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1.
ACS Omega ; 7(15): 12870-12878, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35474802

RESUMO

In this article, we developed a new composite gel for plugging dominant fluid flow channels in offshore oilfields. The composite gel was synthesized by organic and inorganic gel networks interpenetrating into a compact three-dimensional spatial network structure, resulting in a good plugging effect. The performance of the composite gel was evaluated from the aspects of gelling characteristics and gel microstructure, while the plugging effect was evaluated through core experiments. The results showed that the influencing order of each component on gelling was acrylamide > cross-linking agent > urea > initiator > polyaluminum chloride. The initial viscosity of the composite gel was about 5-6 mPa·s, and it had good plugging abilities in different permeability cores. In comparison with inorganic gels (plugging ratio of 77.2%) or organic gels (84.8%), the composite gel system has a plugging ratio of up to 99.5% using a core with water permeability of 4300 mD. Besides, the reservoir applicability of the composite gel was studied, and the results suggested that the composite gel system had good resistance to dilution, mechanical shear, oil corrosion, and aging and could be quickly removed after plugging.

2.
ACS Omega ; 5(49): 32112-32122, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33344866

RESUMO

To meet the technical requirements of deep fluid diversion in Bohai oilfield, the swelling property, plugging effect, transport characteristics of polymer microspheres, and fluid diversion effect in heterogeneous cores are studied in this paper. There are two kinds of polymer microspheres including core-shell microspheres and traditional microspheres. The instruments used in this study include a biomicroscope, a metallurgical microscope, a scanning electron microscope, and core displacement experimental devices. The results of microscopes indicated that the core-shell microspheres were successfully synthesized, and the microspheres had good hydration expansion effect. The expanded microspheres could attract each other through the electrostatic force of anions and cations to achieve the purpose of coalescence. Compared with traditional microspheres (initial particle size is 3.8 µm), the initial particle size of the synthesized core-shell microspheres is close to 3.3 µm, but the particle size distribution is more concentrated, so the injection performance is close to that of traditional microspheres (initial particle size is 3.8 µm). After 8 days of hydration expansion, although the expansion multiple is small, it can coalesce and enhance the plugging effect, which can adapt to a wider range of permeability, ranging from 200 × 10-3 to 3000 × 10-3 µm2 (200 × 10-3-1500 × 10-3 µm2 for traditional microspheres). Under the same conditions (heterogeneous core), compared with the traditional microspheres, the core-shell microspheres have the characteristics of coalescence. Therefore, its fluid diversion effect is better, and the oil recovery is increased by 5.5%. Nevertheless, there is the "end effect" during the injection process, which weakens the steering effect of deep liquid flow. The results show that the "end effect" can be effectively reduced by alternate injection of microspheres and water. Meanwhile, the effect of deep fluid diversion is improved, and the increase of oil recovery is increased by 2.06%.

3.
Microb Cell Fact ; 17(1): 147, 2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30227873

RESUMO

BACKGROUND: Microbial biosynthesis of natural products holds promise for preclinical studies and treating diseases. For instance, pinocembrin is a natural flavonoid with important pharmacologic characteristics and is widely used in preclinical studies. However, high yield of natural products production is often limited by the intracellular cofactor level, including adenosine triphosphate (ATP). To address this challenge, tailored modification of ATP concentration in Escherichia coli was applied in efficient pinocembrin production. RESULTS: In the present study, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was performed for screening several ATP-related candidate genes, where metK and proB showed its potential to improve ATP level and increased pinocembrin production. Subsequently, the repression efficiency of metK and proB were optimized to achieve the appropriate levels of ATP and enhancing the pinocembrin production, which allowed the pinocembrin titer increased to 102.02 mg/L. Coupled with the malonyl-CoA engineering and optimization of culture and induction condition, a final pinocembrin titer of 165.31 mg/L was achieved, which is 10.2-fold higher than control strains. CONCLUSIONS: Our results introduce a strategy to approach the efficient biosynthesis of pinocembrin via ATP level strengthen using CRISPR interference. Furthermore coupled with the malonyl-CoA engineering and induction condition have been optimized for pinocembrin production. The results and engineering strategies demonstrated here would hold promise for the ATP level improvement of other flavonoids by CRISPRi system, thereby facilitating other flavonoids production.


Assuntos
Trifosfato de Adenosina/metabolismo , Flavanonas/biossíntese , Engenharia Metabólica/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Engenharia Genética , Metionina Adenosiltransferase/química , Metionina Adenosiltransferase/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/química , Fosfotransferases (Aceptor do Grupo Carboxila)/genética
4.
Sci Rep ; 6: 32640, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27586788

RESUMO

Microbial biosynthesis of pinocembrin is of great interest in the area of drug research and human healthcare. Here we found that the accumulation of the pathway intermediate cinnamic acid adversely affected pinocembrin production. Hence, a stepwise metabolic engineering strategy was carried out aimed at eliminating this pathway bottleneck and increasing pinocembrin production. The screening of gene source and the optimization of gene expression was first employed to regulate the synthetic pathway of cinnamic acid, which showed a 3.53-fold increase in pinocembrin production (7.76 mg/L) occurred with the alleviation of cinnamic acid accumulation in the engineered E. coli. Then, the downstream pathway that consuming cinnamic acid was optimized by the site-directed mutagenesis of chalcone synthase and cofactor engineering. S165M mutant of chalcone synthase could efficiently improve the pinocembrin production, and allowed the product titer of pinocembrin increased to 40.05 mg/L coupled with the malonyl-CoA engineering. With a two-phase pH fermentation strategy, the cultivation of the optimized strain resulted in a final pinocembrin titer of 67.81 mg/L. The results and engineering strategies demonstrated here would hold promise for the titer improvement of other flavonoids.


Assuntos
Cinamatos/metabolismo , Escherichia coli/metabolismo , Flavanonas/biossíntese , Aciltransferases/metabolismo , Técnicas de Cultura Celular por Lotes , Biomassa , Escherichia coli/efeitos dos fármacos , Fermentação/efeitos dos fármacos , Flavanonas/química , Dosagem de Genes , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Malonil Coenzima A/metabolismo , Engenharia Metabólica , Mutagênese Sítio-Dirigida , Fenilalanina/farmacologia , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Fatores de Tempo
5.
J Ind Microbiol Biotechnol ; 43(4): 557-66, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26733394

RESUMO

The development of efficient microbial processes for pinocembrin production has attracted considerable attention. However, pinocembrin biosynthetic efficiency is greatly limited by the low availability of the malonyl-CoA cofactor in Escherichia coli. Fatty acid biosynthesis is the only metabolic process in E. coli that consumes malonyl-CoA; therefore, we overexpressed the fatty acid biosynthetic pathway enzymes ß-ketoacyl-ACP synthase III (FabH) and ß-ketoacyl-ACP synthase II (FabF) alone and in combination, and investigated the effect on malonyl-CoA. Interestingly, overexpressing FabH, FabF or both enzymes in E. coli BL21 (DE3) decreased fatty acid synthesis and increased cellular malonyl-CoA levels 1.4-, 1.6-, and 1.2-fold, respectively. Furthermore, pinocembrin production was increased 10.6-, 31.8-, and 5.87-fold in recombinant strains overexpressing FabH, FabF and both enzymes, respectively. Overexpression of FabF, therefore, triggered the highest pinocembrin production and malonyl-CoA levels. The addition of cerulenin further increased pinocembrin production in the FabF-overexpressing strain, from 25.8 to 29.9 mg/L. These results demonstrated that overexpressing fatty acid synthases can increase malonyl-CoA availability and improve pinocembrin production in a recombinant E. coli host. This strategy may hold promise for the production of other important natural products in which cellular malonyl-CoA is rate limiting.


Assuntos
Reatores Biológicos , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/biossíntese , Flavanonas/biossíntese , Engenharia Metabólica , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Vias Biossintéticas/genética , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Malonil Coenzima A/metabolismo
6.
Bioresour Technol ; 202: 152-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26708482

RESUMO

In this study, Actinobacillus succinogenes NJ113 microbial electrolysis cells (MECs) were used to enhance the reducing power responsible for succinic acid production from corncob hydrolysate. During corncob hydrolysate fermentation, electric MECs resulted in a 1.31-fold increase in succinic acid production and a 1.33-fold increase in the reducing power compared with those in non-electric MECs. When the hydrolysate was detoxified by combining Ca(OH)2, NaOH, and activated carbon, succinic acid production increased from 3.47 to 6.95 g/l. Using a constant potential of -1.8 V further increased succinic acid production to 7.18 g/l. A total of 18.09 g/l of succinic acid and a yield of 0.60 g/g total sugar were obtained after a 60-h fermentation when NaOH was used as a pH regulator. The improved succinic acid yield from corncob hydrolysate fermentation using A. succinogenes NJ113 in electric MECs demonstrates the great potential of using biomass as a feedstock to cost-effectively produce succinate.


Assuntos
Actinobacillus/metabolismo , Fontes de Energia Bioelétrica , Carbono/farmacologia , Eletrólise/métodos , Ácido Succínico/metabolismo , Zea mays/química , Actinobacillus/efeitos dos fármacos , Biomassa , Eletricidade , Fermentação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrólise
7.
Sci Rep ; 5: 15630, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26490441

RESUMO

Although the routes of de novo pyridoxal 5'-phosphate (PLP) biosynthesis have been well described, studies of the engineering of an intracellular PLP supply are limited, and the effects of cellular PLP levels on PLP-dependent enzyme-based whole-cell biocatalyst activity have not been described. To investigate the effects of PLP cofactor availability on whole-cell biocatalysis, the ribose 5-phosphate (R5P)-dependent pathway genes pdxS and pdxT of Bacillus subtilis were introduced into the lysine decarboxylase (CadA)-overexpressing Escherichia coli strain BL-CadA. This strain was then used as a whole-cell biocatalyst for cadaverine production from L-lysine. Co-expression strategies were evaluated, and the culture medium was optimised to improve the biocatalyst performance. As a result, the intracellular PLP concentration reached 1144 nmol/gDCW, and a specific cadaverine productivity of 25 g/gDCW/h was achieved; these values were 2.4-fold and 2.9-fold higher than those of unmodified BL-CadA, respectively. Additionally, the resulting strain AST3 showed a cadaverine titre (p = 0.143, α = 0.05) similar to that of the BL-CadA strain with the addition of 0.1 mM PLP. These approaches for improving intracellular PLP levels to enhance whole-cell lysine bioconversion activity show great promise for the engineering of a PLP cofactor to optimise whole-cell biocatalysis.


Assuntos
Biocatálise , Cadaverina/biossíntese , Engenharia Metabólica , Fosfato de Piridoxal/biossíntese , Bacillus subtilis/genética , Cadaverina/metabolismo , Carboxiliases/genética , Escherichia coli/genética , Fosfato de Piridoxal/química , Fosfato de Piridoxal/genética
8.
J Biosci Bioeng ; 120(1): 36-40, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25553973

RESUMO

Nitrogen source optimization combined with phased exponential L-tyrosine feeding was employed to enhance L-phenylalanine production by a tyrosine-auxotroph strain, Escherichia coli YP1617. The absence of (NH4)2SO4, the use of corn steep powder and yeast extract as composite organic nitrogen source were more suitable for cell growth and L-phenylalanine production. Moreover, the optimal initial L-tyrosine level was 0.3 g L(-1) and exponential L-tyrosine feeding slightly improved L-phenylalanine production. Nerveless, L-phenylalanine production was greatly enhanced by a strategy of phased exponential L-tyrosine feeding, where exponential feeding was started at the set specific growth rate of 0.08, 0.05, and 0.02 h(-1) after 12, 32, and 52 h, respectively. Compared with exponential L-tyrosine feeding at the set specific growth rate of 0.08 h(-1), the developed strategy obtained a 15.33% increase in L-phenylalanine production (L-phenylalanine of 56.20 g L(-1)) and a 45.28% decrease in L-tyrosine supplementation.


Assuntos
Escherichia coli/metabolismo , Nitrogênio/metabolismo , Fenilalanina/biossíntese , Tirosina/metabolismo , Técnicas de Cultura Celular por Lotes , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Nitrogênio/farmacologia , Tirosina/farmacologia
9.
Biotechnol Lett ; 37(4): 799-806, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25515797

RESUMO

The effect of fusing the PelB signal sequence to lysine/cadaverine antiporter (CadB) on the bioconversion of L-lysine to cadaverine was investigated. To construct a whole-cell biocatalyst for cadaverine production, four expression plasmids were constructed for the co-expression of lysine decarboxylase (CadA) and lysine/cadaverine antiporter (CadB) in Escherichia coli. Expressing CadB with the PelB signal sequence increased cadaverine production by 12%, and the optimal expression plasmid, pETDuet-pelB-CadB-CadA, contained two T7 promoter-controlled genes, CadA and the PelB-CadB fusion protein. Based on pETDuet-pelB-CadB-CadA, a whole-cell system for the bioconversion of L-lysine to cadaverine was constructed, and three strategies for L-lysine feeding were evaluated to eliminate the substrate inhibition problem. A cadaverine titer of 221 g l(-1) with a molar yield of 92% from lysine was obtained.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Cadaverina/metabolismo , Carboxiliases/metabolismo , Escherichia coli/metabolismo , Lisina/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas , Sistemas de Transporte de Aminoácidos/genética , Antiporters/genética , Proteínas de Bactérias/genética , Bacteriófago T7/genética , Carboxiliases/genética , Escherichia coli/genética , Expressão Gênica , Plasmídeos , Regiões Promotoras Genéticas
10.
Bioresour Technol ; 135: 574-7, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23010211

RESUMO

Efficient biosynthesis of succinate from a renewable biomass resource by engineered Escherichia coli is reported in this paper. Fermentation of sugarcane bagasse hydrolysate by engineered E. coli BA204, a pflB, ldhA, and ppc deletion strain overexpressing the ATP-forming phosphoenolpyruvate carboxykinase from Bacillus subtilis 168, produced a final succinate concentration of 15.85 g L(-1) with a high yield of 0.89 g L(-1) total sugar under anaerobic conditions. During dual-phase fermentations, initial aerobic growth facilitated subsequent anaerobic succinate production, with a final succinate concentration of 18.88 g L(-1) and a yield of 0.96 g g(-1) total sugar after 24 h of anaerobic fermentation. The high succinate yield from sugarcane bagasse hydrolysate demonstrated a great potential application of renewable biomass as a feedstock for the economical production of succinate using metabolically engineered E. coli.


Assuntos
Carbono/farmacologia , Celulose/farmacologia , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Saccharum/química , Ácido Succínico/metabolismo , Anaerobiose/efeitos dos fármacos , Técnicas de Cultura Celular por Lotes , Reatores Biológicos/microbiologia , Carboidratos/farmacologia , Escherichia coli/efeitos dos fármacos , Fermentação/efeitos dos fármacos , Hidrólise/efeitos dos fármacos
11.
Sheng Wu Gong Cheng Xue Bao ; 29(11): 1692-5, 2013 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-24701836

RESUMO

Escherichia coli AFP111 is a spontaneous mutant with mutations in the glucose specific phosphotransferase system (ptsG) in NZN111 (delta pflAB deltaldhA). In AFP111, conversion of xylose to succinic acid generates 1.67 molecule of ATP per xylose. However, the strain needs 2.67 molecule ATP for xylose metabolism. Therefore, AFP111 cannot use xylose due to insufficient ATP under anaerobic condition. Through an atmospheric and room temperature plasma (ARTP) jet, we got a mutant strain named DC111 that could use xylose under anaerobic condition in M9 medium to produce succinic acid. After 72 h, DC111 consumed 10.52 g/L xylose to produce 6.46 g/L succinic acid, and the yield was 0.78 mol/mol. Furthermore, the reaction catalyzed by the ATP-generating PEP-carboxykinase (PCK) was enhanced. The specific activity of PCK was 19.33-fold higher in DC111 than that in AFP111, which made the strain have enough ATP to converse xylose to succinic acid.


Assuntos
Escherichia coli/genética , Engenharia Metabólica , Mutação , Ácido Succínico/metabolismo , Xilose/metabolismo , Atmosfera , Escherichia coli/metabolismo , Fermentação , Microbiologia Industrial , Gases em Plasma/farmacologia , Temperatura
12.
Sheng Wu Gong Cheng Xue Bao ; 29(12): 1855-9, 2013 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-24660633

RESUMO

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Pentosiltransferases/biossíntese , Piruvato Carboxilase/biossíntese , Ácido Succínico/metabolismo , Anaerobiose , Escherichia coli/enzimologia , Fermentação , Engenharia Genética , Glucose/metabolismo , Microbiologia Industrial , Lactococcus lactis/enzimologia , NAD/metabolismo , Pentosiltransferases/genética , Piruvato Carboxilase/genética
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