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1.
Sheng Wu Gong Cheng Xue Bao ; 36(10): 2113-2125, 2020 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-33169576

RESUMO

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.


Assuntos
Ciclo do Ácido Cítrico , Ácido Glutâmico , Redes e Vias Metabólicas , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácido Glutâmico/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética
2.
Bioresour Technol ; 318: 124064, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32905949

RESUMO

One challenge in metabolic engineering for industrial applications is the construction of highly efficient microbial cell factories. For this purpose, dynamic regulation of metabolic flux may be indispensable. In this study, an auto-regulated Corynebacterium glutamicum chassis for 5-aminolevulinic acid (5-ALA) biosynthesis was constructed. First, the expression of critical genes involved in 5-ALA synthesis and cofactor regeneration was precisely modulated. Furthermore, odhA expression was controlled using the strategies of static metabolic engineering (SME, with a weak promoter), dynamic metabolic engineering (DME, with a temperature-sensitive plasmid), and auto-inducible metabolic engineering (AME, with a growth-related promoter). The AME strategy showed the best effect and dynamically balanced the tradeoff between cell growth and 5-ALA synthesis. Additionally, the expression of exporter-encoding rhtA was regulated using AME strategy by the two-component system HrrSA in response to extracellular heme. The final strain A30 achieved the highest 5-ALA production (3.16 g/L) ever reported in C. glutamicum through C5 pathway.


Assuntos
Corynebacterium glutamicum , Ácido Aminolevulínico , Corynebacterium glutamicum/genética , Heme , Engenharia Metabólica , Plasmídeos
3.
PLoS One ; 15(8): e0231560, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822353

RESUMO

The dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum encoded by the qsuB gene is related to the previously described QuiC1 protein (39.9% identity) from Pseudomonas putida. Both QuiC1 and QsuB are two-domain bacterial DSDs. The N-terminal domain provides dehydratase activity, while the C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate dioxygenase. Here, the QsuB protein and its N-terminal domain (N-QsuB) were expressed in the T7 system, purified and characterized. QsuB was present mainly in octameric form (60%), while N-QsuB had a predominantly monomeric structure (80%) in aqueous buffer. Both proteins possessed DSD activity with one of the following cofactors (listed in the order of decreasing activity): Co2+, Mg2+, Mn2+. The Km and kcat values for the QsuB enzyme (Km ~ 1 mM, kcat ~ 61 s-1) were two and three times higher than those for N-QsuB. 3,4-DHBA inhibited QsuB (Ki ~ 0.38 mM, Ki' ~ 0.96 mM) and N-QsuB (Ki ~ 0.69 mM) enzymes via mixed and noncompetitive inhibition mechanism, respectively. E. coli MG1655ΔaroEPlac‒qsuB strain produced three times more 3,4-DHBA from glucose in test tube fermentation than the MG1655ΔaroEPlac‒n-qsuB strain. The C-terminal domain activity towards 3,4-DHBA was not established in vitro. This domain was proposed to promote protein oligomerization for maintaining structural stability of the enzyme. The dimer formation of QsuB protein was more predictable (ΔG = ‒15.8 kcal/mol) than the dimerization of its truncated version N-QsuB (ΔG = ‒0.4 kcal/mol).


Assuntos
Biotecnologia , Corynebacterium glutamicum/enzimologia , Hidroliases/metabolismo , Hidroxibenzoatos/metabolismo , Corynebacterium glutamicum/genética , DNA Recombinante/genética , Escherichia coli/metabolismo , Hidroliases/química , Hidroliases/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Estrutura Quaternária de Proteína
4.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 820-828, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567265

RESUMO

Corynebacterium glutamicum, an important microorganism to produce amino acids and organic acids, has been widely applied in food and medicine fields. Therefore, using editing tools to study the function of unknown genes in C. glutamicum has great significance for systematic development of industrial strain with efficient and novel production capability. Recently, gene editing has been greatly developed. Traditional gene editing based on homologous recombination and gene editing mediated by nuclease are successfully applied in C. glutamicum. Among these, the CRISPR system has been developed to be a main tool used for gene knockout of C. glutamicum due to its advantages of efficiency, simplicity and good target specificity. However, more efficient and reliable knockout system is still urgently demanded, to help develop high-performing strains in industrial application.


Assuntos
Corynebacterium glutamicum , Edição de Genes , Microbiologia Industrial , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Corynebacterium glutamicum/genética , Edição de Genes/tendências , Ácido Glutâmico , Microbiologia Industrial/tendências
5.
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
6.
Microb Cell Fact ; 19(1): 102, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398078

RESUMO

BACKGROUND: Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce (3R)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses. RESULTS: In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting (3R)-acetoin surpassed 95%. CONCLUSION: To the best of our knowledge, this is the highest titer of highly enantiomerically enriched (3R)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of (3R)-acetoin via microbial fermentation in the near future.


Assuntos
Acetoína/metabolismo , Corynebacterium glutamicum/metabolismo , Engenharia Metabólica/métodos , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Corynebacterium glutamicum/genética , Fermentação , Glucose/metabolismo , Redes e Vias Metabólicas , Óperon
7.
Nucleic Acids Res ; 48(12): 6547-6562, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32453397

RESUMO

Heme is a multifaceted molecule. While serving as a prosthetic group for many important proteins, elevated levels are toxic to cells. The complexity of this stimulus has shaped bacterial network evolution. However, only a small number of targets controlled by heme-responsive regulators have been described to date. Here, we performed chromatin affinity purification and sequencing to provide genome-wide insights into in vivo promoter occupancy of HrrA, the response regulator of the heme-regulated two-component system HrrSA of Corynebacterium glutamicum. Time-resolved profiling revealed dynamic binding of HrrA to more than 200 different genomic targets encoding proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. By repression of the extracytoplasmic function sigma factor sigC, which activates the cydABCD operon, HrrA prioritizes the expression of genes encoding the cytochrome bc1-aa3 supercomplex. This is also reflected by a significantly decreased activity of the cytochrome aa3 oxidase in the ΔhrrA mutant. Furthermore, our data reveal that HrrA also integrates the response to heme-induced oxidative stress by activating katA encoding the catalase. These data provide detailed insights in the systemic strategy that bacteria have evolved to respond to the versatile signaling molecule heme.


Assuntos
Proteínas de Bactérias/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Óperon , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Fator sigma/metabolismo
8.
Nat Commun ; 11(1): 1970, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327663

RESUMO

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 µM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L-1 of SA with the maximum productivity of 21.3 g L-1 h-1, demonstrating the importance of enzyme optimization in strain development.


Assuntos
Proteínas de Bactérias/genética , Malato Desidrogenase/genética , Pasteurellaceae/metabolismo , Ácido Succínico/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Fermentação , Cinética , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Engenharia Metabólica , Ácido Oxaloacético/metabolismo , Pasteurellaceae/enzimologia , Pasteurellaceae/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
9.
Nat Commun ; 11(1): 1515, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251291

RESUMO

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.


Assuntos
Evolução Molecular Direcionada/métodos , Escherichia coli/enzimologia , Depuradores de Radicais Livres/metabolismo , Engenharia Metabólica , Álcool Feniletílico/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Vias Biossintéticas/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Estudos de Viabilidade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Álcool Feniletílico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido Vanílico/metabolismo
10.
Nat Commun ; 11(1): 1641, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242019

RESUMO

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.


Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/citologia , Corynebacterium glutamicum/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Divisão Celular , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Dimerização , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Alinhamento de Sequência
11.
Enzyme Microb Technol ; 135: 109505, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146930

RESUMO

Amylosucrase (ASase) has great industrial potential owing to its multifunctional activities, including transglucosylation, polymerization, and isomerization. In the present study, the properties of Deinococcus geothermalis ASase (DGAS) expressed in Corynebacterium glutamicum (cDGAS) and purified via Ni-NTA affinity chromatography were compared to those of DGAS expressed in Escherichia coli (eDGAS). The pH profile of cDGAS was similar to that of eDGAS, whereas the temperature profile of cDGAS was lower than that of eDGAS. The melting temperature of both enzymes did not differ significantly. Interestingly, polymerization activity was slightly lower in cDGAS than in eDGAS, whereas luteolin (an acceptor molecule) transglucosylation activity in cDGAS was 10 % higher than that in eDGAS. Analysis of protein secondary structure via circular dichroism spectroscopy revealed that cDGAS had a lower strand/helix ratio than eDGAS. The present results indicate that cDGAS is of greater industrial significance than eDGAS.


Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Deinococcus/enzimologia , Glucosídeos/biossíntese , Glucosiltransferases/metabolismo , Luteolina/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Deinococcus/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Glucosiltransferases/química , Glucosiltransferases/genética , Engenharia Metabólica
12.
Appl Environ Microbiol ; 86(8)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32060028

RESUMO

PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria.IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.


Assuntos
Arginina/metabolismo , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Proteínas PII Reguladoras de Nitrogênio/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Corynebacterium glutamicum/química , Corynebacterium glutamicum/metabolismo , Proteínas PII Reguladoras de Nitrogênio/química , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Alinhamento de Sequência
13.
mBio ; 11(1)2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019787

RESUMO

Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria. In this study, we systematically analyzed the in vivo constraints underlying silencing and counter-silencing of the Lsr2-like protein CgpS in Corynebacterium glutamicum Genome-wide analysis revealed binding of CgpS to regions featuring a distinct drop in GC profile close to the transcription start site (TSS) but also identified an overrepresented motif with multiple A/T steps at the nucleation site of the nucleoprotein complex. Binding of specific transcription factors (TFs) may oppose XS activity, leading to counter-silencing. Following a synthetic counter-silencing approach, target gene activation was realized by inserting operator sites of an effector-responsive TF within various CgpS target promoters, resulting in increased promoter activity upon TF binding. Analysis of reporter constructs revealed maximal counter-silencing when the TF operator site was inserted at the position of maximal CgpS coverage. This principle was implemented in a synthetic toggle switch, which features a robust and reversible response to effector availability, highlighting the potential for biotechnological applications. Together, our results provide comprehensive insights into how Lsr2 silencing and counter-silencing shape evolutionary network expansion in this medically and biotechnologically relevant bacterial phylum.IMPORTANCE In actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XS) of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant as a potential drug target and for the identification of novel bioactive compounds. Here, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and counter-silencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum Our results demonstrated that CgpS distinguishes between self and foreign by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks.


Assuntos
Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Inativação Gênica , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Transferência Genética Horizontal , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Microb Cell Fact ; 19(1): 2, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906967

RESUMO

BACKGROUND: L-ornithine is a valuable amino acid with a wide range of applications in the pharmaceutical and food industries. However, the production of L-ornithine by fermentation cannot compete with other methods, because of the low titers produced with this technique. Development of fermentation techniques that result in a high yield of L-ornithine and efficient strategies for improving L-ornithine production are essential. RESULTS: This study demonstrates that tween 40, a surfactant promoter of the production of glutamate and arginine, improves L-ornithine production titers in engineered C. glutamicum S9114. The intracellular metabolism under tween 40 triggered fermentation conditions was explored using a quantitative proteomic approach, identifying 48 up-regulated and 132 down-regulated proteins when compared with the control. Numerous proteins were identified as membrane proteins or functional proteins involved in the biosynthesis of the cell wall. Modulation of those genes revealed that the overexpression of CgS9114_09558 and the deletion of CgS9114_13845, CgS9114_02593, and CgS9114_02058 improved the production of L-ornithine in the engineered strain of C. glutamicum Orn8. The final strain with all the exploratory metabolic engineering manipulations produced 25.46 g/L of L-ornithine, and a yield of 0.303 g L-ornithine per g glucose, which was 30.6% higher than that produced by the original strain (19.5 g/L). CONCLUSION: These results clearly demonstrate the positive effect of tween 40 addition on L-ornithine accumulation. Proteome analysis was performed to examine the impact of tween 40 addition on the physiological changes in C. glutamicum Orn8 and the results showed several promising modulation targets for developing L-ornithine-producing strains.


Assuntos
Corynebacterium glutamicum/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados/metabolismo , Ornitina/biossíntese , Polissorbatos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/genética , Genes Bacterianos , Genoma Bacteriano , Proteoma/metabolismo , Proteômica
15.
Biosci Biotechnol Biochem ; 84(3): 463-470, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31752618

RESUMO

Lycopene is a highly valued carotenoid with wide applications in various industries. The market demand for lycopene promotes research in metabolic engineering of heterologous hosts for lycopene. In this study, Pichia pastoris strain GS115 was genetically engineered to produce lycopene by integrating the heterologous lycopene biosynthesis genes from Corynebacterium glutamicum ATCC13032. The resulting strain, L1, produced 0.115 mg/g cell dry weight (DCW) lycopene. Through optimization by promoter selection, improving the precursor supply and expanding the Geranylgeranyl diphosphate (GGPP) pool, ultimately, the lycopene yield of the final optimal strain was 6.146 mg/g DCW with shake flask fermentation and 9.319 mg/g DCW (0.714 g/L) in a 3 L fermenter. The lycopene yield in this study is the highest yield of lycopene in P. pastoris reported to date, which demonstrated the potential of P. pastoris in lycopene synthesis and as a candidate host organism for the synthesis of other high value-added terpenoids.


Assuntos
Licopeno/metabolismo , Engenharia Metabólica , Pichia/genética , Reatores Biológicos , Corynebacterium glutamicum/genética , Fermentação
16.
Enzyme Microb Technol ; 132: 109395, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731968

RESUMO

RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance the production of value-added chemicals in Corynebacterium glutamicum as an industrial host. Here, we described the biotransformation of xylose to glycolate using engineered Corynebacterium glutamicum, a well-known industrial amino acid producer. A synthetic pathway involving heterologous D-tagatose 3-epimerase and L-fuculose kinase/aldolase reactions was introduced in C. glutamicum, resulting in 9.9 ±â€¯0.01 g/L glycolate from 20 g/L xylose at a yield of 0.51 g/g (equal to 1.0 mol/mol). Additional glyoxylate reduction pathway developed by CRISPR-Cas12a recombineering has been introduced and attempted to increase the maximum theoretical molar yield of 2.0 (mol/mol). Due to the limitation of the CRISPR-Cas12a recombineering with TTTV PAM sites, advanced CRISPR-Cas systems were suggested for the next-round metabolic engineering for improving the glycolate yield to overcome the current genome-editing tool for metabolic engineering in C. glutamicum.


Assuntos
Sistemas CRISPR-Cas , Corynebacterium glutamicum/metabolismo , Edição de Genes , Glicolatos/metabolismo , Engenharia Metabólica/métodos , Xilose/metabolismo , Estudos de Casos e Controles , Corynebacterium glutamicum/genética , RNA Guia
17.
World J Microbiol Biotechnol ; 36(1): 11, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31879822

RESUMO

Because of their protein cross-linking properties, transglutaminases are widely used in several industrial processes, including the food and pharmaceutical industries. Transglutaminases obtained from animal tissues and organs, the first sources of this enzyme, are being replaced by microbial sources, which are cheaper and easier to produce and purify. Since the discovery of microbial transglutaminase (mTGase), the enzyme has been produced for industrial applications by traditional fermentation process using the bacterium Streptomyces mobaraensis. Several studies have been carried out in this field to increase the enzyme industrial productivity. Researches on gene expression encoding transglutaminase biosynthesis were performed in Streptomyces lividans, Escherichia coli, Corynebacterium glutamicum, Yarrowia lipolytica, and Pichia pastoris. In the first part of this review, we presented an overview of the literature on the origins, types, mediated reactions, and general characterizations of these important enzymes, as well as the studies on recombinant microbial transglutaminases. In this second part, we focus on the application versatility of mTGase in three broad areas: food, pharmacological, and biotechnological industries. The use of mTGase is presented for several food groups, showing possibilities of applications and challenges to further improve the quality of the end-products. Some applications in the textile and leather industries are also reviewed, as well as special applications in the PEGylation reaction, in the production of antibody drug conjugates, and in regenerative medicine.


Assuntos
Biotecnologia , Indústria Alimentícia , Têxteis , Transglutaminases , Animais , Corynebacterium glutamicum/genética , Bases de Dados Factuais , Escherichia coli/genética , Fermentação , Alimentos , Tecnologia de Alimentos , Pichia/genética , Proteínas Recombinantes , Streptomyces/enzimologia , Transglutaminases/biossíntese , Transglutaminases/genética , Yarrowia/genética
18.
Biosci Biotechnol Biochem ; 83(12): 2390-2393, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31671040

RESUMO

We identified L-cysteine exporter candidates of Corynebacterium glutamicum and investigated the effect of overexpression of the potential L-cysteine exporter genes on L-cysteine production in a recombinant strain of C. glutamicum. Overexpression of NCgl2566 and NCgl0580 resulted in enhanced L-cysteine production in an L-cysteine-producing recombinant strain of C. glutamicum.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Corynebacterium glutamicum/genética , Cisteína/biossíntese , Recombinação Genética , Genes Bacterianos
19.
Appl Microbiol Biotechnol ; 103(23-24): 9619-9631, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31686146

RESUMO

Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4'-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.


Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Salicilatos/metabolismo , Escherichia coli/metabolismo , Microbiologia Industrial , Engenharia Metabólica/métodos , Streptomyces antibioticus/enzimologia
20.
Bioengineered ; 10(1): 561-573, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31648597

RESUMO

Currently, the mechanism of temperature-sensitive production of glutamate in Corynebacterium glutamicum has not been clarified. We first found the murA and murB genes were potentially related to temperature-sensitive secretion of glutamate, which were not existed in a temperature-sensitive mutant. When replenishing murA or/and murB in the mutant, the temperature sensitivity was weakened. While, their knockout in a wild-type strain resulted in temperature-sensitive secretion of glutamate. Peptidoglycan analysis showed that deletion of murA and murB decreased the peptidoglycan synthesis. Comparative metabolomics analysis suggested that the variation in cell wall structure resulted in decreased overall cellular metabolism but increased carbon flow to glutamate synthesis, which was a typical metabolism pattern in industrial temperature-sensitive producing strains. This study clarifies the mechanism between murA and murB deletion and the temperature-sensitive secretion of glutamate in C. glutamcium, and provides a reference for the metabolic engineering of cell wall to obtain increased bioproduction of chemicals.


Assuntos
Proteínas de Bactérias/genética , Desidrogenases de Carboidrato/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Deleção de Sequência , Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Corynebacterium glutamicum/química , Corynebacterium glutamicum/metabolismo , Ácido Glutâmico/metabolismo , Peptidoglicano/metabolismo , Temperatura
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