high production CHO cell lines.


Assuntos
Anticorpos Monoclonais/biossíntese , Células CHO , Evolução Clonal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Linhagem da Célula/genética , Cricetulus
20.
Plant Sci ; 273: 50-60, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29907309

RESUMO

The compartmentalization of C4 plants increases photosynthetic efficiency, while constraining how material and energy must flow in leaf tissues. To capture this metabolic phenomenon, a generic plant metabolic reconstruction was replicated into four connected spatiotemporal compartments, namely bundle sheath (B) and mesophyll (M) across the day and night cycle. The C4 leaf model was used to explore how amenable polyhydroxybutyrate (PHB) production is with these four compartments working cooperatively. A strategic pattern of metabolite conversion and exchange emerged from a systems-level network that has very few constraints imposed; mainly the sequential two-step carbon capture in mesophyll, then bundle sheath and photosynthesis during the day only. The building of starch reserves during the day and their mobilization during the night connects day and night metabolism. Flux simulations revealed that PHB production did not require rerouting of metabolic pathways beyond what is already utilised for growth. PHB yield was sensitive to photoassimilation capacity, availability of carbon reserves, ATP maintenance, relative photosynthetic activity of B and M, and type of metabolites exchanged in the plasmodesmata, but not sensitive towards compartmentalization. Hence, the compartmentalization issues currently encountered are likely to be kinetic or thermodynamic limitations rather than stoichiometric.


Assuntos
Hidroxibutiratos/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Poaceae/genética , Ritmo Circadiano , Células do Mesofilo/metabolismo , Análise do Fluxo Metabólico , Modelos Biológicos , Fotossíntese/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Feixe Vascular de Plantas/genética , Feixe Vascular de Plantas/metabolismo , Poaceae/metabolismo
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