scholarly journals Metabolic engineering of Escherichia coli for production of valerenadiene

2017 ◽  
Author(s):  
S. Eric Nybo ◽  
Jacqueline Saunders ◽  
Sean P. McCormick
Keyword(s):  
Escherichia Coli ◽  
Fermentation Medium ◽  
Specific Productivity ◽  
Root Extract ◽  
Valeriana Officinalis ◽  
Mva Pathway ◽  
E Coli ◽  
Production Platform ◽  
Valerenic Acid ◽  
Methyl Erythritol Phosphate

AbstractValeriana officinalis is a medicinal herb which produces a suite of compounds in its root tissue useful for treatment of anxiety and insomnia. The sesquiterpene components of the root extract, valerenic acid and valerena-1,10-diene, are thought to contribute to most of the observed anxiolytic of Valerian root preparations. However, valerenic acid and its biosynthetic intermediates are only produced in low quantities in the roots of V. officinalis. Thus, in this report, Escherichia coli was metabolically engineered to produce substantial quantities of valerena-1,10-diene in shake flask fermentations with decane overlay. Expression of the wildtype valerenadiene synthase gene (pZE-wvds) resulted in production of 12 μg/mL in LB cultures using endogenous FPP metabolism. Expression of a codon-optimized version of the valerenadiene synthase gene (pZE-cvds) resulted in 3-fold higher titers of valerenadiene (32 μg/mL). Co-expression of pZE-cvds with an engineered methyl erythritol phosphate (MEP) pathway improved valerenadiene titers 65-fold to 2.09 mg/L valerenadiene. Optimization of the fermentation medium to include glycerol supplementation enhanced yields by another 5.5-fold (11.0 mg/L valerenadiene). The highest production of valerenadiene resulted from engineering the codon-optimized valerenadiene synthase gene under strong Ptrc and PT7 promoters and via co-expression of an exogenous mevalonate (MVA) pathway. These efforts resulted in an E. coli production strain that produced 62.0 mg/L valerenadiene (19.4 mg/L/OD600 specific productivity). This E. coli production platform will serve as the foundation for the synthesis of novel valerenic acid analogues potentially useful for the treatment of anxiety disorders.

scholarly journals Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9010
Author(s):  
Yonggang Wang ◽  
Jiangqin Wang ◽  
Feifan Leng ◽  
Jianzhong Ma ◽  
Alnoor Bagadi
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Aspergillus Niger ◽  
Pichia Pastoris ◽  
Glucose Oxidase ◽  
Fermentation Medium ◽  
Logarithmic Phase ◽  
Transgenic Strain ◽  
E Coli ◽  
Cell Lysate

The gene encoding glucose oxidase from Aspergillus niger ZM-8 was cloned and transferred to Pichia pastoris GS115, a transgenic strain P. pastoris GS115-His-GOD constructed. The growth curve of P. pastoris GS115-His-GOD was consistent with that of Pichia pastoris GS115-pPIC9K under non-induced culture conditions. Under methanol induction conditions, the growth of the GOD-transgenic strain was significantly lowered than P. pastoris GS115-pPIC9K with the induced-culture time increase, and the optical densities of GOD-transgenic strain reached one-third of that of the P. pastoris GS115-pPIC9K at 51 h. The activity of glucose oxidase in the cell-free supernatant, the supernatant of cell lysate, and the precipitation of cell lysate was 14.3 U/mL, 18.2 U/mL and 0.48 U/mL, respectively. The specific activity of glucose oxidase was 8.3 U/mg, 6.52 U/mg and 0.73 U/mg, respectively. The concentration of hydrogen peroxide formed by glucose oxidase from supernatant of the fermentation medium, the supernatant of the cell lysate, and the precipitation of cell lysate catalyzing 0.2 M glucose was 14.3 μg/mL, 18.2 μg/mL, 0.48 μg/mL, respectively. The combination of different concentrations of glucose oxidase and glucose could significantly inhibit the growth of Agrobacterium and Escherichia coli in logarithmic phase. The filter article containing supernatant of the fermentation medium, supernatant of the cell lysate, and precipitation of cell lysate had no inhibitory effect on Agrobacterium and E. coli. The minimum inhibitory concentration of hydrogen peroxide on the plate culture of Agrobacterium and E. coli was 5.6 × 103 μg/mL and 6.0 × 103 μg/mL, respectively.

scholarly journals A hemolysin secretion pathway-based novel secretory expression platform for efficient manufacturing of tag peptides and anti-microbial peptides in Escherichia coli

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Wen Zhu ◽  
Lifu Hu ◽  
Yang Wang ◽  
Liangyin Lv ◽  
Hui Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Secretory Expression ◽  
Extracellular Medium ◽  
E Coli ◽  
Tev Protease ◽  
Expression Platform ◽  
Fusion Protein Expression ◽  
Production Platform ◽  
Secretory Production

Abstract Background Although Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins. Results In this work, we developed a novel production platform, THHly, based on the HlyA secretion system, and explored its applications in the efficient preparation and quick detection of tag peptides and anti-microbial peptides. In this novel platform the signal sequence of HlyA is fused to the C-terminal of target peptide, with Tobacco Etch Virus (TEV) protease cleavage site and 6*His tag between them. Five tag peptides displayed good secretory properties in E. coli BL21 (DE3), among which T7 tag and S tag were obtained by two rounds of purification steps and TEV cleavage, and maintained their intrinsic immunogenicity. Furthermore, Cecropin A and Melittin, two different types of widely explored anti-microbial peptides, were produced likewise and verified to possess anti-microbial/anti-tumor bioactivities. No significant bacterial growth inhibition was observed during the fusion protein expression, indicating that the fusion form not only mediated the secretion but also decreased the toxicity of anti-microbial peptides (AMPs) to the host bacteria. To the best of our knowledge, this is the first report to achieve the secretory expression of these two AMPs in E. coli with considerable potential for manufacturing and industrialization purposes. Conclusions The results demonstrate that the HlyA based novel production platform of E. coli allowed the efficient secretory production and purification of peptides, thus suggesting a promising strategy for the industrialized production of peptide pharmaceuticals or reagents. Graphical Abstract

scholarly journals Expression of the Corynebacterium glutamicum panD Gene Encoding l-Aspartate-α-Decarboxylase Leads to Pantothenate Overproduction in Escherichia coli

1999 ◽  
Vol 65 (4) ◽  
pp. 1530-1539 ◽  
Author(s):  
Nicole Dusch ◽  
Alfred Pühler ◽  
Jörn Kalinowski
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Specific Productivity ◽  
Decarboxylase Activity ◽  
Coding Region ◽  
E Coli ◽  
Enhanced Expression

ABSTRACT The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panDmutant strain. Sequence analysis revealed that the coding region ofpanD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A definedC. glutamicum panD mutant completely lackedl-aspartate-α-decarboxylase activity and exhibited β-alanine auxotrophy. The C. glutamicum panD(panDC.g. ) as well as the E. coli panD (panDE.c. ) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of l-aspartate-α-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed inE. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of thepanDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of thepanDC.g. gene was sufficient β-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external β-alanine supplementation. The enhanced expression ofpanDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)−1h−1.

scholarly journals One-step fermentation for producing xylo-oligosaccharides from wheat bran by recombinant Escherichia coli containing an alkaline xylanase

2021 ◽  
Author(s):  
Jiawen Liu ◽  
Cong Liu ◽  
Shilei Qiao ◽  
Zhen Dong ◽  
Di Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wheat Bran ◽  
Xylanase Activity ◽  
Maximal Activity ◽  
Fermentation Medium ◽  
Growth Conditions ◽  
Glycoside Hydrolase Family ◽  
E Coli ◽  
Response Surface Optimization ◽  
One Step

Abstract One-step fermentation is a cheap way to produce xylo-oligosaccharides (XOS), where production of xylanases and XOS is integrated into a single process. In spite of having cost advantage, one-step fermentation is still short in yield so far due to the limited exploration. To cope with this issue, production of XOS from wheat bran by recombinant Escherichia coli through one-step fermentation was investigated here. A xylanase gene belonging to glycoside hydrolase family 11 of Bacillus agaradhaerens was employed to construct recombinant E. coli. This xylanase showed maximal activity at 60°C and pH 8.0. Its activity retained more than 60% after incubation at 70°C for 4 hours, showing a good stability. The recombinant E. coli successfully secreted xylanases that directly hydrolyzed wheat bran to XOS in fermentation medium. The generated XOS consisted of xylose, xylobiose and xylotriose accounting for 23.1%, 37.3% and 39.6%, respectively. Wheat bran concentration was found to be the most crucial factor affecting XOS production. The yield reached 5.3 mg/mL at 10% of wheat bran, which is higher than previous reports employing one-step fermentation. Nitrogen source type could also affect XOS yield by changing extracellular xylanase activity, and glycine was found to be the best one for fermentation. Optimal fermentation conditions were finally studied by response surface optimization. The maximal yield emerged at 44.3°C, pH 7.98, which is affected by characteristics of the xylanase and growth conditions of E. coli. This work indicates that the integrated fermentation using recombinant E. coli is highly competitive in cost and yield for production of XOS.

scholarly journals Displacement of Escherichia coli O157:H7 from Rumen Medium Containing Prebiotic Sugars

2002 ◽  
Vol 68 (2) ◽  
pp. 519-524 ◽  
Author(s):  
Albane de Vaux ◽  
Mark Morrison ◽  
Robert W. Hutkins
Keyword(s):  
Escherichia Coli ◽  
Culture System ◽  
Rumen Fluid ◽  
Enteric Bacteria ◽  
Fermentation Medium ◽  
Escherichia Coli O157 ◽  
Anaerobic Culture ◽  
Fluid Medium ◽  
E Coli ◽  
Cell Densities

ABSTRACT A fed-batch, anaerobic culture system was developed to assess the behavior of Escherichia coli O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 1010 and 104 cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of E. coli O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 106 to 109 CFU/ml within 24 h. Most strains of E. coli O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude E. coli O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter, E. coli O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as l-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever E. coli O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of E. coli O157:H7 by the addition of “nonfermentable” sugars and that this approach may be effective as a preharvest strategy for reducing carriage of E. coli O157:H7 in the rumen.

scholarly journals Metabolic Engineering Escherichia coli for the Production of Lycopene

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3136 ◽  
Author(s):  
Zhaobao Wang ◽  
JingXin Sun ◽  
Qun Yang ◽  
Jianming Yang
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Regulatory Networks ◽  
Carbon Sources ◽  
Operation Technique ◽  
Mva Pathway ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Lycopene Production

Lycopene, a potent antioxidant, has been widely used in the fields of pharmaceuticals, nutraceuticals, and cosmetics. However, the production of lycopene extracted from natural sources is far from meeting the demand. Consequently, synthetic biology and metabolic engineering have been employed to develop microbial cell factories for lycopene production. Due to the advantages of rapid growth, complete genetic background, and a reliable genetic operation technique, Escherichia coli has become the preferred host cell for microbial biochemicals production. In this review, the recent advances in biological lycopene production using engineered E. coli strains are summarized: First, modification of the endogenous MEP pathway and introduction of the heterogeneous MVA pathway for lycopene production are outlined. Second, the common challenges and strategies for lycopene biosynthesis are also presented, such as the optimization of other metabolic pathways, modulation of regulatory networks, and optimization of auxiliary carbon sources and the fermentation process. Finally, the future prospects for the improvement of lycopene biosynthesis are also discussed.

scholarly journals Enhanced lycopene Production in Escherichia coli by Expression of Two MEP Pathway Enzymes from Vibrio sp. dhg

2019 ◽  
Author(s):  
Min Jae Kim ◽  
Myung Hyun Noh ◽  
Sunghwa Woo ◽  
Hyun Gyu Lim ◽  
Gyoo Yeol Jung
Keyword(s):  
Escherichia Coli ◽  
Electron Transport Chain ◽  
Farnesyl Diphosphate ◽  
Mep Pathway ◽  
Farnesyl Diphosphate Synthase ◽  
Mva Pathway ◽  
E Coli ◽  
Transport Chain ◽  
Lycopene Production ◽  
Specific Activities

Microbial production is a promising method that can overcome major limitations in conventional methods of lycopene production, such as low yields and variations in product quality. Significant efforts have been made to improve lycopene production by engineering either the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway or mevalonate (MVA) pathway in microorganisms. To further improve lycopene production, it is critical to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are required in lycopene production using MEP pathway. Here, we evaluated the activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. Considering that the MEP pathway is closely related to the cell membrane and electron transport chain, the activities of the two enzymes of Vibrio sp. dhg were expected to be higher than the enzymes of E. coli. We found that Dxs and IspA in Vibrio sp. dhg exhibited 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression improved the specific lycopene production by 1.88-fold. Our findings could be widely utilized to enhance production of lycopene and other carotenoids.

scholarly journals Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

2014 ◽  
Vol 80 (10) ◽  
pp. 3276-3282 ◽  
Author(s):  
Matthew S. Wong ◽  
Mai Li ◽  
Ryan W. Black ◽  
Thao Q. Le ◽  
Sharon Puthli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fermentation Process ◽  
Safety Issue ◽  
Specific Productivity ◽  
Content Type ◽  
E Coli ◽  
Pathway Gene ◽  
Fuels And Chemicals ◽  
Glycerol Uptake ◽  
The Cost

ABSTRACTGlycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol byEscherichia colithat presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genesdhaKLM,gldA, andglpK, as well as the ethanol pathway geneadhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used theadhEandicdpromoters fromE. coliin our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of anE. coli-based glycerol-to-ethanol process.

Enhanced bioproduction of fucosylated oligosaccharide 3-fucosyllactose in engineered Escherichia coli with an improved de novo pathway

2021 ◽  
Author(s):  
Zhijian Ni ◽  
Jinyong Wu ◽  
Zhongkui Li ◽  
Lixia Yuan ◽  
Yu Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Human Milk ◽  
Infant Formula ◽  
De Novo ◽  
Specific Productivity ◽  
Control Strain ◽  
E Coli ◽  
Lower Performance ◽  
Production Strategy ◽  
Engineered Escherichia Coli

Abstract 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2′-FL), are two important fucosylated oligosaccharides in human milk. Extensive studies on 2'-FL enabled its official approval for use in infant formula. However, commercialization of 3-FL seems to be a bit sluggish due to its low content in human milk and poor yield in enlarged production. Here, an α-1,3-fucosyltransferase mutant was expressed in an engineered E. coli able to produce GDP-L-fucose, and gave a promising 3-FL titer in a 5.0-L bioreactor. To increase the availability of cofactors (NADPH and GTP) for optimized 3-FL production, zwf, pntAB, and gsk genes were successively overexpressed, finally resulting in a high 3-FL level with a titer of 35.72 g/L, a yield of 0.82 mol 3-FL/mol lactose, and a specific productivity of 0.46 g/L*h. Unexpectedly, the deletion of pfkA gene led to a much lower performance of 3-FL production than the control strain. Taken together, our production strategy finally achieved the highest 3-FL level in E. coli to date.

scholarly journals Enhanced (−)-α-Bisabolol Productivity by Efficient Conversion of Mevalonate in Escherichia coli

Catalysts ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 432 ◽  
Author(s):  
Soo-Jung Kim ◽  
Seong Keun Kim ◽  
Wonjae Seong ◽  
Seung-Gyun Woo ◽  
Hyewon Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Industrial Application ◽  
Batch Fermentation ◽  
Mevalonate Pathway ◽  
Fed Batch ◽  
Mva Pathway ◽  
E Coli ◽  
Beneficial Effects ◽  
Fed Batch Fermentation ◽  
Skin Healing

(−)-α-Bisabolol, a naturally occurring sesquiterpene alcohol, has been used in pharmaceuticals and cosmetics owing to its beneficial effects on inflammation and skin healing. Previously, we reported the high production of (−)-α-bisabolol by fed-batch fermentation using engineered Escherichia coli (E. coli) expressing the exogenous mevalonate (MVA) pathway genes. The productivity of (−)-α-bisabolol must be improved before industrial application. Here, we report enhancement of initial (−)-α-bisabolol productivity to 3-fold higher than that observed in our previous study. We first harnessed a farnesyl pyrophosphate (FPP)-resistant mevalonate kinase 1 (MvaK1) from an archaeon Methanosarcina mazei (M. mazei) to create a more efficient heterologous MVA pathway that produces (−)-α-bisabolol in the engineered E. coli. The resulting strain produced 1.7-fold higher (−)-α-bisabolol relative to the strain expressing a feedback-inhibitory MvaK1 from Staphylococcus aureus (S. aureus). Next, to efficiently convert accumulated MVA to (−)-α-bisabolol, we additionally overexpressed genes involved in the lower MVA mevalonate pathway in E. coli containing the entire MVA pathway genes. (−)-α-Bisabolol production increased by 1.8-fold with reduction of MVA accumulation, relative to the control strain. Finally, we optimized the fermentation conditions including inducer concentration, aeration and enzymatic cofactor. The strain was able to produce 8.5 g/L of (−)-α-bisabolol with an initial productivity of 0.12 g/L h in the optimal fed-batch fermentation. Thus, the microbial production of (−)-α-bisabolol would be an economically viable bioprocess for its industrial application.

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