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Parenteral nutrition, received by many patients with intestinal failure, can induce hepatobiliary complications, which is termed as parenteral nutrition-associated liver disease (PNALD). The spectrum of PNALD ranges from cholestasis and steatosis to fibrosis and cirrhosis. Although many factors contribute to the pathogenesis of PNALD, the underlying mechanisms remain unclear. In this study, we performed targeted metabolomics to characterize the metabolomic profile in neonatal piglets receiving total parenteral nutrition (TPN) or enteral nutrition (EN) for 1 or 2 weeks. Overall, the metabolomic signature of TPN groups differed from EN groups at both time points. Among the 20 acylcarnitines identified, a majority of them were significantly reduced in TPN groups. KEGG pathway analysis showed that phenylalanine metabolism-associated pathways were dysregulated accompanied by more progressive liver steatosis associated with TPN. Next, we evaluated phenylalanine catabolism and its association with fatty acid oxidation in piglets and rats with PNALD. We showed that the hepatic expression of phenylalanine-degrading enzyme phenylalanine hydroxylase (PAH) was reduced and systemic phenylalanine levels were increased in both animal models of PNALD. Moreover, carnitine palmitoyltransferase 1A, a central regulator of fatty acid oxidation, was downregulated and its expression was negatively correlated with phenylalanine levels in TPN-fed animals. To explore the effects of phenylalanine accumulation on lipid metabolism, we treated HepG2 cells with phenylalanine co-cultured with sodium palmitate or soybean oil emulsion to induce lipid accumulation. We found that phenylalanine treatment exacerbated lipid accumulation by inhibiting fatty acid oxidation without affecting fatty acid synthesis. In summary, our findings establish a pathogenic role of increased phenylalanine levels in driving liver steatosis, linking dysregulation of phenylalanine catabolism with lipid accumulation in the context of PNALD.
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Fígado Gorduroso , Hepatopatias , Animais , Suínos , Ratos , Animais Recém-Nascidos , Nutrição Parenteral Total/efeitos adversos , Fígado/metabolismo , Hepatopatias/patologia , Fígado Gorduroso/metabolismo , Óleo de Soja/efeitos adversos , Óleo de Soja/metabolismo , Ácido Palmítico/farmacologia , MetabolômicaRESUMO
L-leucine is an essential amino acid widely used in food and pharmaceutical industries. However, the relatively low production efficiency limits its large-scale application. In this study, we rationally developed an efficient L-leucine-producing Escherichia coli strain. Initially, the L-leucine synthesis pathway was enhanced by overexpressing feedback-resistant 2-isopropylmalate synthase and acetohydroxy acid synthase both derived from Corynebacterium glutamicum, along with two other native enzymes. Next, the pyruvate and acetyl-CoA pools were enriched by deleting competitive pathways, employing the nonoxidative glycolysis pathway, and dynamically modulating the citrate synthase activity, which significantly promoted the L-leucine production and yield to 40.69 g/L and 0.30 g/g glucose, respectively. Then, the redox flux was improved by substituting the native NADPH-dependent acetohydroxy acid isomeroreductase, branched chain amino acid transaminase, and glutamate dehydrogenase with their NADH-dependent equivalents. Finally, L-leucine efflux was accelerated by precise overexpression of the exporter and deletion of the transporter. Under fed-batch conditions, the final strain LXH-21 produced 63.29 g/L of L-leucine, with a yield and productivity of 0.37 g/g glucose and 2.64 g/(L h), respectively. To our knowledge, this study achieved the highest production efficiency of L-leucine to date. The strategies presented here will be useful for engineering E. coli strains for producing L-leucine and related products on an industrial scale.
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Corynebacterium glutamicum , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Leucina/genética , Leucina/metabolismo , Vias Biossintéticas , Glucose/genética , Glucose/metabolismo , Corynebacterium glutamicum/metabolismoRESUMO
BACKGROUND: trans-4-Hydroxyproline (T-4-HYP) is a promising intermediate in the synthesis of antibiotic drugs. However, its industrial production remains challenging due to the low production efficiency of T-4-HYP. This study focused on designing the key nodes of anabolic pathway to enhance carbon flux and minimize carbon loss, thereby maximizing the production potential of microbial cell factories. RESULTS: First, a basic strain, HYP-1, was developed by releasing feedback inhibitors and expressing heterologous genes for the production of trans-4-hydroxyproline. Subsequently, the biosynthetic pathway was strengthened while branching pathways were disrupted, resulting in increased metabolic flow of α-ketoglutarate in the Tricarboxylic acid cycle. The introduction of the NOG (non-oxidative glycolysis) pathway rearranged the central carbon metabolism, redirecting glucose towards acetyl-CoA. Furthermore, the supply of NADPH was enhanced to improve the acid production capacity of the strain. Finally, the fermentation process of T-4-HYP was optimized using a continuous feeding method. The rate of sugar supplementation controlled the dissolved oxygen concentrations during fermentation, and Fe2+ was continuously fed to supplement the reduced iron for hydroxylation. These modifications ensured an effective supply of proline hydroxylase cofactors (O2 and Fe2+), enabling efficient production of T-4-HYP in the microbial cell factory system. The strain HYP-10 produced 89.4 g/L of T-4-HYP in a 5 L fermenter, with a total yield of 0.34 g/g, the highest values reported by microbial fermentation, the yield increased by 63.1% compared with the highest existing reported yield. CONCLUSION: This study presents a strategy for establishing a microbial cell factory capable of producing T-4-HYP at high levels, making it suitable for large-scale industrial production. Additionally, this study provides valuable insights into regulating synthesis of other compounds with α-ketoglutaric acid as precursor.
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Vias Biossintéticas , Escherichia coli , Hidroxiprolina , Escherichia coli/genética , Escherichia coli/metabolismo , Prolil Hidroxilases/genética , Prolil Hidroxilases/metabolismo , Ciclo do Ácido Cítrico , Engenharia Metabólica/métodos , Carbono/metabolismoRESUMO
Compatible solutes are key for the ability of halophilic bacteria to resist high osmotic stress. They have received wide attention from researchers for their excellent osmotic protection properties. Hydroxyectoine is a particularly important compatible solute, but its production by microbes faces several challenges, including low titer/yield, the presence of the byproduct ectoine, and the requirement of high salinity. Here, we aimed to metabolically engineer Escherichia coli to efficiently produce hydroxyectoine in the absence of osmotic stress without accumulating the byproduct ectoine. First, combinatorial optimization of the expression strength of key genes in the ectoine synthesis module and hydroxyectoine synthesis module was conducted. After optimization of the expression of these genes, 12.12 g/L hydroxyectoine and 0.24 g/L ectoine were obtained at 36 h in shake-flask fermentation with the addition of the co-substrate α-ketoglutarate. Further optimization of the addition of α-ketoglutarate achieved the sole production of hydroxyectoine (i.e., no ectoine accumulation), indicating that the supply of α-ketoglutarate is critically important for sole hydroxyectoine production. Finally, quorum sensing-based auto-regulation of intracellular α-ketoglutarate pool was implemented as an alternative to α-ketoglutarate addition by coupling the expression of sucA with the esaI/esaR circuit, which led to 14.93 g/L hydroxyectoine with a unit cell yield of 1.678 g/g and no ectoine accumulation in the absence of osmotic stress. This is the highest reported titer of sole hydroxyectoine production under salinity-free fermentation to date.
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Diamino Aminoácidos/metabolismo , Escherichia coli , Engenharia Metabólica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Cetoglutáricos/metabolismo , Pressão Osmótica , Percepção de QuorumRESUMO
BACKGROUND: 5-hydroxytryptophan (5-HTP), the direct biosynthetic precursor of the neurotransmitter 5-hydroxytryptamine, has been shown to have unique efficacy in the treatment of a variety of disorders, including depression, insomnia, and chronic headaches, and is one of the most commercially valuable amino acid derivatives. However, microbial fermentation for 5-HTP production continues to face many challenges, including low titer/yield and the presence of the intermediate L-tryptophan (L-Trp), owing to the complexity and low activity of heterologous expression in prokaryotes. Therefore, there is a need to construct an efficient microbial cell factory for 5-HTP production. RESULTS: We describe the systematic modular engineering of wild-type Escherichia coli for the efficient fermentation of 5-HTP from glucose. First, a xylose-induced T7 RNA polymerase-PT7 promoter system was constructed to ensure the efficient expression of each key heterologous pathway in E. coli. Next, a new tryptophan hydroxylase mutant was used to construct an efficient tryptophan hydroxylation module, and the cofactor tetrahydrobiopterin synthesis and regeneration pathway was expressed in combination. The L-Trp synthesis module was constructed by modifying the key metabolic nodes of tryptophan biosynthesis, and the heterologous synthesis of 5-HTP was achieved. Finally, the NAD(P)H regeneration module was constructed by the moderate expression of the heterologous GDHesi pathway, which successfully reduced the surplus of the intermediate L-Trp. The final engineered strain HTP11 was able to produce 8.58 g/L 5-HTP in a 5-L bioreactor with a yield of 0.095 g/g glucose and a maximum real-time productivity of 0.48 g/L/h, the highest values reported by microbial fermentation. CONCLUSION: In this study, we demonstrate the successful design of a cell factory for high-level 5-HTP production, combined with simple processes that have potential for use in industrial applications in the future. Thus, this study provides a reference for the production of high-value amino acid derivatives using a systematic modular engineering strategy and a basis for an efficient engineered strain development of 5-HTP high-value derivatives.
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5-Hidroxitriptofano , Engenharia Metabólica , 5-Hidroxitriptofano/genética , 5-Hidroxitriptofano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , NAD/metabolismo , Neurotransmissores/metabolismo , Serotonina/metabolismo , Triptofano/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Xilose/metabolismoRESUMO
Grey wolf optimization (GWO) is a meta-heuristic algorithm inspired by the hierarchy and hunting behavior of grey wolves. GWO has the superiorities of simpler concept and fewer adjustment parameters, and has been widely used in different fields. However, there are some disadvantages in avoiding prematurity and falling into local optimum. This paper presents an improved grey wolf optimization (IGWO) to ameliorate these drawbacks. Firstly, a modified position update mechanism for pursuing high quality solutions is developed. By designing an ameliorative position update formula, a proper balance between the exploration and exploitation is achieved. Moreover, the leadership hierarchy is strengthened by proposing adaptive weights of α, ß and δ. Then, a dynamic local optimum escape strategy is proposed to reinforce the ability of the algorithm to escape from the local stagnations. Finally, some individuals are repositioned with the aid of the positions of the leaders. These individuals are pulled to new positions near the leaders, helping to accelerate the convergence of the algorithm. To verify the effectiveness of IGWO, a series of contrast experiments are conducted. On the one hand, IGWO is compared with some state-of-the-art GWO variants and several promising meta-heuristic algorithms on 20 benchmark functions. Experimental results indicate that IGWO performs better than other competitors. On the other hand, the applicability of IGWO is verified by a robot global path planning problem, and simulation results demonstrate that IGWO can plan shorter and safer paths. Therefore, IGWO is successfully applied to the path planning as a new method.
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Robótica , Algoritmos , Benchmarking , Simulação por ComputadorRESUMO
In this work, a universal strategy for solid, solution, or gel state organic persistent luminescent materials via radiative energy transfer is proposed. The persistent luminescence (τ>0.7â s) could be remotely regulated between different colors by controlling the isomerization of the energy acceptor. The function relies on the simple radiative energy transfer (reabsorption) mechanism, rather than the complicated communication between the excited state of the molecules such as Förster resonance energy transfer or Dexter energy transfer. And the "apparent lifetime" for the energy acceptor is the same as the lifetime of the energy donor, which was different with a traditional radiative energy transfer process. The simple working principle endows this strategy with huge universality, flexibility, and operability. This work offers a simple, feasible, and universal way to construct various persistent luminescent materials in solid, solution, and gel states.
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INTRODUCTION AND OBJECTIVES: Chronic hepatitis B virus (HBV) infection exerts an impact on lipid metabolism, but its interaction with dysmetabolism-based non-alcoholic fatty liver disease (NAFLD) remains uncertain. The purpose of the study was to investigate the effects of HBV infection on lipid metabolism, hepatic steatosis and related impairments of NAFLD patients. METHODS: Biopsy-proven Chinese NAFLD patients with (NAFLD-HBV group, n = 21) or without chronic HBV infection (NAFLD group, n = 41) were enrolled in the case-control study. Their serum lipidomics was subjected to individual investigation by ultra-performance liquid chromatography-tandem mass spectrometry. Steatosis, activity, and fibrosis (SAF) scoring revealed the NAFLD-specific pathological characteristics. RESULTS: Chronic HBV infection was associated with global alteration of serum lipidomics in NAFLD patients. Upregulation of phosphatidylcholine (PCs), choline plasmalogen (PC-Os) and downregulation of free fatty acids (FFAs), lysophosphatidylcholine (LPCs) dominated the HBV-related lipidomic characteristics. Compared to those of NAFLD group, the levels of serum hepatoxic lipids (FFA16:0, FFA16: 1, FFA18:1, FFA18:2) were significantly lowered in the NAFLD-HBV group. These low-level FFAs demonstrated correlation to statistical improvements in aspartate aminotransferase activity (FFA16:0, r = 0.33; FFA16:1, r = 0.37; FFA18:1, r = 0.32; FFA18:2, r = 0.42), hepatocyte steatosis (FFA16: 1, r = 0.39; FFA18:1, r = 0.39; FFA18:2, r = 0.32), and ballooning (FFA16:0, r = 0.30; FFA16:1, r = 0.45; FFA18:1, r = 0.36; FFA18:2, r = 0.30) (all P < 0.05). CONCLUSION: Chronic HBV infection may impact on the serum lipidomics and steatosis-related pathological characteristics of NAFLD.
Assuntos
Hepatite B Crônica/sangue , Hepatite B Crônica/complicações , Metabolismo dos Lipídeos/fisiologia , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/virologia , Adulto , Estudos de Casos e Controles , China , Ácidos Graxos não Esterificados/sangue , Feminino , Hepatite B Crônica/patologia , Humanos , Lipidômica , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Adulto JovemRESUMO
L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. L-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in L-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of L-theanine in Corynebacterium glutamicum, an industrially safe host. For L-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of L-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an L-glutamate overproducing strain, to produce L-theanine upon ethylamine addition to the hosts. Second, the L-glutamate exporter in host cells was disrupted, which markedly increased the L-theanine titer in GDK-9 cells and almost eliminated the accumulation of L-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated L-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived L-serine/L-alanine decarboxylases; however, these enzymes showed no L-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L L-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of L-theanine achieved using ethylamine supplementation.
Assuntos
Corynebacterium glutamicum/metabolismo , Fermentação , Glutamatos/biossíntese , Engenharia Metabólica/métodos , Trifosfato de Adenosina/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Etilaminas/metabolismo , Ácido Glutâmico/metabolismo , Microbiologia IndustrialRESUMO
Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA fragment is still challenging compared with gene deletion and small fragment integration. Moreover, to guarantee sufficient Cas9-induced double-strand breaks, it is usually necessary to design several gRNAs to select the appropriate one. Accordingly, we established a practical daily routine in the laboratory work, involving multiple-step chromosomal integration of the divided segments from a large DNA fragment. First, we introduced and optimized the protospacers from Streptococcus pyogenes in E. coli W3110. Next, the appropriate fragment size for each round of integration was optimized to be within 3-4 kb. Taking advantage of the optimized protospacer/gRNA pairs, a DNA fragment with a total size of 15.4 kb, containing several key genes for uridine biosynthesis, was integrated into W3110 chromosome, which produced 5.6 g/L uridine in shake flask fermentation. Using this strategy, DNA fragments of virtually any length can be integrated into a suitable genomic site, and two gRNAs can be alternatively used, avoiding the tedious construction of gRNA-expressing plasmids. This study thus presents a useful strategy for large DNA fragment integration into the E. coli chromosome, which can be easily adapted for use in other bacteria.
Assuntos
Sistemas CRISPR-Cas , Cromossomos Bacterianos/genética , Fragmentação do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Sequência de Bases , Clonagem Molecular , Deleção de Genes , Edição de Genes , Genes Bacterianos , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/genéticaRESUMO
Uridine is a kind of pyrimidine nucleoside that has been widely applied in the pharmaceutical industry. Although microbial fermentation is a promising method for industrial production of uridine, an efficient microbial cell factory is still lacking. In this study, we constructed a metabolically engineered Escherichia coli capable of high-yield uridine production. First, we developed a CRISPR/Cas9-mediated chromosomal integration strategy to integrate large DNA into the E. coli chromosome, and a 9.7â¯kb DNA fragment including eight genes in the pyrimidine operon of Bacillus subtilis F126 was integrated into the yghX locus of E. coli W3110. The resultant strain produced 3.3â¯g/L uridine and 4.5â¯g/L uracil in shake flask culture for 32â¯h. Subsequently, five genes involved in uridine catabolism were knocked out, and the uridine titer increased to 7.8â¯g/L. As carbamyl phosphate, aspartate, and 5'-phosphoribosyl pyrophosphate are important precursors for uridine synthesis, we further modified several metabolism-related genes and synergistically improved the supply of these precursors, leading to a 76.9% increase in uridine production. Finally, nupC and nupG encoding nucleoside transport proteins were deleted, and the extracellular uridine accumulation increased to 14.5â¯g/L. After 64â¯h of fed-batch fermentation, the final engineered strain UR6 produced 70.3â¯g/L uridine with a yield and productivity of 0.259â¯g/g glucose and 1.1â¯g/L/h, respectively. To the best of our knowledge, this is the highest uridine titer and productivity ever reported for the fermentative production of uridine.
Assuntos
Escherichia coli , Engenharia Metabólica , Microrganismos Geneticamente Modificados , Uridina/biossíntese , Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Loci Gênicos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Óperon , Uridina/genéticaRESUMO
4-Hydroxyisoleucine (4-HIL) exhibits a unique glucose-dependent insulinotropic activity and is a promising candidate for the treatment of diabetes. Direct fermentation of 4-HIL has been recently studied; however, the expected titre and yield were not achieved. In this study, we initially developed a pathway for the synthesis of 4-HIL in an L-isoleucine producer, C. glutamicum YI, but insufficient supply of α-ketoglutarate was a bottleneck for a strong production. Six genes involved in oxaloacetate and α-ketoglutarate branches were overexpressed or deleted, which increased the production of 4-HIL to 5.12â¯g/L but a considerable amount of L-isoleucine still accumulated in the culture. We then dynamically modulated the activity of the α-ketoglutarate dehydrogenase complex (ODHC) by employing L-isoleucine-responsive transcription or attenuation strategies. The best-engineered strain, HIL18, produced 34.21â¯g/L 4-HIL with a negligible accumulation of byproducts, including approximately 0.6â¯g/L L-isoleucine. This study achieved the highest production and yield of 4-HIL, and optimizing the TCA cycle by dynamically modulating the activity of ODHA can be a powerful strategy to balance the carbon flux and achieve efficient production of α-ketoglutarate and derivatives.
Assuntos
Corynebacterium glutamicum , Isoleucina/análogos & derivados , Engenharia Metabólica , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Isoleucina/biossíntese , Isoleucina/genéticaRESUMO
Ectoine is a protective agent and stabilizer whose synthesis pathway exclusively exists in select moderate halophiles. A novel established process called "bacterial milking" efficiently synthesized ectoine in moderate halophiles, however, this method places high demands on equipment and is cost prohibitive. In this study, we constructed an ectoine producing strain by introducing the ectoine synthesis pathway into Escherichia coli and improved its production capacity. Firstly, the ectABC gene cluster from Halomonas elongata was introduced into E. coli W3110 and the resultant strain synthesized 4.9g/L ectoine without high osmolarity. Subsequently, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was deleted to weaken the competitive l-threonine branch, resulting in an increase of ectoine titer by 109%. Furthermore, a feedback resistant lysC from Corynebacterium glutamicum encoding the aspartate kinase was introduced to complement the enzymatic activity deficiency caused by thrA deletion and a 9% increase of ectoine titer was obtained. Finally, the promoter of ppc that encodes phosphoenolpyruvate carboxylase was replaced by a trc promoter, and iclR, a glyoxylate shunt transcriptional repressor gene, was deleted. The oxaloacetate pool, was thus reinforced and ectoine titer increased by 21%. The final engineered strain ECT05 (pTrcECT, pSTVLysC-CG) produced 25.1g/L ectoine by fed-batch fermentation in low salt concentration with glucose as a carbon source. The specific ectoine production and productivity was 0.8g/g DCW and 0.84gL(-)(1)h(-)(1) respectively. The overall ectoine yield was 0.11g/g of glucose.
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Diamino Aminoácidos/biossíntese , Vias Biossintéticas/fisiologia , Escherichia coli/fisiologia , Fermentação/fisiologia , Melhoramento Genético/métodos , Glucose/metabolismo , Engenharia Metabólica/métodos , Diamino Aminoácidos/genética , Diamino Aminoácidos/isolamento & purificação , Redes e Vias Metabólicas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Alpha-ketobutyrate has been widely used in medicine and food additive industry. Because chemical and enzymatic methods are associated with many deficiencies, the recent focus shifted to fermentation for the production of α-ketobutyrate. In this study, a genetically engineered strain THRDΔrhtAΔilvIH/pWSK29-ilvA was constructed, starting from an L-threonine-producing strain, by overexpressing threonine dehydratase (TD), reducing α-ketobutyrate catabolism and L-threonine export. The shake flask cultivation of THRDΔrhtAΔilvIH/pWSK29-ilvA allowed the production of 16.2 g/L α-ketobutyrate. Accumulation of α-ketobutyrate severely inhibited the cell growth. To develop a better TD expression system and avoid the usage of the expensive inducer IPTG, a temperature-induced plasmid pBV220-ilvA was selected to transform the strain THRDΔrhtAΔilvIH for α-ketobutyrate production. The initial temperature was maintained at 35°C to guarantee normal cell growth, and then elevated to 40°C to induce the expression of TD. Under optimized conditions, the α-ketobutyrate titer reached 40.8 g/L after 28 h of fermentation, with a productivity of 1.46 g/L/h and a yield of 0.19 g/g glucose, suggesting large-scale production potential. Biotechnol. Bioeng. 2016;113: 2054-2059. © 2016 Wiley Periodicals, Inc.
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Butiratos/metabolismo , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Engenharia Metabólica/métodos , Técnicas de Cultura Celular por Lotes , Butiratos/análise , Escherichia coli/genética , Fermentação , Redes e Vias Metabólicas , Temperatura , Treonina DesidrataseRESUMO
Production of L-glutamate using a biotin-deficient strain of Corynebacterium glutamicum has a long history. The process is achieved by controlling biotin at suboptimal dose in the initial fermentation medium, meanwhile feeding NH4OH to adjust pH so that α-ketoglutarate (α-KG) can be converted to L-glutamate. In this study, we deleted glutamate dehydrogenase (gdh1 and gdh2) of C. glutamicum GKG-047, an L-glutamate overproducing strain, to produce α-KG that is the direct precursor of L-glutamate. Based on the method of L-glutamate fermentation, we developed a novel double-phase pH and biotin control strategy for α-KG production. Specifically, NH4OH was added to adjust the pH at the bacterial growth stage and NaOH was used when the cells began to produce acid; besides adding an appropriate amount of biotin in the initial medium, certain amount of additional biotin was supplemented at the middle stage of fermentation to maintain a high cell viability and promote the carbon fixation to the flux of α-KG production. Under this control strategy, 45.6 g/L α-KG accumulated after 30-h fermentation in a 7.5-L fermentor and the productivity and yield achieved were 1.52 g/L/h and 0.42 g/g, respectively.
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Biotina/metabolismo , Corynebacterium glutamicum/metabolismo , Ácidos Cetoglutáricos/metabolismo , Corynebacterium glutamicum/enzimologia , Meios de Cultura , Fermentação , Glutamato Desidrogenase/genética , Concentração de Íons de HidrogênioRESUMO
Perovskite film generally has rough surface morphology due to the voids between the grain domains. Smoothed interface contact between the perovskite layer and the top electrode is critical for planar perovskite solar cells. We reported high efficiency bromine-iodine based perovskite solar cells with a flattening cathode interface by incorporating a solution-processed bathocuproine (sBCP) interfacial layer at the cathode side. Compared with vacuum evaporated bathocuproine (eBCP), sBCP demonstrated an excellent surface modification effect at the cathode side with very smaller charge transfer resistance. Accordingly, a high fill factor exceeding 85% and a power conversion efficiency exceeding 13% in CH3NH3PbI3-xBrx based perovskite solar cells were achieved. The largely improved fill factor was attributed to the smooth film morphology and full surface coverage of perovskite films modified by the solution-processed BCP layer.
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OBJECTIVES: To rationally identify targets for enhancing adenosine production, transcription level of genes involved in adenosine synthesis of Bacillus subtilis XGL was detected during the fermentation process, complemented with metabolite pool analysis. RESULTS: PurR-regulated genes (pur operon and purA) and prs were down-regulated and 5-phosphoribosyl 1-pyrophosphate (PRPP) decreased considerably after 24 h when adenosine significantly accumulated. Since PRPP could strongly antagonize the binding of PurR to its targets, it was inferred that down-regulation of pur operon and purA might be due to a low PRPP pool, which was confirmed by metabolite analysis. So desensitized prs responsible for PRPP synthesis was overexpressed, resulting in increased PRPP concentration and pur operon transcription. To further enhance the adenosine production, desensitized purF and prs were co-overexpressed with integrating additional copy of purA to B. subtilis XGL genome, resulting in 24.3 % (1.29 g/g DCW) higher adenosine production than that by B. subtilis XG. CONCLUSIONS: Overexpression of prs, purF and purA under the guidance of transcriptional and metabolite pool analysis significantly increased adenosine production. Strategies used in this study have potential applications for rational modification of industrial microorganisms.
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Adenosina/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Adenosina/análise , Adenosina/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Proteínas Repressoras/metabolismoRESUMO
Fed-batch cultivations of L-isoleucine-producing Escherichia coli TRFP (SGr, α-ABAr, with a pTHR101 plasmid containing a thr operon and ilvA) were carried out on different carbon sources: glucose, sucrose, fructose, maltose and glycerol. The results indicated that sucrose was the best initial carbon source for L-isoleucine production and then sucrose concentration of 30 g·L-1 was determined in the production medium. The results of different carbon sources feeding showed that the glucose solution was the most suitable feeding media. The dissolved oxygen (DO) of L-isoleucine fermentation was maintained at 5%, 15% and 30% with DO-stat feeding, respectively. The results indicated that when the DO level was maintained at 30%, the highest biomass and L-isoleucine production were obtained. The accumulation of acetate was decreased and the production of L-isoleucine was increased markedly, when the glucose concentration was maintained at 0.15 g·L-1 by using glucose-stat feeding. Finally, the glucose concentration was maintained at 0.10 g·L-1 and the DO level was controlled at approximately 30% during the whole fermentation period, using the combined feeding strategy of glucose-stat feeding and DO feedback feeding. The acetate accumulation was decreased to 7.23 g·L-1, and biomass and production of L-isoleucine were increased to 46.8 and 11.95 g·L-1, respectively.
RESUMO
High concentrations of acetate, the main by-product of Escherichia coli (E. coli) high cell density culture, inhibit bacterial growth and L-threonine production. Since metabolic overflux causes acetate accumulation, we attempted to reduce acetate production by redirecting glycolysis flux to the pentose phosphate pathway by deleting the genes encoding phosphofructokinase (pfk) and/or pyruvate kinase (pyk) in an L-threonine-producing strain of E. coli, THRD. pykF, pykA, pfkA, and pfkB deletion mutants produced less acetate (9.44 ± 0.83, 3.86 ± 0.88, 0.30 ± 0.25, and 6.99 ± 0.85 g/l, respectively) than wild-type THRD cultures (19.75 ± 0.93 g/l). THRDΔpykF and THRDΔpykA produced 11.05 and 5.35 % more L-threonine, and achieved a 10.91 and 5.60 % higher yield on glucose, respectively. While THRDΔpfkA grew more slowly and produced less L-threonine than THRD, THRDΔpfkB produced levels of L-threonine (102.28 ± 2.80 g/l) and a yield on glucose (0.34 g/g) similar to that of THRD. The dual deletion mutant THRDΔpfkBΔpykF also achieved low acetate (7.42 ± 0.81 g/l) and high L-threonine yields (111.37 ± 2.71 g/l). The level of NADPH in THRDΔpfkA cultures was depressed, whereas all other mutants produced more NADPH than THRD did. These results demonstrated that modification of glycolysis in E. coli THRD reduced acetate production and increased accumulation of L-threonine.
Assuntos
Escherichia coli/genética , Glicólise , Treonina/biossíntese , Escherichia coli/metabolismo , Fermentação , Deleção de Genes , Glucose/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fosfofrutoquinases/genética , Piruvato Quinase/genéticaRESUMO
Estimation of distribution algorithm (EDA) is an intelligent optimization algorithm based on the probability statistics theory. A fast elitism Gaussian estimation of distribution algorithm (FEGEDA) is proposed in this paper. The Gaussian probability model is used to model the solution distribution. The parameters of Gaussian come from the statistical information of the best individuals by fast learning rule. A fast learning rule is used to enhance the efficiency of the algorithm, and an elitism strategy is used to maintain the convergent performance. The performances of the algorithm are examined based upon several benchmarks. In the simulations, a one-dimensional benchmark is used to visualize the optimization process and probability model learning process during the evolution, and several two-dimensional and higher dimensional benchmarks are used to testify the performance of FEGEDA. The experimental results indicate the capability of FEGEDA, especially in the higher dimensional problems, and the FEGEDA exhibits a better performance than some other algorithms and EDAs. Finally, FEGEDA is used in PID controller optimization of PMSM and compared with the classical-PID and GA.