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1.
Nat Commun ; 11(1): 4050, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792485

RESUMO

Regulatory networks describe the hierarchical relationship between transcription factors, associated proteins, and their target genes. Regulatory networks respond to environmental and genetic perturbations by reprogramming cellular metabolism. Here we design, construct, and map a comprehensive regulatory network library containing 110,120 specific mutations in 82 regulators expected to perturb metabolism. We screen the library for different targeted phenotypes, and identify mutants that confer strong resistance to various inhibitors, and/or enhanced production of target compounds. These improvements are identified in a single round of selection, showing that the regulatory network library is universally applicable and is convenient and effective for engineering targeted phenotypes. The facile construction and mapping of the regulatory network library provides a path for developing a more detailed understanding of global regulation in E. coli, with potential for adaptation and use in less-understood organisms, expanding toolkits for future strain engineering, synthetic biology, and broader efforts.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Engenharia Metabólica/métodos , Biologia Sintética/métodos , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia
2.
Sci Adv ; 6(30): eaba6884, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32832666

RESUMO

More than 1050 clinical trials are registered at FDA.gov that explore multipotent mesenchymal stromal cells (MSCs) for nearly every clinical application imaginable, including neurodegenerative and cardiac disorders, perianal fistulas, graft-versus-host disease, COVID-19, and cancer. Several companies have or are in the process of commercializing MSC-based therapies. However, most of the clinical-stage MSC therapies have been unable to meet primary efficacy end points. The innate therapeutic functions of MSCs administered to humans are not as robust as demonstrated in preclinical studies, and in general, the translation of cell-based therapy is impaired by a myriad of steps that introduce heterogeneity. In this review, we discuss the major clinical challenges with MSC therapies, the details of these challenges, and the potential bioengineering approaches that leverage the unique biology of MSCs to overcome the challenges and achieve more potent and versatile therapies.


Assuntos
Betacoronavirus , Infecções por Coronavirus/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Pneumonia Viral/terapia , Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Infecções por Coronavirus/virologia , Doença Enxerto-Hospedeiro/terapia , Humanos , Engenharia Metabólica/métodos , Pandemias , Pneumonia Viral/virologia , Transplantados
3.
PLoS Comput Biol ; 16(8): e1008125, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776925

RESUMO

In the growing field of metabolic engineering, where cells are treated as 'factories' that synthesize industrial compounds, it is essential to consider the ability of the cells' native metabolism to accommodate the demands of synthetic pathways, as these pathways will alter the homeostasis of cellular energy and electron metabolism. From the breakdown of substrate, microorganisms activate and reduce key co-factors such as ATP and NAD(P)H, which subsequently need to be hydrolysed and oxidized, respectively, in order to restore cellular balance. A balanced supply and consumption of such co-factors, here termed co-factor balance, will influence biotechnological performance. To aid the strain selection and design process, we used stoichiometric modelling (FBA, pFBA, FVA and MOMA) and the Escherichia coli (E.coli) core stoichiometric model to investigate the network-wide effect of butanol and butanol precursor production pathways differing in energy and electron demand on product yield. An FBA-based co-factor balance assessment (CBA) algorithm was developed to track and categorise how ATP and NAD(P)H pools are affected in the presence of a new pathway. CBA was compared to the balance calculations proposed by Dugar et al. (Nature Biotechnol. 29 (12), 1074-1078). Predicted solutions were compromised by excessively underdetermined systems, displaying greater flexibility in the range of reaction fluxes than experimentally measured by 13C-metabolic flux analysis (MFA) and the appearance of unrealistic futile co-factor cycles. With the assumption that futile cycles are tightly regulated in reality, the FBA models were manually constrained in a step-wise manner. Solutions with minimal futile cycling diverted surplus energy and electrons towards biomass formation. As an alternative, the use of loopless FBA or constraining the models with measured flux ranges were tried but did not prevent futile co-factor cycles. The results highlight the need to account for co-factor imbalance and confirm that better-balanced pathways with minimal diversion of surplus towards biomass formation present the highest theoretical yield. The analysis also suggests that ATP and NAD(P)H balancing cannot be assessed in isolation from each other, or even from the balance of additional co-factors such as AMP and ADP. We conclude that, through revealing the source of co-factor imbalance CBA can facilitate pathway and host selection when designing new biocatalysts for implementation by metabolic engineering.


Assuntos
Simulação por Computador , Escherichia coli , Engenharia Metabólica/métodos , Algoritmos , Biomassa , Butanóis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Análise do Fluxo Metabólico , Redes e Vias Metabólicas , Modelos Biológicos
4.
PLoS Comput Biol ; 16(7): e1008110, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716928

RESUMO

The concept of minimal cut sets (MCS) provides a flexible framework for analyzing properties of metabolic networks and for computing metabolic intervention strategies. In particular, it has been used to support the targeted design of microbial strains for bio-based production processes. Herein we present a number of major extensions that generalize the existing MCS approach and broaden its scope for applications in metabolic engineering. We first introduce a modified approach to integrate gene-protein-reaction associations (GPR) in the metabolic network structure for the computation of gene-based intervention strategies. In particular, we present a set of novel compression rules for GPR associations, which effectively speedup the computation of gene-based MCS by a factor of up to one order of magnitude. These rules are not specific for MCS and as well applicable to other computational strain design methods. Second, we enhance the MCS framework by allowing the definition of multiple target (undesired) and multiple protected (desired) regions. This enables precise tailoring of the metabolic solution space of the designed strain with unlimited flexibility. Together with further generalizations such as individual cost factors for each intervention, direct combinations of reaction/gene deletions and additions as well as the possibility to search for substrate co-feeding strategies, the scope of the MCS framework could be broadly extended. We demonstrate the applicability and performance benefits of the described developments by computing (gene-based) Escherichia coli strain designs for the bio-based production of 2,3-butanediol, a chemical, that has recently received much attention in the field of metabolic engineering. With our extended framework, we could identify promising strain designs that were formerly unpredictable, including those based on substrate co-feeding.


Assuntos
Escherichia coli/genética , Deleção de Genes , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Trifosfato de Adenosina/química , Aerobiose , Algoritmos , Butileno Glicóis/farmacologia , Simulação por Computador , Microbiologia Industrial , Modelos Biológicos , Modelos Estatísticos , Oxirredução , Processos Estocásticos
5.
Nat Commun ; 11(1): 3313, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620759

RESUMO

Ectoine, a compatible solute synthesized by many halophiles for hypersalinity resistance, has been successfully produced by metabolically engineered Halomonas bluephagenesis, which is a bioplastic poly(3-hydroxybutyrate) producer allowing open unsterile and continuous conditions. Here we report a de novo synthesis pathway for ectoine constructed into the chromosome of H. bluephagenesis utilizing two inducible systems, which serve to fine-tune the transcription levels of three clusters related to ectoine synthesis, including ectABC, lysC and asd based on a GFP-mediated transcriptional tuning approach. Combined with bypasses deletion, the resulting recombinant H. bluephagenesis TD-ADEL-58 is able to produce 28 g L-1 ectoine during a 28 h fed-batch growth process. Co-production of ectoine and PHB is achieved to 8 g L-1 ectoine and 32 g L-1 dry cell mass containing 75% PHB after a 44 h growth. H. bluephagenesis demonstrates to be a suitable co-production chassis for polyhydroxyalkanoates and non-polymer chemicals such as ectoine.


Assuntos
Diamino Aminoácidos/biossíntese , Halomonas/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Biomassa , Vias Biossintéticas/genética , Cromatografia Líquida/métodos , Halomonas/genética , Halomonas/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Poli-Hidroxialcanoatos/química , Poli-Hidroxialcanoatos/metabolismo , Espectrometria de Massas em Tandem/métodos
6.
Gene ; 759: 144993, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32717311

RESUMO

Plants generate many secondary metabolites, so called phyto-metabolites, which can be used as toxins, dyes, drugs, and insecticides in bio-warfare plus bio-terrorism, industry, medicine, and agriculture, respectively. To 2013, the first generation metabolic engineering approaches like miRNA-based manipulation were widely adopted by researchers in biosciences. However, the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) genome editing system revolutionized metabolic engineering due to its unique features so that scientists could manipulate the biosynthetic pathways of phyto-metabolites through approaches like miRNA-mediated CRISPR-Cas9. According to the increasing importance of the genome editing in plant sciences, we discussed the current findings on CRISPR-based manipulation of phyto-metabolites in plants, especially medicinal ones, and suggested the ideas to phyto-metabolic editing.


Assuntos
Sistemas CRISPR-Cas , Engenharia Metabólica/métodos , Melhoramento Vegetal/métodos , Edição de Genes/métodos
7.
Nat Commun ; 11(1): 3120, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561727

RESUMO

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L-1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum, changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L-1. Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers.


Assuntos
Corynebacterium glutamicum/enzimologia , Glucose/metabolismo , Ácido Hialurônico/biossíntese , Engenharia Metabólica/métodos , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Metabolismo dos Carboidratos/genética , Corynebacterium glutamicum/genética , Meios de Cultura/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Hialuronoglucosaminidase/metabolismo , Redes e Vias Metabólicas/genética , Polissacarídeos Bacterianos/biossíntese
8.
Nat Commun ; 11(1): 3138, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561745

RESUMO

Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits.


Assuntos
Proteínas de Bactérias/genética , Engenharia Metabólica/métodos , Proteoma/genética , Biologia Sintética/métodos , Proteínas de Bactérias/metabolismo , Sistema Livre de Células/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Redes e Vias Metabólicas/genética , Biossíntese de Proteínas/genética , Proteoma/metabolismo
9.
PLoS One ; 15(5): e0233492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469948

RESUMO

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.


Assuntos
Kluyveromyces/genética , Kluyveromyces/metabolismo , Engenharia Metabólica/métodos , Polissacarídeos/biossíntese , Polissacarídeos/química , Biotecnologia , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Fúngicos , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Manosidases/genética , Manosidases/metabolismo , Manosiltransferases/antagonistas & inibidores , Manosiltransferases/genética , Manosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
10.
Gene ; 753: 144813, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32470504

RESUMO

Microbial production of bio-based ingredients often requires metabolically engineered bacterial strains with the edited genome. Genome editing tools are also essential for gene identification and investigating genotype-phenotype connections. Currently, one of the most common tools of genome editing is based on a natural bacterial adaptive immune system known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) due to its simple, rapid, and efficient activities. Although successful in some in vitro systems, its application as an approach of metabolic engineering and genome editing is still not so extensive. Here, we discuss existing barriers and challenges of the CRISPR/Cas9 editing tool for in vitro systems. Firstly, we aim to briefly introduce the CRISPR/Cas9 method as an in vitro gene editing tool. Next, we discuss existing obstacles to CRISPR-based editing in bacterial and in vitro model systems and offer guidelines to help achieve editing in an expanded range of in vitro systems.


Assuntos
Edição de Genes/métodos , Edição de Genes/tendências , Engenharia Metabólica/tendências , Animais , Bactérias/genética , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma Bacteriano/genética , Humanos , Engenharia Metabólica/métodos
11.
PLoS Comput Biol ; 16(4): e1007795, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282794

RESUMO

Synthetic microbial consortia have been increasingly utilized in biotechnology and experimental evidence shows that suitably engineered consortia can outperform individual species in the synthesis of valuable products. Despite significant achievements, though, a quantitative understanding of the conditions that make this possible, and of the trade-offs due to the concurrent growth of multiple species, is still limited. In this work, we contribute to filling this gap by the investigation of a known prototypical synthetic consortium. A first E. coli strain, producing a heterologous protein, is sided by a second E. coli strain engineered to scavenge toxic byproducts, thus favoring the growth of the producer at the expense of diverting part of the resources to the growth of the cleaner. The simplicity of the consortium is ideal to perform an in depth-analysis and draw conclusions of more general interest. We develop a coarse-grained mathematical model that quantitatively accounts for literature data from different key growth phenotypes. Based on this, assuming growth in chemostat, we first investigate the conditions enabling stable coexistence of both strains and the effect of the metabolic load due to heterologous protein production. In these conditions, we establish when and to what extent the consortium outperforms the producer alone in terms of productivity. Finally, we show in chemostat as well as in a fed-batch scenario that gain in productivity comes at the price of a reduced yield, reflecting at the level of the consortium resource allocation trade-offs that are well-known for individual species.


Assuntos
Engenharia Metabólica/métodos , Microbiota , Proteínas Recombinantes , Biologia Sintética/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Microbiota/genética , Microbiota/fisiologia , Modelos Biológicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Nat Commun ; 11(1): 1908, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313013

RESUMO

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.


Assuntos
Engenharia Metabólica/métodos , Proteínas Recombinantes/biossíntese , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Produtos Biológicos , Células CHO , Cromatografia , Cricetulus , Técnicas de Inativação de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Rituximab , Biologia Sintética
13.
PLoS One ; 15(4): e0231770, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298377

RESUMO

The Warburg effect, a hallmark of cancer, has recently been identified as a metabolic limitation of Chinese Hamster Ovary (CHO) cells, the primary platform for the production of monoclonal antibodies (mAb). Metabolic engineering approaches, including genetic modifications and feeding strategies, have been attempted to impose the metabolic prevalence of respiration over aerobic glycolysis. Their main objective lies in decreasing lactate production while improving energy efficiency. Although yielding promising increases in productivity, such strategies require long development phases and alter entangled metabolic pathways which singular roles remain unclear. We propose to apply drugs used for the metabolic therapy of cancer to target the Warburg effect at different levels, on CHO cells. The use of α-lipoic acid, a pyruvate dehydrogenase activator, replenished the Krebs cycle through increased anaplerosis but resulted in mitochondrial saturation. The electron shuttle function of a second drug, methylene blue, enhanced the mitochondrial capacity. It pulled on anaplerotic pathways while reducing stress signals and resulted in a 24% increase of the maximum mAb production. Finally, the combination of both drugs proved to be promising for stimulating Krebs cycle activity and mitochondrial respiration. Therefore, drugs used in metabolic therapy are valuable candidates to understand and improve the metabolic limitations of CHO-based bioproduction.


Assuntos
Anticorpos Monoclonais/biossíntese , Ciclo do Ácido Cítrico/fisiologia , Glicólise/efeitos dos fármacos , Engenharia Metabólica/métodos , Azul de Metileno/farmacologia , Ácido Tióctico/farmacologia , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Glucose/metabolismo , Glutamina/metabolismo , Glicólise/fisiologia , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Azul de Metileno/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Respiração , Ácido Tióctico/metabolismo
14.
Microbiol Res ; 236: 126455, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32179389

RESUMO

Echinenone and canthaxanthin are important carotenoid pigments with food and industrial applications. Biosynthesis of echinenone and/or canthaxanthin is catalyzed by ß-carotene ketolase (CrtO), with ß-carotene as the substrate. In this study, we generated transgenic Nostoc sp. PCC 7120 overexpressing a heterologous crtO gene from Nostoc flagelliforme and evaluated the productivity of both pigments. Normal (BG11 medium, 30 °C) and osmotic stress (BG11 medium supplemented with 0.4 M mannitol, 30 °C) conditions were used for cultivation. As compared to control strain, production of echinenone and canthaxanthin in transgenic strain were respectively increased by more than 16 % and 80 %, under either normal or osmotic stress conditions. Especially upon the stress condition, higher proportion of echinenone and canthaxanthin in total pigments was achieved, which should be beneficial for downstream separation and purification. In addition, transgenic strain showed drought tolerance and could revive from desiccation treatment after rewetting. Thus, this study provided technical clues for production of both pigments in engineered cyanobacteria as well as for cyanobacterial anhydrobiotic engineering.


Assuntos
Nostoc/genética , Organismos Geneticamente Modificados/crescimento & desenvolvimento , Organismos Geneticamente Modificados/metabolismo , Oxigenases/genética , Adaptação Fisiológica , Proteínas de Bactérias/genética , Cantaxantina/biossíntese , Carotenoides/metabolismo , Clonagem Molecular , Secas , Genes Bacterianos , Engenharia Metabólica/métodos , Nostoc/crescimento & desenvolvimento , Nostoc/metabolismo , Organismos Geneticamente Modificados/genética , Oxigenases/metabolismo , beta Caroteno/biossíntese
15.
World J Microbiol Biotechnol ; 36(3): 46, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32140791

RESUMO

Azotobacter vinelandii is a microorganism with biotechnological potential because its ability to produce alginate and polyhydroxybutyrate. Large-scale biotechnological processes are oriented to sustainable production by using biomass hydrolysates that are mainly composed by glucose and xylose. In the present study, it was observed that A. vinelandii was unable to consume xylose as the sole carbon source and that glucose assimilation in the presence of xylose was negatively affected. Adaptive Laboratory Evolution (ALE) was used as a metabolic engineering tool in A. vinelandii, to improve both carbohydrate assimilation. As a result of ALE process, the CT387 strain was obtained. The evolved strain (CT387) grown in shaken flask cultivations with xylose (8 g L-1) and glucose (2 g L-1), showed an increase of its specific growth rate (µ), as well as of its glucose and xylose uptake rates of 2, 6.45 and 3.57-fold, respectively, as compared with the parental strain. At bioreactor level, the µ, the glucose consumption rate and the relative expression of gluP that codes for the glucose permease in the evolved strain were also higher than in the native strain (1.53, 1.29 and 18-fold, respectively). Therefore, in the present study, we demonstrated the potential of ALE as a metabolic engineering tool for improving glucose and xylose consumption in A. vinelandii.


Assuntos
Azotobacter vinelandii/metabolismo , Glucose/metabolismo , Engenharia Metabólica/métodos , Xilose/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Reatores Biológicos , Meios de Cultura/química , Fermentação , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação
16.
World J Microbiol Biotechnol ; 36(3): 49, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157439

RESUMO

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.


Assuntos
Glicerol/metabolismo , Engenharia Metabólica/métodos , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Aquaporinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capsicum/microbiologia , China , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Glicerolfosfato Desidrogenase/genética , Redes e Vias Metabólicas/genética , Fenazinas/metabolismo , Proteínas Repressoras/genética , Rizosfera
17.
Proc Natl Acad Sci U S A ; 117(11): 6003-6013, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32111691

RESUMO

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.


Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/genética , Pectinas/metabolismo , Polissacarídeos/metabolismo , Fatores de Transcrição/metabolismo , Biocombustíveis , Biomassa , Repressão Catabólica , Parede Celular/química , Regulação Fúngica da Expressão Gênica , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Neurospora crassa/metabolismo , RNA-Seq
18.
Nat Commun ; 11(1): 1098, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107375

RESUMO

The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.


Assuntos
Proteínas de Bactérias/genética , Reatores Biológicos , Caulobacter crescentus/genética , Engenharia Metabólica/métodos , Modelos Químicos , Proteínas de Bactérias/metabolismo , Biocombustíveis , Metabolismo dos Carboidratos/genética , Caulobacter crescentus/enzimologia , Simulação por Computador , Hidroliases/genética , Hidroliases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Redes e Vias Metabólicas/genética , NADP/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Xilose/metabolismo
19.
BMC Bioinformatics ; 21(1): 69, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093622

RESUMO

BACKGROUND: In this paper, we explore the concept of multi-objective optimization in the field of metabolic engineering when both continuous and integer decision variables are involved in the model. In particular, we propose a multi-objective model that may be used to suggest reaction deletions that maximize and/or minimize several functions simultaneously. The applications may include, among others, the concurrent maximization of a bioproduct and of biomass, or maximization of a bioproduct while minimizing the formation of a given by-product, two common requirements in microbial metabolic engineering. RESULTS: Production of ethanol by the widely used cell factory Saccharomyces cerevisiae was adopted as a case study to demonstrate the usefulness of the proposed approach in identifying genetic manipulations that improve productivity and yield of this economically highly relevant bioproduct. We did an in vivo validation and we could show that some of the predicted deletions exhibit increased ethanol levels in comparison with the wild-type strain. CONCLUSIONS: The multi-objective programming framework we developed, called MOMO, is open-source and uses POLYSCIP (Available at http://polyscip.zib.de/). as underlying multi-objective solver. MOMO is available at http://momo-sysbio.gforge.inria.fr.


Assuntos
Engenharia Metabólica/métodos , Software , Biomassa , Etanol/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
20.
Mol Biotechnol ; 62(4): 219-227, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32103426

RESUMO

The ribosome is an essential organelle in charge of the translational processes in all kinds of cells. Currently, the scenario of its function has been significantly expanded from the classic machine for protein synthesis to a regulatory platform for quality control to maintain the protein homeostasis in a living cell. The ribosome is much more than a mechanical device with a static structure: it is inherently dynamic in structure and function, especially in response to the environmental fluctuations. Considerable effort has been made to regulate its structure and physiological function by engineering the components of a ribosome. The findings of the pioneering studies significantly deepened our understanding of a ribosome and exemplified how a ribosome could be engineered for biotechnology purposes in the era of synthetic biology. The engineering of ribosome offered highly accessible methods capable of comprehensively optimizing the performance of strains of industrial importance. In this article, the relevant recent advances were systematically reviewed.


Assuntos
Aminoácidos/química , Biotecnologia/métodos , Biossíntese de Proteínas , Ribossomos/química , Ribossomos/enzimologia , Biologia Sintética/métodos , Aminoácidos/síntese química , Aminoácidos/metabolismo , Códon sem Sentido/química , Códon sem Sentido/genética , Farmacorresistência Bacteriana/genética , Engenharia Metabólica/métodos , Engenharia de Proteínas/métodos , RNA Catalítico/biossíntese , RNA Catalítico/química , RNA Catalítico/genética , RNA Ribossômico/química , RNA de Transferência/química , RNA de Transferência/genética , Ribossomos/metabolismo
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