Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 76
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Microb Cell Fact ; 23(1): 264, 2024 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-39367476

RESUMEN

BACKGROUND: Whey, which has high biochemical oxygen demand and chemical oxygen demand, is mass-produced as a major by-product of the dairying industry. Microbial fermentation using whey as the carbon source may convert this potential pollutant into value-added products. This study investigated the potential of using whey powder to produce α-ketoisovalerate, an important platform chemical. RESULTS: Klebsiella oxytoca VKO-9, an efficient L-valine producing strain belonging to Risk Group 1 organism, was selected for the production of α-ketoisovalerate. The leucine dehydrogenase and branched-chain α-keto acid dehydrogenase, which catalyzed the reductive amination and oxidative decarboxylation of α-ketoisovalerate, respectively, were inactivated to enhance the accumulation of α-ketoisovalerate. The production of α-ketoisovalerate was also improved through overexpressing α-acetolactate synthase responsible for pyruvate polymerization and mutant acetohydroxyacid isomeroreductase related to α-acetolactate reduction. The obtained strain K. oxytoca KIV-7 produced 37.3 g/L of α-ketoisovalerate from lactose, the major utilizable carbohydrate in whey. In addition, K. oxytoca KIV-7 also produced α-ketoisovalerate from whey powder with a concentration of 40.7 g/L and a yield of 0.418 g/g. CONCLUSION: The process introduced in this study enabled efficient α-ketoisovalerate production from low-cost substrate whey powder. Since the key genes for α-ketoisovalerate generation were integrated in genome of K. oxytoca KIV-7 and constitutively expressed, this strain is promising in stable α-ketoisovalerate fermentation and can be used as a chassis strain for α-ketoisovalerate derivatives production.


Asunto(s)
Fermentación , Hemiterpenos , Klebsiella oxytoca , Ingeniería Metabólica , Suero Lácteo , Klebsiella oxytoca/metabolismo , Klebsiella oxytoca/genética , Suero Lácteo/metabolismo , Ingeniería Metabólica/métodos , Hemiterpenos/metabolismo , Polvos , Acetolactato Sintasa/metabolismo , Acetolactato Sintasa/genética , Cetoácidos
2.
Molecules ; 28(7)2023 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-37049692

RESUMEN

A new series of ternary metal complexes, including Co(II), Ni(II), Cu(II), and Zn(II), were synthesized and characterized by elemental analysis and diverse spectroscopic methods. The complexes were synthesized from respective metal salts with Schiff's-base-containing amino acids, salicylaldehyde derivatives, and heterocyclic bases. The amino acids containing Schiff bases showed promising pharmacological properties upon complexation. Based on satisfactory elemental analyses and various spectroscopic techniques, these complexes revealed a distorted, square pyramidal geometry around metal ions. The molecular structures of the complexes were optimized by DFT calculations. Quantum calculations were performed with the density functional method for which the LACVP++ basis set was used to find the optimized molecular structure of the complexes. The metal complexes were subjected to an electrochemical investigation to determine the redox behavior and oxidation state of the metal ions. Furthermore, all complexes were utilized for catalytic assets of a multi-component Mannich reaction for the preparation of -amino carbonyl derivatives. The synthesized complexes were tested to determine their antibacterial activity against E. coli, K. pneumoniae, and S. aureus bacteria. To evaluate the cytotoxic effects of the Cu(II) complexes, lung cancer (A549), cervical cancer (HeLa), and breast cancer (MCF-7) cells compared to normal cells, cell lines such as human dermal fibroblasts (HDF) were used. Further, the docking study parameters were supported, for which it was observed that the metal complexes could be effective in anticancer applications.


Asunto(s)
Complejos de Coordinación , Humanos , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Bases de Schiff/farmacología , Bases de Schiff/química , Valina , Escherichia coli , Staphylococcus aureus , Metales/química , Antibacterianos/farmacología , Antibacterianos/química , Ligandos , Cobre/química
3.
Molecules ; 28(14)2023 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-37513312

RESUMEN

As a follow-up to our effort to establish reliable thermodynamic data for amino acids, the heat capacity and phase behavior are reported for N-acetyl glycine amide (CAS RN: 2620-63-5), N-acetyl-L-alanine amide (CAS RN: 15962-47-7), N-acetyl-L-valine amide (CAS RN: 37933-88-3), N-acetyl-L-isoleucine amide (CAS RN: 56711-06-9), and N-acetyl-L-leucine amide (CAS RN: 28529-34-2). Prior to heat capacity measurement, thermogravimetric analysis and X-ray powder diffraction were performed to determine decomposition temperatures and initial crystal structures, respectively. The crystal heat capacities of the five N-acetyl amino acid amides were measured by Tian-Calvet calorimetry in the temperature interval (266-350 K), by power compensation DSC in the temperature interval (216-471 K), and by relaxation (heat-pulse) calorimetry in the temperature interval (2-268 K). As a result, reference heat capacities and thermodynamic functions for the crystalline phase from 0 K up to 470 K were developed.


Asunto(s)
Isoleucina , Valina , Leucina/metabolismo , Isoleucina/metabolismo , Valina/metabolismo , Amidas , Calor , Aminoácidos , Alanina , Glicina
4.
Chirality ; 34(3): 462-472, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35060196

RESUMEN

Chiral covalent organic framework materials have many excellent properties, which have received much attention in the field of separation. Synthesized the covalent organic framework COF-TpBD (NH2 )2 modified, respectively, by L-valine trifluoroacetyl derivative, L-hydroxyproline, and (1S)-(+)-10-camphorsulfonyl chloride, three capillary columns of chiral covalent organic framework materials were obtained for gas chromatography. Those columns are able to separate some chiral compounds, positional isomers, n-alkanes, n-alcohols, aromatic hydrocarbon mixture, and Grob's reagents. They are complementary to other chiral capillary columns and are possible for potential applications.


Asunto(s)
Estructuras Metalorgánicas , Alcoholes , Cromatografía de Gases/métodos , Isomerismo , Estructuras Metalorgánicas/química , Estereoisomerismo
5.
World J Microbiol Biotechnol ; 38(5): 81, 2022 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-35348886

RESUMEN

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4 g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55 h of cultivation, with a substrate conversion ratio of 0.27 mol/mol glucose.


Asunto(s)
Klebsiella pneumoniae , Valina , Vías Biosintéticas/genética , Butileno Glicoles/metabolismo , Klebsiella pneumoniae/genética
6.
Amino Acids ; 53(9): 1301-1312, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34401958

RESUMEN

L-valine is an essential branched-chain amino acid that cannot be synthesized by the human body and has a wide range of applications in food, medicine and feed. Market demand has stimulated people's interest in the industrial production of L-valine. At present, the mutagenized or engineered Corynebacterium glutamicum is an effective microbial cell factory for producing L-valine. Because the biosynthetic pathway and metabolic network of L-valine are intricate and strictly regulated by a variety of key enzymes and genes, highly targeted metabolic engineering can no longer meet the demand for efficient biosynthesis of L-valine. In recent years, the development of omics technology has promoted the upgrading of traditional metabolic engineering to systematic metabolic engineering. This whole-cell-scale transformation strategy has become a productive method for developing L-valine producing strains. This review provides an overview of the biosynthesis and regulation mechanism of L-valine, and summarizes the current metabolic engineering techniques and strategies for constructing L-valine high-producing strains. Finally, the opinion of constructing a cell factory for efficiently biosynthesizing L-valine was proposed.


Asunto(s)
Vías Biosintéticas , Corynebacterium glutamicum/metabolismo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas , Valina/metabolismo , Corynebacterium glutamicum/crecimiento & desarrollo , Fermentación , Humanos
7.
Microb Cell Fact ; 20(1): 172, 2021 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-34461907

RESUMEN

L-valine is an essential amino acid that has wide and expanding applications with a suspected growing market demand. Its applicability ranges from animal feed additive, ingredient in cosmetic and special nutrients in pharmaceutical and agriculture fields. Currently, fermentation with the aid of model organisms, is a major method for the production of L-valine. However, achieving the optimal production has often been limited because of the metabolic imbalance in recombinant strains. In this review, the constrains in L-valine biosynthesis are discussed first. Then, we summarize the current advances in engineering of microbial cell factories that have been developed to address and overcome major challenges in the L-valine production process. Future prospects for enhancing the current L-valine production strategies are also discussed.


Asunto(s)
Bacterias , Ingeniería Metabólica/métodos , Valina/biosíntesis , Bacterias/genética , Bacterias/metabolismo
8.
Microb Cell Fact ; 20(1): 111, 2021 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-34082758

RESUMEN

BACKGROUND: Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2. RESULTS: Supplemented with L-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post L-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type. CONCLUSIONS: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.


Asunto(s)
Macrólidos/metabolismo , Policétidos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Factores de Transcripción/genética , Valina/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/genética , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/metabolismo , Proteínas Bacterianas/genética , Vías Biosintéticas , Perfilación de la Expresión Génica/métodos , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Microbiología Industrial , Metaboloma , Familia de Multigenes , Mutación
9.
Development ; 144(6): 1018-1024, 2017 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-28219950

RESUMEN

Liver bud progenitors experience a transient amplification during the early organ growth phase, yet the mechanism responsible is not fully understood. Collective evidence highlights the specific requirements in stem cell metabolism for expanding organ progenitors during organogenesis and regeneration. Here, transcriptome analyses show that progenitors of the mouse and human liver bud growth stage specifically express the gene branched chain aminotransferase 1, encoding a known breakdown enzyme of branched-chain amino acids (BCAAs) for energy generation. Global metabolome analysis confirmed the active consumption of BCAAs in the growing liver bud, but not in the later fetal or adult liver. Consistently, maternal dietary restriction of BCAAs during pregnancy significantly abrogated the conceptus liver bud growth capability through a striking defect in hepatic progenitor expansion. Under defined conditions, the supplementation of L-valine specifically among the BCAAs promoted rigorous growth of the human liver bud organoid in culture by selectively amplifying self-renewing bi-potent hepatic progenitor cells. These results highlight a previously underappreciated role of branched-chain amino acid metabolism in regulating mouse and human liver bud growth that can be modulated by maternal nutrition in vivo or cultural supplement in vitro.


Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Hígado/embriología , Hígado/metabolismo , Fenómenos Fisiológicos de la Nutrición , Transaminasas/metabolismo , Animales , Feto/efectos de los fármacos , Feto/embriología , Feto/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/efectos de los fármacos , Ratones Endogámicos C57BL , Fenómenos Fisiológicos de la Nutrición/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Valina/farmacología
10.
Metab Eng ; 62: 198-206, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32961297

RESUMEN

L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3'-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.


Asunto(s)
Corynebacterium glutamicum , Escherichia coli , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Ingeniería Metabólica , Valina/genética
11.
Appl Microbiol Biotechnol ; 104(1): 391-403, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31745574

RESUMEN

Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and L-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward L-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in L-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the L-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis.


Asunto(s)
Bacillus licheniformis/genética , Sistemas CRISPR-Cas , Silenciador del Gen , Ingeniería Metabólica/métodos , Bacillus licheniformis/enzimología , Proteínas Bacterianas/genética , Carboxiliasas/genética , Leucina-Deshidrogenasa/genética , Redes y Vías Metabólicas , Valina/análisis , Valina/metabolismo
12.
J Bacteriol ; 201(20)2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31358612

RESUMEN

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate, yielding acetyl coenzyme A (acetyl-CoA) and CO2 The PDHC-deficient Corynebacterium glutamicum ΔaceE strain therefore lacks an important decarboxylation step in its central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15-h lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates, such as acetate. Growth was successfully restored by deletion of ptsG, encoding the glucose-specific permease of the phosphotransferase system (PTS), thereby linking the observed phenotype to the increased sensitivity of the ΔaceE Δpyc strain to glucose catabolism. In this work, the ΔaceE Δpyc strain was used to systematically study the impact of perturbations of the intracellular CO2/HCO3- pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3- levels, such as external addition of HCO3-, increasing the pH, or rerouting metabolic flux via the pentose phosphate pathway, at least partially eliminated the lag phase of the ΔaceE Δpyc strain on glucose medium. In accordance with these results, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3- pool, led to an even longer lag phase, accompanied by the excretion of l-valine and l-alanine. Transcriptome analysis, as well as an adaptive laboratory evolution experiment with the ΔaceE Δpyc strain, revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose-acetate mixtures. Taken together, our results highlight the significant impact of the intracellular CO2/HCO3- pool on metabolic flux distribution, which becomes especially evident in engineered strains exhibiting low endogenous CO2 production rates, as exemplified by PDHC-deficient strains.IMPORTANCE CO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3- pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which additionally lacked pyruvate carboxylase (ΔaceE Δpyc). This strain featured a >15-h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3- pool on growth in glucose-acetate medium. Remarkably, all measures enhancing CO2/HCO3- levels successfully restored growth. These results emphasize the strong impact of the intracellular CO2/HCO3- pool on metabolic flux, especially in strains exhibiting low endogenous CO2 production rates.


Asunto(s)
Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Corynebacterium glutamicum/crecimiento & desarrollo , Complejo Piruvato Deshidrogenasa/genética , Proteínas Bacterianas/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Fenotipo
13.
Amino Acids ; 51(4): 717-726, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30798466

RESUMEN

This study was conducted to determine the catabolism of L-valine in porcine mammary epithelial cells (PMECs) and its role in stimulating protein synthesis in these cells. PMECs were incubated with 0.05-, 0.10-, 0.25-, 0.5-, and 1.0-mM L-valine at 37 oC for 2 h. Cell viability and expressions of α-lactalbumin and ß-casein were measured after culture with L-valine for 3 days. L-[1-14C]valine was used to study valine catabolism, whereas [3H]phenylalanine was employed as a tracer to determine protein synthesis and degradation in PMECs. The abundances of proteins involved in the mTOR signaling pathway and the mRNA levels for the related key genes were determined using the western blot and RT-PCR techniques, respectively. Cell numbers and the synthesis of proteins (including α-lactalbumin and ß-casein) were greater (P < 0.05) in the presence of 0.5-mM L-valine, compared with 0.05- or 0.1-mM L-valine. L-Valine at 0.5 mM also enhanced (P < 0.05) the production of α-lactalbumin by PMECs, in comparison with 0.25 mM L-valine. Increasing the extracellular concentration of L-valine from 0.05 to 0.5 mM stimulated protein synthesis in a concentration-dependent manner without affecting proteolysis. Although L-valine was actively transaminated in PMECs, its α-ketoacid product (α-ketoisovalerate) at 0.05-0.2 mM did not affect protein synthesis or degradation in the cells. Thus, the effect of L-valine on protein synthesis was independent of its metabolism to yield α-ketoisovalerate. At the molecular level, 0.5-mM L-valine increased (P < 0.05) the mRNA levels for Ras, ERK1/2, and p70S6K, and the abundances of mTOR, p-4EBP1, total 4EBP1, p-ERK1/2, and total ERK1/2 proteins. These findings establish the critical role of L-valine in enhancing PMEC growth and milk protein synthesis possibly by regulating the mTOR and Ras/ERK signaling pathways. Further studies are warranted to understand how L-valine regulates gene expression and mTOR activation in PMECs.


Asunto(s)
Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Biosíntesis de Proteínas , Valina/farmacología , Animales , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Fosforilación , Transducción de Señal , Porcinos
14.
Metab Eng ; 47: 31-41, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29522826

RESUMEN

Evolutionary approaches are often undirected and mutagen-based yielding numerous mutations, which need elaborate screenings to identify relevant targets. We here apply Metabolic engineering to Guide Evolution (MGE), an evolutionary approach evolving and identifying new targets to improve microbial producer strains. MGE is based on the idea to impair the cell's metabolism by metabolic engineering, thereby generating guided evolutionary pressure. It consists of three distinct phases: (i) metabolic engineering to create the evolutionary pressure on the applied strain followed by (ii) a cultivation phase with growth as straightforward screening indicator for the evolutionary event, and (iii) comparative whole genome sequencing (WGS), to identify mutations in the evolved strains, which are eventually re-engineered for verification. Applying MGE, we evolved the PEP and pyruvate carboxylase-deficient strain C. glutamicum Δppc Δpyc to grow on glucose as substrate with rates up to 0.31 ±â€¯0.02 h-1 which corresponds to 80% of the growth rate of the wildtype strain. The intersection of the mutations identified by WGS revealed isocitrate dehydrogenase (ICD) as consistent target in three independently evolved mutants. Upon re-engineering in C. glutamicum Δppc Δpyc, the identified mutations led to diminished ICD activities and activated the glyoxylate shunt replenishing oxaloacetate required for growth. Intracellular relative quantitative metabolome analysis showed that the pools of citrate, isocitrate, cis-aconitate, and L-valine were significantly higher compared to the WT control. As an alternative to existing L-valine producer strains based on inactivated or attenuated pyruvate dehydrogenase complex, we finally engineered the PEP and pyruvate carboxylase-deficient C. glutamicum strains with identified ICD mutations for L-valine production by overexpression of the L-valine biosynthesis genes. Among them, C. glutamicum Δppc Δpyc ICDG407S (pJC4ilvBNCE) produced up to 8.9 ±â€¯0.4 g L-valine L-1, with a product yield of 0.22 ±â€¯0.01 g L-valine per g glucose.


Asunto(s)
Corynebacterium glutamicum , Evolución Molecular Dirigida/métodos , Ingeniería Metabólica/métodos , Valina , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Valina/biosíntesis , Valina/genética
15.
Biotechnol Bioeng ; 115(11): 2778-2792, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-29981237

RESUMEN

Bacillus subtilis has been commonly applied to industrial enzyme production due to its genetic tractability, "generally recognized as safe (GRAS)" status, and robust growth characteristics. In spite of its ideal attributes as a biomanufacturing platform, B. subtilis has seen limited use in the production of other value-added biochemicals. Here, we report the derivation of engineered strains of B. subtilis for l-valine overproduction using our recently developed CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated [protein] 9) toolkit. We first manipulate the native l-valine biosynthetic pathway by relieving transcriptional and allosteric regulation, resulting in a >14-fold increase in the l-valine titer, compared to the wild-type strain. We subsequently identify and eliminate factors limiting l-valine overproduction, specifically increasing pyruvate availability and blocking the competing l-leucine and l-isoleucine biosynthetic pathways. By inactivating (a) pdhA, encoding the E1α subunit of the pyruvate dehydrogenase complex, to increase the intracellular pyruvate pool, and (b) leuA and ilvA, respectively encoding 2-isopropylmalate synthase and l-threonine dehydratase, to abolish the competing pathways, the l-valine titer reached 4.61 g/L in shake flask cultures. Our engineered l-valine-overproducing strains of B. subtilis are devoid of plasmids and do not sporulate due to the inactivation of sigF, encoding the sporulation-specific transcription factor σ F , making them attractive for large-scale l-valine production. However, acetate dissimilation was identified as limiting l-valine overproduction in ΔpdhA B. subtilis strains, and improving acetate dissimilation or identifying alternate modes of increasing pyruvate pools to enhance l-valine-overproduction should be explored.


Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Valina/biosíntesis , Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dosificación de Gen , Edición Génica/métodos , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Vectores Genéticos , Plásmidos , Activación Transcripcional
16.
Microb Cell Fact ; 17(1): 185, 2018 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-30474553

RESUMEN

BACKGROUND: Promoters are commonly used to regulate the expression of specific target genes or operons. Although a series of promoters have been developed in Corynebacterium glutamicum, more precise and unique expression patterns are needed that the current selection of promoters cannot produce. RNA-Seq technology is a powerful tool for helping us to screen out promoters with expected transcriptional strengths. RESULTS: The promoter PCP_2836 of an aldehyde dehydrogenase coding gene from Corynebacterium glutamicum CP was identified via RNA-seq and RT-PCR as a growth-regulated promoter. Comparing with the strong constitutive promoter Ptuf, the transcriptional strength of PCP_2836 showed a significant decrease that from about 75 to 8% in the stationary phase. By replacing the native promoters of the aceE and gltA genes with PCP_2836 in the C. glutamicum ATCC 13032-derived L-valine-producing strain AN02, the relative transcriptional levels of the aceE and gltA genes decreased from 1.2 and 1.1 to 0.35 and 0.3, and the activity of their translation products decreased to 43% and 35%, respectively. After 28 h flask fermentation, the final cell density of the obtained strains, GRaceE and GRgltA, exhibited a 7-10% decrease. However, L-valine production increased by 23.9% and 27.3%, and the yield of substrate to product increased 43.8% and 62.5%, respectively. In addition, in the stationary phase, the intracellular citrate levels in GRaceE and GRgltA decreased to 27.0% and 33.6% of AN02, and their intracellular oxaloacetate levels increased to 2.7 and 3.0 times that of AN02, respectively. CONCLUSIONS: The PCP_2836 promoter displayed a significant difference on its transcriptional strength in different cell growth phases. With using PCP_2836 to replace the native promoters of aceE and gltA genes, both the transcriptional levels of the aceE and gltA genes and the activity of their translation products demonstrated a significant decrease in the stationary phase. Thus, the availability of pyruvate was significantly increased for the synthesis of L-valine without any apparent irreversible negative impacts on cell growth. Use of this promoter can enhance the selectivity and control of gene expression and could serve as a useful research tool for metabolic engineering.


Asunto(s)
Corynebacterium glutamicum/crecimiento & desarrollo , Corynebacterium glutamicum/genética , Regiones Promotoras Genéticas , Valina/biosíntesis , Secuencia de Bases , Carbono/metabolismo , Ácidos Carboxílicos/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Reproducibilidad de los Resultados
17.
Appl Microbiol Biotechnol ; 102(10): 4319-4330, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29594358

RESUMEN

L-Valine is one of the three branched-chain amino acids (valine, leucine, and isoleucine) essential for animal health and important in metabolism; therefore, it is widely added in the products of food, medicine, and feed. L-Valine is predominantly produced through microbial fermentation, and the production efficiency largely depends on the quality of microorganisms. In recent years, continuing efforts have been made in revealing the mechanisms and regulation of L-valine biosynthesis in Corynebacterium glutamicum, the most utilitarian bacterium for amino acid production. Metabolic engineering based on the metabolic biosynthesis and regulation of L-valine provides an effective alternative to the traditional breeding for strain development. Industrially competitive L-valine-producing C. glutamicum strains have been constructed by genetically defined metabolic engineering. This article reviews the global metabolic and regulatory networks responsible for L-valine biosynthesis, the molecular mechanisms of regulation, and the strategies employed in C. glutamicum strain engineering.


Asunto(s)
Corynebacterium glutamicum , Microbiología Industrial/tendencias , Ingeniería Metabólica/tendencias , Valina/biosíntesis , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Fermentación , Regulación Bacteriana de la Expresión Génica , Valina/genética
18.
Bioprocess Biosyst Eng ; 41(1): 87-96, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29026998

RESUMEN

Acetolactate synthase catalyzes two molecules of pyruvates to form α-acetolactate, which is further converted to acetoin and 2,3-butanediol. In this study, by heterologous expression in Escherichia coli, the enzymatic properties of acetolactate synthase (AlsS) from Bacillus licheniformis WX-02 were characterized. Its K m and k cat for pyruvate were 3.96 mM and 514/s, respectively. It has the optimal activity at pH 6.5, 37 °C and was feedback inhibited by L-valine, L-leucine and L-isoleucine. Furthermore, the alsS-deficient strain could not produce acetoin, 2,3-butanediol, and L-valine, while the complementary strain was able to restore these capacities. The alsS overexpressing strain produced higher amounts of acetoin/2,3-butanediol (57.06 g/L) and L-valine (2.68 mM), which were 10.90 and 92.80% higher than those of the control strain, respectively. This is the first report regarding the in-depth understanding of AlsS enzymatic properties and its functions in B. licheniformis, and overexpression of AlsS can effectively improve acetoin/2,3-butanediol and L-valine production in B. licheniformis. We envision that this AlsS can also be applied in the improvement of acetoin/2,3-butanediol and L-valine production in other microbes.


Asunto(s)
Acetoína/metabolismo , Acetolactato Sintasa , Bacillus licheniformis/genética , Proteínas Bacterianas , Butileno Glicoles/metabolismo , Escherichia coli , Valina/metabolismo , Acetolactato Sintasa/biosíntesis , Acetolactato Sintasa/genética , Bacillus licheniformis/enzimología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
19.
World J Microbiol Biotechnol ; 34(8): 121, 2018 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-30039311

RESUMEN

L-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for L-valine grows rapidly, there is an increasing interest to develop an efficient L-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield L-valine strains to replace the traditional L-valine assay. L-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six L-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an L-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of L-valine (0.598 mol L-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m3 fermentor, this strain produced > 66.8 g/L L-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of L-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.


Asunto(s)
Brevibacterium flavum/genética , Brevibacterium flavum/metabolismo , Barajamiento de ADN/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Valina/biosíntesis , Valina/genética , Aziridinas/farmacología , Técnicas de Cultivo Celular por Lotes , Biomasa , Reactores Biológicos/microbiología , Brevibacterium flavum/efectos de los fármacos , Brevibacterium flavum/efectos de la radiación , Fermentación , Genoma Bacteriano , Inestabilidad Genómica , Glucosa/metabolismo , Microbiología Industrial , Fusión de Membrana , Mutagénesis , Mutación/genética , Protoplastos/efectos de los fármacos , Protoplastos/efectos de la radiación , Factores de Tiempo , Rayos Ultravioleta
20.
Chemistry ; 22(33): 11826-36, 2016 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-27412571

RESUMEN

A series of newly synthesized dicyanoplatinum(II) 2,2'-bipyridine complexes exhibits self-assembly properties in solution after the incorporation of the l-valine amino units appended with various hydrophobic motifs. These l-valine-derived substituents were found to have critical control over the aggregation behaviors of the complexes in the solution state. On one hand, one of the complexes was found to exhibit interesting circularly polarized luminescence (CPL) signals at low temperature due to the formation of chiral spherical aggregates in the temperature-dependent studies. On the other hand, systematic transformation from less uniform aggregates to well-defined fibrous and rod-like structures via Pt⋅⋅⋅Pt and π-π stacking interactions has also been observed in the mixed-solvent studies. These changes were monitored by UV/Vis absorption, emission, circular dichroism (CD), and CPL spectroscopies, and morphologies were studied by electron microscopy.

SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA