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We investigated a fatal case of primary amoebic meningoencephalitis from an indoor surfing center in Taiwan. The case was detected through encephalitis syndromic surveillance. Of 56 environmental specimens, 1 was positive for Naegleria fowleri ameba. This report emphasizes the risk for N. fowleri infection from inadequately disinfected recreational waters, even indoors.
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Infecções Protozoárias do Sistema Nervoso Central , Naegleria fowleri , Humanos , Naegleria fowleri/isolamento & purificação , Naegleria fowleri/genética , Taiwan/epidemiologia , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/diagnóstico , Infecções Protozoárias do Sistema Nervoso Central/epidemiologia , Evolução Fatal , Masculino , Meningoencefalite/parasitologia , Meningoencefalite/diagnóstico , Amebíase/diagnóstico , Amebíase/parasitologia , AdultoRESUMO
BACKGROUND: D-allulose, a hexulose monosaccharide with low calorie content and high sweetness, is commonly used as a functional sugar in food and nutrition. However, enzyme preparation of D-allulose from D-frutose was severely hindered by the non-enzymatic browning under alkaline and high-temperature, and the unnecessary by-products further increased the difficulties in separation and extraction for industrial applications. Here, to address the above issue during the production process, a tandem D-allulose 3-epimerase (DPEases) isomerase synergistic expression strategy and an auto-inducible promoter engineering were levered in Bacillus subtilis 168 (Bs168) for efficient synthesis of D-allulose under the acidic conditions without browning. RESULTS: First, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 (DSdpe) and Clostridium cellulolyticum H10 (RCdpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs168/pMA5-DSdpe-RCdpe increases enzyme activity to 18.9 U/mL at acidic conditions (pH 6.5), much higher than 17.2 and 16.7 U/mL of Bs168/pMA5-DSdpe and Bs168/pMA5-RCdpe, respectively. Subsequently, six recombinant strains based on four constitutive promoters were constructed in variable expression cassettes for improving the expression level of protein. Among those engineered strains, Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe exhibited the highest enzyme activity with 480.1 U/mL on fed-batch fermentation process in a 5 L fermenter at pH 6.5, about 2.1-times higher than the 228.5 U/mL of flask fermentation. Finally, the maximum yield of D-allulose reached as high as 163.5 g/L at the fructose concentration (50% w/v) by whole-cell biocatalyst. CONCLUSION: In this work, the engineered recombinant strain Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe was demonstrated as an effective microbial cell factory for the high-efficient synthesis of D-allulose without browning under acidic conditions. Based on the perspectives from this research, this strategy presented here also made it possible to meet the requirements of the industrial hyper-production of other rare sugars under more acidic conditions in theory.
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Bacillus subtilis , Racemases e Epimerases , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Fermentação , Frutose/metabolismo , Racemases e Epimerases/metabolismoRESUMO
Methanol is considered as a potential hazard in the methanol-induced yeast expression of food-related enzymes. To increase the production efficiency of recombinant proteins in Pichia pastoris without methanol induction, a novel dual-plasmid system was constructed, for the first time, by a combining the strategies of genomic integration and episomal expression. To obtain a high copy number of the target gene, the autonomously replicating sequence derived from Kluyveromyces lactis (PARS) was used to construct episomal vectors carrying the constitutive promoters PGAP and PGCW14. In addition, an integrative vector carrying the PGCW14 promoter was constructed by replacing the PGAP promoter sequence with a partial PGCW14 promoter. Next, using xylanase XynA from Streptomyces sp. FA1 as the model enzyme, recombination strains were transformed with different combinations of integrating and episomal vectors that were constructed to investigate the changes in the protein yield. Results in shake flasks indicated that the highest enzyme yield was achieved when integrated PGAP and episomal PGCW14 were simultaneously transformed into the host strain. Meanwhile, the copy number of xynA increased from 1.14 ± 0.46 to 3.06 ± 0.35. The yield of XynA was successfully increased to 3925 U·mL-1 after 102 h of fermentation in a 3.6 L fermenter, which was 16.7-fold and 2.86-fold of the yields that were previously reported for the constitutive expression and methanol-induced expression of the identical protein, respectively. Furthermore, the high-cell-density fermentation period was shortened from 132 h to 102 h compared to that of methanol-induced system. Since the risk of methanol toxicity is removed, this novel expression system would be suitable for the production of proteins related to the food and pharmaceutical industries.
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Endo-1,4-beta-Xilanases/biossíntese , Pichia/genética , Pichia/metabolismo , Plasmídeos/genética , Streptomyces/enzimologia , Endo-1,4-beta-Xilanases/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Vetores Genéticos , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossínteseRESUMO
Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that impairs synthesis of dopamine and serotonin. Children with AADC deficiency exhibit severe motor, behavioral, and autonomic dysfunctions. We previously generated an IVS6+4A>T knock-in mouse model of AADC deficiency (Ddc(KI) mice) and showed that gene therapy at the neonatal stage can rescue this phenotype. In the present study, we extended this treatment to systemic therapy on young mice. After intraperitoneal injection of AADC viral vectors into 7-day-old Ddc(KI) mice, the treated mice exhibited improvements in weight gain, survival, motor function, autonomic function, and behavior. The yfAAV9/3-Syn-I-mAADC-treated mice showed greater neuronal transduction and higher brain dopamine levels than AAV9-CMV-hAADC-treated mice, whereas AAV9-CMV-hAADC-treated mice exhibited hyperactivity. Therefore, neurotransmitter-deficient animals can be rescued at a young age using systemic gene therapy, although a vector for preferential neuronal expression may be necessary to avoid hyperactivity caused by this treatment.
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Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/terapia , Descarboxilases de Aminoácido-L-Aromático/deficiência , Descarboxilases de Aminoácido-L-Aromático/genética , Terapia Genética , Neurônios/metabolismo , Neurotransmissores/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico por imagem , Erros Inatos do Metabolismo dos Aminoácidos/mortalidade , Erros Inatos do Metabolismo dos Aminoácidos/fisiopatologia , Animais , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Comportamento Animal , Pressão Sanguínea/genética , Encéfalo/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Dopamina/metabolismo , Ativação Enzimática , Fluordesoxiglucose F18 , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Frequência Cardíaca , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Atividade Motora , Especificidade de Órgãos/genética , Tomografia por Emissão de Pósitrons , Transdução Genética , Aumento de Peso/genéticaRESUMO
Lactoferrin is an iron-binding glycoprotein with antibacterial, antitumor, and immunomodulatory functions derived from milk and mucosal secretions. Lactoferrin is used in various products, such as infant formula milk powder, nutritional supplements, and cosmetics. Researchers have developed new technologies to produce lactoferrin because there are limitations in the separation and purification of lactoferrin from milk that cannot compensate for the market demand. Therefore, synthetic systems of lactoferrin have been developed with the development of genetic engineering, and the structure of lactoferrin expressed in heterologous systems is very similar to that of natural lactoferrin. The structure and functions of lactoferrin and the design and construction of synthetic lactoferrin biological systems, especially microbial synthetic systems, including prokaryotic and eukaryotic host-expression systems, are described. On the basis of these results, we summarize the challenges and solutions for constructing systems of high-yield lactoferrin. The development directions of recombinant lactoferrin are discussed in this review. Overall, the design and development of these synthetic biological systems have allowed us to explore the great potential of the industrial large-scale preparation of lactoferrin.
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The production of androst-4-ene-3,17-dione (AD) by the steroidal microbial cell factory requires transcription factors (TFs) to participate in metabolic regulation. However, microbial cell factory lacks effective TFs that can respond to AD in its metabolic pathway. Additionally, finding and obtaining natural TFs that specifically respond to AD is a complex and onerous task. In this study, we devised an artificial TF that responds to AD, termed AdT, based on structure-guided molecular dynamics (MD) simulation. According to MD analysis of the conformational changes of AdT after binding to AD, an LBD in which the N- and C-termini exhibited convergence tendencies was used as a microswitch to guide the assembly of a DNA-binding domain lexA, a linker (GGGGS)2, and a transcription activation domain B42 into an artificial TF. As a proof of design, a AD biosensor was designed and constructed in yeast on the basis of the ligand-binding domain (LBD) of hormone receptor. In addition, the transcription factor activity of AdT was increased by 1.44-fold for its variant F320Y. Overall, we created non-natural TF elements for AD microbial cell factory, and expected that the design TF strategy will be applied to running in parallel to the signaling machinery of the host cell.
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Phospholipase A1 (PLA1) is a kind of specific phospholipid hydrolase widely used in food, medical, textile. However, limitations in its expression and enzymatic activity have prompted the investigation of the phospholipase-assisting protein PlaS. In this study, we elucidate the role of PlaS in enhancing the expression and activity of PlaA1 through N-terminal truncation. Our research demonstrates that truncating the N-terminal region of PlaS effectively overcomes its inhibitory effect on host cells, resulting in improved cell growth and increased protein solubility of the protein. The yeast two-hybrid assay confirms the interaction between PlaA1 and N-terminal truncated PlaS (∆N27 PlaS), highlighting their binding capabilities. Furthermore, in vitro studies using Biacore analysis reveal a concentration-dependent and specific binding between PlaA1 and ∆N27 PlaS, exhibiting high affinity. Molecular docking analysis provides insights into the hydrogen bond interactions between ∆N27 PlaS and PlaA1, identifying key amino acid residues crucial for their binding. Finally, the enzyme activity of PLA1 was boost to 8.4 U/mL by orthogonal test. Study significantly contributes to the understanding of the interaction mechanism between PlaS and PlaA1, offering potential strategies for enhancing PlaA1 activity through protein engineering approaches.
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Aromatic l-amino acid decarboxylase (AADC) is responsible for the syntheses of dopamine and serotonin. Children with AADC deficiency exhibit compromised development, particularly with regard to their motor functions. Currently, no animal model of AADC deficiency exists. We inserted an AADC gene mutation (IVS6+4A>T) and a neomycin-resistance gene into intron 6 of the mouse AADC (Ddc) gene. In the brains of homozygous knock-in (KI) mice (Ddc(IVS6/IVS6)), AADC mRNA lacked exon 6, and AADC activity was <0.3% of that in wild-type mice. Half of the KI mice were born alive but grew poorly and exhibited severe dyskinesia and hindlimb clasping after birth. Two-thirds of the live-born KI mice survived the weaning period, with subsequent improvements in their growth and motor functions; however, these mice still displayed cardiovascular dysfunction and behavioral problems due to serotonin deficiencies. The brain dopamine levels in the KI mice increased from 9.39% of the levels in wild-type mice at 2weeks of age to 37.86% of the levels in wild-type mice at 8weeks of age. Adult KI mice also exhibited an exaggerated response to apomorphine and an elevation of striatal c-Fos expression, suggesting post-synaptic adaptations. Therefore, we generated an AADC deficient mouse model, in which compensatory regulation allowed the mice to survive to adulthood. This mouse model will be useful both for developing gene therapies for AADC deficiency and for designing treatments for diseases associated with neurotransmitter deficiency.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Dopamina/metabolismo , Discinesias/metabolismo , Neostriado/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Descarboxilases de Aminoácido-L-Aromático/deficiência , Descarboxilases de Aminoácido-L-Aromático/genética , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Modelos Animais de Doenças , Dopamina/genética , Discinesias/genética , Técnicas de Introdução de Genes , CamundongosRESUMO
BACKGROUND: (R)-mandelic acid (R-MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R-MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains. RESULTS: Herein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R-MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans. With a new screened strong endogenous promoter P12780, the production of R-MA was improved to 10.26 g/L compared to 7.36 g/L of using Plac. As R-MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R-MA tolerance. The adapted strain that can tolerate 6 g/L R-MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R-MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R-MA stress condition, we identified five critical genes that were associated with R-MA tolerance, among which AcrA overexpression could further improve R-MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate. CONCLUSIONS: The microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.
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l-arginine is a semi-essential amino acid that is broadly used as food additives and pharmaceutical intermediates. The synthesis of l-arginine is restricted by complex metabolic mechanisms and suboptimal fermentation conditions. Initially, a mutant strain that accumulated 19.4 g/L l-arginine was generated by random mutagenesis. Subsequently, a mutation of the repressor protein (argRG159D) in the l-arginine operon and glutamate synthase (gltD) with 532-fold upregulation were identified to be vital for l-arginine production by multi-omic analysis. Systematic metabolic engineering was used to modify the strain, which included interfering with α-ketoglutarate dehydrogenase complex (ODHC) activity by knocking out serine/threonine-protein kinase (pknG), enhancing the expression of multiple key enzymes in the l-arginine synthesis pathway, and increasing the availability of intracellular cofactor (NADPH) and energy (ATP). Finally, C. glutamicum ARG12 produced 71.3 g/L l-arginine, with a yield of 0.43 g/g glucose by fermentation optimization. This study provides new ideas to boost l-arginine production.
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D-mannitol is widely used in the pharmaceutical and medical industries as an important precursor of antitumor drugs and immune stimulants. However, the cost of the current enzymatic process for D-mannitol synthesis is high, thus not suitable for commercialization. To address this issue, an efficient mannitol dehydrogenase LpGDH used for the conversion and a glucose dehydrogenase BaGDH used for NADH regeneration were screened, respectively. These two enzymes were co-expressed in Escherichia coli BL21(DE3) to construct a two-enzyme cascade catalytic reaction for the efficient synthesis of d-mannitol, with a conversion rate of 59.7% from D-fructose achieved. The regeneration of cofactor NADH was enhanced by increasing the copy number of Bagdh, and a recombinant strain E. coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh was constructed to address the imbalance between cofactor amount and key enzyme expression level in the two-enzyme cascade catalytic reaction. An optimized whole cell transformation process was conducted under 30 â, initial pH 6.5, cell mass (OD600) 30, 100 g/L D-fructose substrate and an equivalent molar concentration of glucose. The highest yield of D-mannitol was 81.9 g/L with a molar conversion rate of 81.9% in 5 L fermenter under the optimal conversion conditions. This study provides a green and efficient biotransformation method for future large-scale production of D-mannitol, which is also of great importance for the production of other sugar alcohols.
Assuntos
Escherichia coli , Manitol , Escherichia coli/metabolismo , Frutose , Manitol/metabolismo , Manitol Desidrogenases/química , Manitol Desidrogenases/genética , Manitol Desidrogenases/metabolismo , NAD/metabolismoRESUMO
Sucrose isomerase (SI), catalyzing sucrose to isomaltulose, has been widely used in isomaltulose production, but its poor thermostability is still resisted in sustainable batches production. Here, protein engineering and one-step immobilized cell strategy were simultaneously coupled to maintain steady state for long-term operational stabilities. First, rational design of Pantoea dispersa SI (PdSI) for improving its thermostability by predicting and substituting the unstable amino acid residues was investigated using computational analysis. After screening mutagenesis library, two single mutants (PdSIV280L and PdSIS499F) displayed favorable characteristics on thermostability, and further study found that the double mutant PdSIV280L/S499F could stabilize PdSIWT better. Compared with PdSIWT, PdSIV280L/S499F displayed a 3.2°C-higher T m , and showed a ninefold prolonged half-life at 45°C. Subsequently, a one-step simplified immobilization method was developed for encapsulation of PdSIV280L/S499F in food-grade Corynebacterium glutamicum cells to further enhance the recyclability of isomaltulose production. Recombinant cells expressing combinatorial mutant (RCSI2) were successfully immobilized in 2.5% sodium alginate without prior permeabilization. The immobilized RCSI2 showed that the maximum yield of isomaltulose by batch conversion reached to 453.0 g/L isomaltulose with a productivity of 41.2 g/l/h from 500.0 g/L sucrose solution, and the conversion rate remained 83.2% after 26 repeated batches.
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L-valine is a valuable amino acid in mammals that is used as the main component of feed additives. The low efficiency of the fermentation titer limits the industrial application of L-valine. Here, an L-valine-producing strain of Escherichia coli was obtained using a multi-modular strategy. Initially, a chassis strain was generated by mutagenesis and high-throughput screening. The L-valine biosynthetic pathway and transport module were modified to improve the L-valine titer. Subsequently, the transcription factors associated with L-valine biosynthesis were investigated. Overexpression of PdhR and inhibition of the expression of RpoS promoted L-valine synthesis. Finally, the NADPH supply was enhanced after the introduction of the heterologous Entner-Doudoroff (ED) pathway from Zymomonas mobilis. The strain VAL38 produced 92 g/L L-valine in a 5-L bioreactor with a yield of 0.34 g/g glucose. This strategy is provided as a reference for improving the production performance of cell factories for L-valine and its derivatives.
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Proteínas de Escherichia coli , Escherichia coli , Engenharia Metabólica , Valina , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Fermentação , Engenharia Metabólica/métodos , NADP/metabolismo , Valina/biossínteseRESUMO
l-glutamine is a semi-essential amino acid widely used in the food and pharmaceutical industries. The microbial synthesis of l-glutamine is limited by lack of effective strains with high titer and safety. First, ARTP mutagenesis combined with high-throughput screening generated an l-glutamine-producing strain of Corynebacterium glutamicum with titer of 25.7 ± 2.7 g/L. Subsequently, a series of rational metabolic approaches were used to further improve l-glutamine production, which included increasing the carbon flow to l-glutamine (proB and NCgl1221 knockout), enhancing the catalytic efficiency of the key enzyme (glnE knockout and glnA screening and overexpression) and reinforcement of ATP regeneration (ppk overexpression and RBS optimization). Finally, we proposed a two-stage pH control strategy to address the inconsistent effect of pH on cell growth and l-glutamine production. These combined strategies led to a 186.0% increase of l-glutamine titer compared to that of the initial strain, reaching 73.5 ± 3.1 g/L with a yield of 0.368 ± 0.034 g/g glucose.
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Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Fermentação , Glucose , Glutamina , Concentração de Íons de Hidrogênio , Engenharia MetabólicaRESUMO
D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.
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Bacillus subtilis , Racemases e Epimerases , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Carboidratos Epimerases/genética , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Frutose , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
d-tagatose is a popular functional monosaccharide produced from lactose by ß-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for ß-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing ß-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars.
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Riboflavin, an essential vitamin for animals, is used widely in the pharmaceutical industry and as a food and feed additive. The microbial synthesis of riboflavin requires a large amount of oxygen, which limits the industrial-scale production of the vitamin. In this study, a metabolic engineering strategy based on transcriptome analysis was identified as effective in increasing riboflavin production. First, transcriptional profiling revealed that hypoxia affects purine, and nitrogen metabolism. Next, the precursor supply pool was increased by purR knockout and tnrA and glnR knockdown to balance intracellular nitrogen metabolism. Finally, increased oxygen utilization was achieved by dynamically regulating vgb. Fed-batch fermentation of the engineered strain in a 5-liter bioreactor produced 10.71 g/l riboflavin, a 45.51% higher yield than that obtained with Bacillus subtilis RF1. The metabolic engineering strategy described herein is useful for alleviating the oxygen limitation of bacterial strains used for the industrial production of riboflavin and related products.
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Bacillus subtilis , Engenharia Metabólica , Animais , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Fermentação , Oxigênio , RiboflavinaRESUMO
2,5-Dimethylpyrazine (2,5-DMP) is an important pharmaceutical intermediate and an important essence. Conventional chemical synthesis methods are often accompanied by toxic substances as by-products, and the biosynthesis efficiency of 2,5-DMP is insufficient for industrial applications. In this study, the tdh and soaao genes were overexpressed to enhance enzymatic and nonenzymatic reactions in metabolic pathways, and kbl was knocked out to block competitive branching carbon flow metabolic pathways. Finally, a genetically engineered Escherichia coli strain with the highest carbon recovery rate (30.18%) and the highest yield reported to date was successfully constructed, and 9.21 g·L-1 threonine was able to produce 1682 mg·L-1 2,5-DMP after 24 h. At the same time, an expression regulation strategy and whole-cell biocatalysis helped to eliminate the damage to cells caused by 2,5-DMP, aminoacetone, and reactive oxygen species generated by aminoacetone oxidase from S. oligofermentans, and the negative effect of 2-amino-3-ketobutyrate CoA ligase on the yield of 2,5-DMP in E. coli was also demonstrated.
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Carbono , Escherichia coli , Escherichia coli/genética , Engenharia Metabólica , Pirazinas , TreoninaRESUMO
Trans-4-hydroxy-l-proline is produced by trans-proline-4-hydroxylase with l-proline through glucose fermentation. Here, we designed a thorough "from A to Z" strategy to significantly improve trans-4-hydroxy-l-proline production. Through rare codon selected evolution, Escherichia coli M1 produced 18.2 g L-1 l-proline. Metabolically engineered M6 with the deletion of putA, proP, putP, and aceA, and proB mutation focused carbon flux to l-proline and released its feedback inhibition. It produced 15.7 g L-1 trans-4-hydroxy-l-proline with 10 g L-1 l-proline retained. Furthermore, a tunable circuit based on quorum sensing attenuated l-proline hydroxylation flux, resulting in 43.2 g L-1 trans-4-hydroxy-l-proline with 4.3 g L-1 l-proline retained. Finally, rationally designed l-proline hydroxylase gave 54.8 g L-1 trans-4-hydroxy-l-proline in 60 hours almost without l-proline remaining-the highest production to date. The de novo engineering carbon flux through rare codon selected evolution, dynamic precursor modulation, and metabolic engineering provides a good technological platform for efficient hydroxyl amino acid synthesis.