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1.
Zhongguo Zhong Yao Za Zhi ; 45(16): 3819-3825, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32893576

RESUMO

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in ß-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the ß-amyrin production. Compared with the control strain, the production of ß-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the ß-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of ß-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.


Assuntos
Engenharia Metabólica , Saccharomyces cerevisiae/genética , Etanol , Fermentação
2.
Nat Commun ; 11(1): 4202, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826900

RESUMO

Antibiotic biosynthetic gene clusters (BGCs) produce bioactive metabolites that impart a fitness advantage to their producer, providing a mechanism for natural selection. This selection drives antibiotic evolution and adapts BGCs for expression in different organisms, potentially providing clues to improve heterologous expression of antibiotics. Here, we use phage-assisted continuous evolution (PACE) to achieve bioactivity-dependent adaptation of the BGC for the antibiotic bicyclomycin (BCM), facilitating improved production in a heterologous host. This proof-of-principle study demonstrates that features of natural bioactivity-dependent evolution can be engineered to access unforeseen routes of improving metabolic pathways and product yields.


Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Família Multigênica , Produtos Biológicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Clonagem Molecular , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo
3.
Bioresour Technol ; 317: 123991, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32805480

RESUMO

Squalene is the precursor for triterpene-based natural products and steroids-based drugs. It has been widely used as pharmaceutical intermediates and personal care products. The aim of this work is to test the feasibility of engineering Yarrowia lipolytica as a potential host for squalene production. The bottleneck of the pathway was removed by overexpressing native HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase. With the recycling of NADPH from the mannitol cycle, the engineered strain produced about 180.3 mg/L and 188.2 mg/L squalene from glucose or acetate minimal media. By optimizing the C/N ratio, controlling the media pH and mitigating acetyl-CoA flux competition from lipogenesis, the engineered strain produced 502.7 mg/L squalene, a 28-fold increase over the parental strain (17.2 mg/L). This work may serve as a baseline to harness Y. lipolytica as an oleaginous cell factory for sustainable production of squalene or terpenoids-based chemicals and natural products.


Assuntos
Yarrowia , Acetilcoenzima A , Engenharia Metabólica , Esqualeno , Terpenos , Yarrowia/genética
4.
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
5.
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
6.
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
7.
PLoS Comput Biol ; 16(8): e1008137, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804944

RESUMO

Genome-scale metabolic models have been utilized extensively in the study and engineering of the organisms they describe. Here we present the analysis of a published dataset from pooled transposon mutant fitness experiments as an approach for improving the accuracy and gene-reaction associations of a metabolic model for Zymomonas mobilis ZM4, an industrially relevant ethanologenic organism with extremely high glycolytic flux and low biomass yield. Gene essentiality predictions made by the draft model were compared to data from individual pooled mutant experiments to identify areas of the model requiring deeper validation. Subsequent experiments showed that some of the discrepancies between the model and dataset were caused by polar effects, mis-mapped barcodes, or mutants carrying both wild-type and transposon disrupted gene copies-highlighting potential limitations inherent to data from individual mutants in these high-throughput datasets. Therefore, we analyzed correlations in fitness scores across all 492 experiments in the dataset in the context of functionally related metabolic reaction modules identified within the model via flux coupling analysis. These correlations were used to identify candidate genes for a reaction in histidine biosynthesis lacking an annotated gene and highlight metabolic modules with poorly correlated gene fitness scores. Additional genes for reactions involved in biotin, ubiquinone, and pyridoxine biosynthesis in Z. mobilis were identified and confirmed using mutant complementation experiments. These discovered genes, were incorporated into the final model, iZM4_478, which contains 747 metabolic and transport reactions (of which 612 have gene-protein-reaction associations), 478 genes, and 616 unique metabolites, making it one of the most complete models of Z. mobilis ZM4 to date. The methods of analysis that we applied here with the Z. mobilis transposon mutant dataset, could easily be utilized to improve future genome-scale metabolic reconstructions for organisms where these, or similar, high-throughput datasets are available.


Assuntos
Aptidão Genética/genética , Genoma Bacteriano/genética , Modelos Genéticos , Mutação/genética , Zymomonas , Anaerobiose , Engenharia Metabólica , Zymomonas/genética , Zymomonas/metabolismo
8.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1293-1304, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748587

RESUMO

Cordycepin as the main active ingredient of Cordyceps militaris, a traditional medicinal fungus in China, has many physiological functions such as anti-cancer, anti-tumor and anti-virus activity. The most potential route for effective cordycepin production has been considered as liquid fermentation of C. militaris though with low productivity at present. Thus, it is urgent to apply both process engineering strategy and metabolic engineering strategy to enhance the productivity of cordycepin. In this review, the effects of medium components (i.e. the carbon/nitrogen source, precursor substances and metal ions) and operation factors (i.e. pH, dissolved oxygen and light) on cordycepin biosynthesis in liquid fermentation system are summarized. Besides, separation of cordycepin, the gene cluster involved and predicted biosynthesis pathways of cordycepin are also discussed, providing possible solutions of finally realizing efficient production of cordycepin.


Assuntos
Biotecnologia , Cordyceps , Desoxiadenosinas , Biotecnologia/tendências , China , Desoxiadenosinas/biossíntese , Desoxiadenosinas/genética , Fermentação , Engenharia Metabólica/tendências
9.
Nat Commun ; 11(1): 3327, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620863

RESUMO

Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebrosidase activity. Its hallmark manifestations are attributed to infiltration and inflammation by macrophages. Current therapies for Gaucher disease include life-long intravenous administration of recombinant glucocerebrosidase and orally-available glucosylceramide synthase inhibitors. An alternative approach is to engineer the patient's own hematopoietic system to restore glucocerebrosidase expression, thereby replacing the affected cells, and constituting a potential one-time therapy for this disease. Here, we report an efficient CRISPR/Cas9-based approach that targets glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe-harbor locus in human hematopoietic stem and progenitor cells. The targeted cells generate glucocerebrosidase-expressing macrophages and maintain long-term repopulation and multi-lineage differentiation potential with serial transplantation. The combination of a safe-harbor and a lineage-specific promoter establishes a universal correction strategy and circumvents potential toxicity of ectopic glucocerebrosidase in the stem cells. Furthermore, it constitutes an adaptable platform for other lysosomal enzyme deficiencies.


Assuntos
Edição de Genes/métodos , Glucosilceramidase/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/enzimologia , Macrófagos/enzimologia , Monócitos/enzimologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Doença de Gaucher/genética , Doença de Gaucher/terapia , Glucosilceramidase/genética , Células HEK293 , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Macrófagos/metabolismo , Engenharia Metabólica , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Monócitos/metabolismo , Transplante Autólogo
10.
PLoS One ; 15(7): e0236294, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32716960

RESUMO

Xylose, the second most abundant sugar in lignocellulosic biomass hydrolysates, can be fermented by Saccharomyces cerevisiae expressing one of two heterologous xylose pathways: a xylose oxidoreductase pathway and a xylose isomerase pathway. Depending on the type of the pathway, its optimization strategies and the fermentation efficiencies vary significantly. In the present study, we constructed two isogenic strains expressing either the oxidoreductase pathway (XYL123) or the isomerase pathway (XI-XYL3), and delved into simple and reproducible ways to improve the resulting strains. First, the strains were subjected to the deletion of PHO13, overexpression of TAL1, and adaptive evolution, but those individual approaches were only effective in the XYL123 strain but not in the XI-XYL3 strain. Among other optimization strategies of the XI-XYL3 strain, we found that increasing the copy number of the xylose isomerase gene (xylA) is the most promising but yet preliminary strategy for the improvement. These results suggest that the oxidoreductase pathway might provide a simpler metabolic engineering strategy than the isomerase pathway for the development of efficient xylose-fermenting strains under the conditions tested in the present study.


Assuntos
Engenharia Metabólica , Redes e Vias Metabólicas , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Aldose-Cetose Isomerases/metabolismo , Evolução Biológica , Fermentação , Deleção de Genes , Transcrição Genética
11.
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
12.
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
13.
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
14.
Nature ; 584(7819): 148-153, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32699417

RESUMO

Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.


Assuntos
Vias Biossintéticas , Colchicina/biossíntese , Engenharia Metabólica , Vias Biossintéticas/genética , Colchicaceae/enzimologia , Colchicaceae/genética , Colchicaceae/metabolismo , Colchicina/química , Colchicina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Metabolômica , Fenilalanina/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transcriptoma , Tirosina/metabolismo
15.
Bioresour Technol ; 314: 123726, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32622278

RESUMO

Xylose-inducible modules simultaneously expressing xylose utilization and naringenin biosynthesis pathways were developed in Yarrowia lipolytica to produce naringenin from a mixture of glucose and xylose. The naringenin synthetic pathway was constructed using a constitutive expression to yield 239.1 ± 5.1 mg/L naringenin. Furthermore, the introduction of an inducible pathway realized the dual function of xylose as a substrate and synthetic inducer, which coupled the xylose utilization with naringenin biosynthesis and increased production. Interestingly, the simultaneous enhancement of xylose reductase and xylose transporter expression along with that of xylitol dehydrogenase and xylulokinase can further improve the xylose utilization ability of Y. lipolytica. As expected, xylose-inducible synthesis of naringenin could achieved a titer of 715.3 ± 12.8 mg/L through the shake-flask cultivation level. Therefore, xylose-induced activation of both the xylose utilization and product biosynthesis pathway is considered to be an effective strategy for the biosynthesis of xylose-derived chemicals in yeast.


Assuntos
Yarrowia/genética , Flavanonas , Glucose , Engenharia Metabólica , Xilose
17.
J Biosci Bioeng ; 130(3): 290-294, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32482608

RESUMO

Aeromonas hydrophila 4AK4 normally produces the copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) using lauric acid as the carbon source. In this study we reported the metabolic engineering of A. hydrophila 4AK4 for the production of polyhydroxyalkanoate (PHA) using acetate as a main carbon source. Recombinant A. hydrophila overexpressing ß-ketothiolase and acetoacetyl-CoA reductase could accumulate poly-3-hydroxybutyrate (PHB) from acetate with a polymer content of 1.39 wt%. Further overexpression of acetate kinase/phosphotransacetylase and acetyl-CoA synthetase improved PHB content to 8.75 wt% and 19.82 wt%, respectively. When acetate and propionate were simultaneously supplied as carbon sources, the engineered A. hydrophila overexpressing ß-ketothiolase, acetoacetyl-CoA reductase, and acetyl-CoA synthetase was found able to produce the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV). The recombinant grew to 3.79 g/L cell dry weight (CDW) containing 15.02 wt% PHBV. Our proposed metabolic engineering strategies illustrate the feasibility for producing PHA from acetate by A. hydrophila.


Assuntos
Acetatos/metabolismo , Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Engenharia Metabólica , Poli-Hidroxialcanoatos/biossíntese , Ácido 3-Hidroxibutírico/metabolismo , Acetil-CoA C-Aciltransferase/genética , Oxirredutases do Álcool/genética , Ácidos Pentanoicos/metabolismo
18.
J Biosci Bioeng ; 130(3): 272-282, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32546403

RESUMO

The industrially relevant biopolymer poly-γ-glutamic acid (γ-PGA) is commonly synthesized using glycerol, citrate, and glutamic acid as carbon sources. In this study, two strains capable of utilizing glucose as sole carbon source for γ-PGA synthesis were constructed. Efficient γ-PGA production was achieved with derivatives of the well-investigated laboratory strain Bacillus subtilis 168, by replacing the native promoter of the PGA synthetase operon with the strong constitutive promoter Pveg or with the xylose-inducible promoter Pxyl. The carbon yield for γ-PGA increased by 129% to 0.131 C-mol C-mol-1 when using glucose as the sole substrate compared to the conventional carbon source mixture glycerol, citrate, and glutamic acid. The characterization of the produced γ-PGA demonstrated a time-dependent molecular weight of 1180-1850 kDa and a d-glutamic acid monomer content of 49-62%. To elucidate the consequences of γ-PGA production, we characterized the engineered strain by metabolomics. While the metabolite concentrations in the TCA cycle leading up to 2-oxoglutarate decreased in γ-PGA producer strains, the glutamic acid concentration was constant, despite the drastic increase in glutamic acid demand. The results are discussed in the context of metabolic regulation and future metabolic engineering strategies to enhance precursor supply for γ-PGA synthesis from glucose.


Assuntos
Bacillus subtilis/metabolismo , Glucose/metabolismo , Metabolômica , Ácido Poliglutâmico/análogos & derivados , Bacillus subtilis/genética , Ciclo do Ácido Cítrico , Engenharia Metabólica , Peso Molecular , Óperon/genética , Ácido Poliglutâmico/biossíntese , Ácido Poliglutâmico/química
19.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1101-1112, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597060

RESUMO

As an important platform compound, 3-hydroxypropionic acid (3-HP) can be used as a substrate to synthesize a variety of biological products with commercial potential. The titer of 3-HP by wild-type bacteria is low, which severely limits the large-scale application and production of 3-HP. By modifying the genes related to the metabolic pathway, engineered bacteria using cheap substrates as carbon sources are constructed, the aim of reducing production cost and increasing output is realized. In this paper, the recent progress in the synthesis of 3-HP by metabolic engineering at home and abroad is reviewed. The advantages and disadvantages of glycerol pathway, malonyl-CoA pathway and beta-alanine pathway for synthesis of 3-HP are also summarized and analyzed, and the future development of 3-HP is prospected.


Assuntos
Microbiologia Industrial , Ácido Láctico/análogos & derivados , Engenharia Metabólica , Glicerol/metabolismo , Microbiologia Industrial/tendências , Ácido Láctico/biossíntese , Redes e Vias Metabólicas/genética
20.
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
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