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1.
Chinese Journal of Biotechnology ; (12): 3273-3289, 2023.
Artigo em Chinês | WPRIM | ID: wpr-1007957

RESUMO

L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using Corynebacterium glutamicum G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (alaT), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.


Assuntos
Ácido Glutâmico , Corynebacterium glutamicum/genética , Ácidos Cetoglutáricos , Engenharia Metabólica , Alanina
2.
Chinese Journal of Biotechnology ; (12): 3253-3272, 2023.
Artigo em Chinês | WPRIM | ID: wpr-1007956

RESUMO

As a branched chain amino acid, L-valine is widely used in the medicine and feed sectors. In this study, a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies. First, precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products. Subsequently, the key enzyme in the L-valine synthesis pathway, acetylhydroxylate synthase, was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain. Moreover, promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway. Furthermore, cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH. The engineered strain C. glutamicum K020 showed a significant increase in L-valine titer, yield and productivity in 5 L fed-batch bioreactor, up to 110 g/L, 0.51 g/g and 2.29 g/(L‧h), respectively.


Assuntos
Valina , Corynebacterium glutamicum/genética , Engenharia Metabólica , Aminoácidos de Cadeia Ramificada , Reatores Biológicos
3.
Chinese Journal of Biotechnology ; (12): 1677-1696, 2021.
Artigo em Chinês | WPRIM | ID: wpr-878661

RESUMO

Fermentative production of amino acids is one of the pillars of the fermentation industry in China. Recently, with the fast development of metabolic engineering and synthetic biology technologies, the metabolic engineering for production of amino acids has been flourishing. Conventional forward metabolic engineering, reversed metabolic engineering based on omics data and in silico simulation, and evolutionary metabolic engineering mimicking the natural evolution, have shown increasingly promising applications. A series of highly efficient and robust amino acids-producing strains have been developed and applied in the industrial production of amino acids. The increasingly fierce market competition has put forward new requirements for strain breeding and selection, such as developing high value-added amino acids, dynamic regulation of cellular metabolism, and adapting to the requirements of new process. This review summarizes the advances and prospects in metabolic engineering for the production of amino acids.


Assuntos
Aminoácidos , China , Corynebacterium glutamicum/genética , Engenharia Metabólica , Biologia Sintética
4.
Chinese Journal of Biotechnology ; (12): 1603-1618, 2021.
Artigo em Chinês | WPRIM | ID: wpr-878657

RESUMO

Corynebacterium glutamicum is an important workhorse of industrial biotechnology, especially for amino acid bioindustry. This bacterium is being used to produce various amino acids at a level of over 6 million tons per year. In recent years, enabling technologies for C. glutamicum metabolic engineering have been developed and improved, which accelerated construction and optimization of microbial cell factoriers, expanding spectra of substrates and products, and facilitated basic researches on C. glutamicum. With these technologies, C. glutamicum has become one of the ideal microbial chasses. This review summarizes recent key technological developments of enabling technologies for C. glutamicum metabolic engineering and focuses on establishment and applications of CRISPR-based genome editing, gene expression regulation, adaptive laboratory evolution, and biosensor technologies.


Assuntos
Aminoácidos , Biotecnologia , Corynebacterium glutamicum/genética , Edição de Genes , Engenharia Metabólica
5.
Chinese Journal of Biotechnology ; (12): 860-873, 2021.
Artigo em Chinês | WPRIM | ID: wpr-878601

RESUMO

Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.


Assuntos
Corynebacterium glutamicum/genética , Escherichia coli/genética , Engenharia Metabólica , Redes e Vias Metabólicas/genética
6.
Chinese Journal of Biotechnology ; (12): 2250-2259, 2020.
Artigo em Chinês | WPRIM | ID: wpr-878483

RESUMO

Amino acids are important compounds with a wide range of applications in the food, medicine and chemical industries. Corynebacterium glutamicum is a powerful workhorse commonly used in industrial amino acid production, with the scale of more than one million tons. In addition to its efficient anabolism, the effective exporters also ensure the high amino acid production by C. glutamicum. In this review, the research progress of amino acid exporter of C. glutamicum is summarized, to provide the foundation for further improving amino acid production by C. glutamicum via metabolic engineering.


Assuntos
Aminoácidos , Corynebacterium glutamicum/genética , Engenharia Metabólica
7.
Braz. j. microbiol ; 45(4): 1477-1483, Oct.-Dec. 2014. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-741303

RESUMO

L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO) is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions.


Assuntos
Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/metabolismo , Deleção de Genes , Ácido Glutâmico/metabolismo , L-Lactato Desidrogenase/genética , Ácido Láctico/metabolismo , Engenharia Metabólica , Corynebacterium glutamicum/genética , Oxigênio/metabolismo , Deleção de Sequência
8.
Biol. Res ; 41(3): 349-358, 2008. tab
Artigo em Inglês | LILACS | ID: lil-511924

RESUMO

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral médium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constituvely expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli Ml-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.


Assuntos
Corynebacterium glutamicum/genética , Proteínas de Escherichia coli/genética , Gluconatos/metabolismo , Clonagem Molecular , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/crescimento & desenvolvimento , DNA Bacteriano , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Genet. mol. res. (Online) ; 5(4): 773-789, 2006.
Artigo em Inglês | LILACS | ID: lil-482081

RESUMO

Corynebacterium glutamicum is a gram-positive soil microorganism able to utilize a large variety of aromatic compounds as the sole carbon source. The corresponding catabolic routes are associated with multiple ring-fission dioxygenases and among other channeling reactions, include the gentisate pathway, the protocatechuate and catechol branches of the beta-ketoadipate pathway and two potential hydroxyquinol pathways. Genes encoding the enzymatic machinery for the bioconversion of aromatic compounds are organized in several clusters in the C. glutamicum genome. Expression of the gene clusters is under specific transcriptional control, apparently including eight DNA-binding proteins belonging to the AraC, IclR, LuxR, PadR, and TetR families of transcriptional regulators. Expression of the gentisate pathway involved in the utilization of 3-hydroxybenzoate and gentisate is positively regulated by an IclR-type activator. The metabolic channeling of ferulate, vanillin and vanillate into the protocatechuate branch of the beta-ketoadipate pathway is controlled by a PadR-like repressor. Regulatory proteins of the IclR and LuxR families participate in transcriptional regulation of the branches of the beta-ketoadipate pathway that are involved in the utilization of benzoate, 4-hydroxybenzoate and protocatechuate. The channeling of phenol into this pathway may be under positive transcriptional control by an AraC-type activator. One of the potential hydroxyquinol pathways of C. glutamicum is apparently repressed by a TetR-type regulator. This global analysis revealed that transcriptional regulation of aromatic compound utilization is mainly controlled by single regulatory proteins sensing the presence of aromatic compounds, thus representing single input motifs within the transcriptional regulatory network of C. glutamicum.


Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Hidroxibenzoatos , Hidrocarbonetos Aromáticos/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Transcrição Gênica/genética , Biodegradação Ambiental
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