Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli.
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ABSTRACT:
SUBMITTER: Dresler K
PROVIDER: S-EPMC1705536 | biostudies-other | 2006 Aug
REPOSITORIES: biostudies-other
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Project description:BACKGROUND:Salicylate can be biosynthesized from the common metabolic intermediate shikimate and has found applications in pharmaceuticals and in the bioplastics industry. While much metabolic engineering work focused on the shikimate pathway has led to the biosynthesis of a variety of aromatic compounds, little is known about how the relative expression levels of pathway components influence salicylate biosynthesis. Furthermore, some host strain gene deletions that improve salicylate production may be impossible to predict. Here, a salicylate-responsive transcription factor was used to optimize the expression levels of shikimate/salicylate pathway genes in recombinant E. coli, and to screen a chromosomal transposon insertion library for improved salicylate production. RESULTS:A high-throughput colony screen was first developed based on a previously designed salicylate-responsive variant of the E. coli AraC regulatory protein ("AraC-SA"). Next, a combinatorial library was constructed comprising a series of ribosome binding site sequences corresponding to a range of predicted protein translation initiation rates, for each of six pathway genes (>?38,000 strain candidates). Screening for improved salicylate production allowed for the rapid identification of optimal gene expression patterns, conferring up to 123% improved production of salicylate in shake-flask culture. Finally, transposon mutagenesis and screening revealed that deletion of rnd (encoding RNase D) from the host chromosome further improved salicylate production by 27%. CONCLUSIONS:These results demonstrate the effectiveness of the salicylate sensor-based screening platform to rapidly identify beneficial gene expression patterns and gene knockout targets for improving production. Such customized high-throughput tools complement other cell factory engineering strategies. This approach can be generalized for the production of other shikimate-derived compounds.
| S-EPMC6350385 | biostudies-literature
Project description:BackgroundLipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product.ResultsAs an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels.ConclusionsThis paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.
| S-EPMC4404585 | biostudies-literature
Project description:In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.
| S-EPMC5981079 | biostudies-literature
Project description:Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable alpha-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting alpha-amylase activity. The alpha-amylase beads were assessed with respect to alpha-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized alpha-amylase showed Michaelis-Menten enzyme kinetics exerting a V(max) of about 506 mU/mg of bead protein with a K(m) of about 5 microM, consistent with that of free alpha-amylase. The stability of the enzyme at 85 degrees C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized alpha-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.
| S-EPMC2663195 | biostudies-literature
Project description:Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]). Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP) tagged with CusF, using Ag(I) metal affinity chromatography.
| S-EPMC4792856 | biostudies-literature
Project description:BACKGROUND: Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO). RESULTS: Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from approximately 500 up to approximately 25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies. CONCLUSIONS: Comparison of our results with those published on non-covalent (type I) COs expressed in recombinant form (either in E. coli or Streptomyces spp.), shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.
| S-EPMC2890692 | biostudies-literature
Project description:Matrix metalloproteinase 9 (MMP9) is involved in several aspects of the pathology of cancer, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant scFv-type anti-MMP9 antibody in soluble form using Escherichia coli, purified it, and confirmed its antigen-binding ability. The convenient, rapid, inexpressive system used in this study for producing recombinant antibody fragments needs only five days, and thus can be used for the efficient production of scFv against MMP9, which can be used in a range of applications and industrial fields, including diagnosis and treatment of inflammatory and cancer-related diseases.
| S-EPMC8468072 | biostudies-literature
Project description:A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a "top value-added chemical" from biomass.
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Project description:Background(R)-(+)-perillyl alcohol is a naturally oxygenated monoterpene widely used as the natural flavor additives, insecticides, jet fuels and anti-cancer therapies. It was also readily available monoterpene precursors. However, this natural product is present at low concentrations from plant sources which are not economically viable. Therefore, alternative microbial production methods are rapidly emerging as an attractive alternative to make (R)-(+)-perillyl alcohol production more sustainable and environmentally friendly.ResultsWe engineered Escherichia coli to possess a heterologous mevalonate (MVA) pathway, including limonene synthase, P-cymene monoxygenase hydroxylase and P-cymene monoxygenase reductase for the production of (R)-(+)-perillyl alcohol. The concentration of (R)-(+)-limonene (the monoterpene precursor to (R)-(+)-perillyl alcohol) reached 45 mg/L from glucose. Enhanced (R)-(+)-perillyl alcohol production was therefore achieved. The strain produced (R)-(+)-perillyl alcohol at a titer of 87 mg/L and a yield of 1.5 mg/g glucose in a 5 L bioreactor fed batch system.ConclusionsThese datas highlight the efficient production of (R)-(+)-perillyl alcohol through the mevalonate pathway from glucose. This method serves as a platform for the future production of other monoterpenes.
| S-EPMC7791655 | biostudies-literature
Project description:Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) family proteins are anti-inflammatory. They bind and destabilize some AU-rich element-containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was overexpressed in E. coli, purified, and used for polyclonal antibody production in rabbits. The antiserum recognized nanograms of the antigen on immunoblots. This antiserum and another antiserum developed against recombinant mouse TTP were used to detect ZFP36L1 and TTP in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. Immunoblotting showed that ZFP36L1 was stably expressed with a size corresponding to the lower mass size of ZFP36L1 expressed in transfected human embryonic kidney 293 cells, but TTP was induced by cinnamon extract and not by lipopolysaccharide (LPS) in adipocytes. In contrast, ZFP36L1 was undetectable, but TTP was strongly induced in LPS-stimulated RAW cells. Quantitative real-time polymerase chain reaction confirmed the higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW cells. Low levels of ZFP36L1 expression were also confirmed by Northern blotting in mouse embryonic fibroblasts. These results demonstrate that ZFP36L1 antiserum is useful in the detection of this protein and that TTP and ZFP36L1 are differentially expressed and regulated at the mRNA and protein levels in mouse adipocytes and macrophages.
| S-EPMC2674335 | biostudies-literature