Enhanced Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Coexpression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Escherichia coli

Two genes involved in δ-guaiene biosynthesis in Aquilaria microcarpa, δ-guaiene synthase (GS) and farnesyl diphosphate synthase (FPS), were overexpressed in Escherichia coli cells. Immunoblot analysis revealed that the concentration of GS-translated protein was rather low in the cells transformed by solely GS while appreciable accumulation of the recombinant protein was observed when GS was coexpressed with FPS GS-transformed cells liberated only a trace amount of δ-guaiene (0.004 μg/mL culture), however, the concentration of the compound elevated to 0.08 μg/mL culture in the cells transformed by GS plus FPS δ-Guaiene biosynthesis was markedly activated when E. coli cells coexpressing GS and FPS were incubated in enriched Terrific broth, and the content of the compound increased to approximately 0.6 μg/mL culture. These results suggest that coexpression of FPS and GS in E. coli is required for efficient 6-guaiene production in the bacterial cells, and the sesquiterpene-producing activity of the transformant is appreciably enhanced in the nutrients-enriched medium.

Download Full-text

Expression of a Gene Encoding the Carrot HSP17.7 in Escherichia coli Enhances Cell Viability and Protein Solubility Under Heat Stress

HortScience ◽

10.21273/hortsci.44.3.866 ◽

2009 ◽

Vol 44 (3) ◽

pp. 866-869 ◽

Cited By ~ 8

Author(s):

Hyesoon Kim ◽

Yeh-Jin Ahn

Keyword(s):

Escherichia Coli ◽

Heat Stress ◽

Protein Solubility ◽

Small Heat Shock Protein ◽

Immunoblot Analysis ◽

Transformed Cells ◽

Bacterial Cells ◽

Native Page ◽

E Coli ◽

Inducible Protein

DcHSP17.7, a small heat shock protein from carrot (Daucus carota L.), was expressed in Escherichia coli to examine its functional mechanism under heat stress. When transformed cells expressing DcHSP17.7 were exposed to 50 °C for 1 h, the number of viable cells was ≈4-fold higher than that of control. When the amount of soluble proteins was compared, it was more than twofold higher in transformed cells expressing DcHSP17.7 than that in control, suggesting that DcHSP17.7 may function as a molecular chaperone preventing heat-inducible protein degradation. Native-PAGE followed by immunoblot analysis showed that in transformed E. coli, DcHSP17.7 was present in an oligomeric complex, ≈300 kDa in molecular mass, on isopropyl b-D-thiogalactopyranoside treatment. However, the complex rapidly disappeared when bacterial cells were exposed to heat stress. In carrot, DcHSP17.7 was found in the similar-sized complex (≈300 kDa), but only during heat stress (40 °C), suggesting that the functional structure of DcHSP17.7 may be different in transformed E. coli from that in carrot.

Download Full-text

Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01402-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 7291-7293 ◽

Cited By ~ 44

Author(s):

Gopal Prasad Ghimire ◽

Hei Chan Lee ◽

Jae Kyung Sohng

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Isopentenyl Diphosphate ◽

Isopentenyl Diphosphate Isomerase ◽

Streptomyces Peucetius ◽

E Coli ◽

Expression Of Genes ◽

Genes Encoding

ABSTRACT Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

Download Full-text

RNase E Maintenance of Proper FtsZ/FtsA Ratio Required for Nonfilamentous Growth of Escherichia coli Cells but Not for Colony-Forming Ability

Journal of Bacteriology ◽

10.1128/jb.00367-06 ◽

2006 ◽

Vol 188 (14) ◽

pp. 5145-5152 ◽

Cited By ~ 30

Author(s):

Masaru Tamura ◽

Kangseok Lee ◽

Christine A. Miller ◽

Christopher J. Moore ◽

Yukio Shirako ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Liquid Culture ◽

Bacterial Cells ◽

Rnase E ◽

Null Mutant ◽

E Coli ◽

Escherichia Coli Cells ◽

Solid Media ◽

Rnase G

ABSTRACT Inactivation or deletion of the RNase E-encoding rne gene of Escherichia coli results in the growth of bacterial cells as filamentous chains in liquid culture (K. Goldblum and D. Apirion, J. Bacteriol. 146:128-132, 1981) and the loss of colony-forming ability (CFA) on solid media. RNase E dysfunction is also associated with abnormal processing of ftsQAZ transcripts (K. Cam, G. Rome, H. M. Krisch, and J.-P. Bouché, Nucleic Acids Res. 24:3065-3070, 1996), which encode proteins having a central role in septum formation during cell division. We show here that RNase E regulates the relative abundances of FtsZ and FtsA proteins and that RNase E depletion results in decreased FtsZ, increased FtsA, and consequently an altered FtsZ/FtsA ratio. However, while restoration of the level of FtsZ to normal in rne null mutant bacteria reverses the filamentation phenotype, it does not restore CFA. Conversely, overexpression of a related RNase, RNase G, in rne-deleted bacteria restores CFA, as previously reported, without affecting FtsZ abundance. Our results demonstrate that RNase E activity is required to maintain a proper cellular ratio of the FtsZ and FtsA proteins in E. coli but that FtsZ deficiency does not account for the nonviability of cells lacking RNase E.

Download Full-text

Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates

Synthetic Biology ◽

10.1093/synbio/ysz003 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 36

Author(s):

Quentin M Dudley ◽

Connor J Nash ◽

Michael C Jewett

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Enzymatic Synthesis ◽

Biosynthetic Pathway ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Design Criteria ◽

Acetyl Coa ◽

E Coli ◽

Enzymatic Pathways

Abstract Isoprenoids are an attractive class of metabolites for enzymatic synthesis from renewable substrates. However, metabolic engineering of microorganisms for monoterpenoid production is limited by the need for time-consuming, and often non-intuitive, combinatorial tuning of biosynthetic pathway variations to meet design criteria. Towards alleviating this limitation, the goal of this work was to build a modular, cell-free platform for construction and testing of monoterpenoid pathways, using the fragrance and flavoring molecule limonene as a model. In this platform, multiple Escherichia coli lysates, each enriched with a single overexpressed pathway enzyme, are mixed to construct the full biosynthetic pathway. First, we show the ability to synthesize limonene from six enriched lysates with mevalonate substrate, an adenosine triphosphate (ATP) source, and cofactors. Next, we extend the pathway to use glucose as a substrate, which relies on native metabolism in the extract to convert glucose to acetyl-CoA along with three additional enzymes to convert acetyl-CoA to mevalonate. We find that the native E. coli farnesyl diphosphate synthase (IspA) is active in the lysate and diverts flux from the pathway intermediate geranyl pyrophospahte to farnesyl pyrophsophate and the byproduct farnesol. By adjusting the relative levels of cofactors NAD+, ATP and CoA, the system can synthesize 0.66 mM (90.2 mg l−1) limonene over 24 h, a productivity of 3.8 mg l−1 h−1. Our results highlight the flexibility of crude lysates to sustain complex metabolism and, by activating a glucose-to-limonene pathway with 9 heterologous enzymes encompassing 20 biosynthetic steps, expands an approach of using enzyme-enriched lysates for constructing, characterizing and prototyping enzymatic pathways.

Download Full-text

Assessment of correlation between physiological states of Escherichia coli cells and their susceptibility to chlorine using flow cytometry

Water Science & Technology Water Supply ◽

10.2166/ws.2013.083 ◽

2013 ◽

Vol 13 (4) ◽

pp. 1056-1062 ◽

Cited By ~ 3

Author(s):

Saeid Rezaeinejad ◽

Volodymyr Ivanov

Keyword(s):

Escherichia Coli ◽

Flow Cytometry ◽

Growth Rates ◽

Specific Growth ◽

Bacterial Cells ◽

Tetrazolium Chloride ◽

E Coli ◽

Escherichia Coli Cells ◽

Cell Subpopulations ◽

Specific Growth Rates

The physiological differences of individual cells of bacterial population may imply the existence of cell subpopulations with different sensitivity to chlorine, which may affect the efficiency of drinking water disinfection. The susceptibility of individual bacterial cells to chlorine was examined using flow cytometry. The inactivation of Escherichia coli cells by chlorine in the populations with specific growth rates of 0.2 and 0.9 h−1 was assessed using various viability indicators. Viability of bacterial cells was evaluated using membrane integrity propidium iodide (PI) dye, respiratory activity indicator of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and membrane potential probe of DiBAC4(3). It was found that there were cell subpopulations of E. coli with different levels of susceptibility to chlorine. E. coli cell population with higher specific growth rate was more susceptible to chlorine. The CT values for inactivation of 99% of cells (CT99) in populations of E. coli with specific growth rates of 0.9 and 0.2 h−1 were 0.06 and 0.09 mg min l−1, respectively. Flow cytometry could be used to study the sensitivity of bacterial cells to the chemical agents.

Download Full-text

Enhanced Production of Recombinant Mycobacterium tuberculosis Antigens in Escherichia coli by Replacement of Low-Usage Codons

Infection and Immunity ◽

10.1128/iai.68.1.233-238.2000 ◽

2000 ◽

Vol 68 (1) ◽

pp. 233-238 ◽

Cited By ~ 48

Author(s):

David L. Lakey ◽

Rama K. R. Voladri ◽

Kathryn M. Edwards ◽

Cynthia Hager ◽

Buka Samten ◽

...

Keyword(s):

Escherichia Coli ◽

Superoxide Dismutase ◽

Mycobacterium Tuberculosis ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Recombinant Antigen ◽

E Coli ◽

Enhanced Production ◽

Antigen 85B

ABSTRACT A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins. To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E. coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase. Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E. coli 54-fold. The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes. Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, suggesting that the enhanced protein production was due primarily to enhanced efficiency of translation. Codon replacement in the genes encoding antigen 85A and superoxide dismutase yielded four- to sixfold increases in recombinant protein production, suggesting that this strategy may be generally applicable to overexpression of mycobacterial genes in E. coli.

Download Full-text

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

10.20944/preprints201910.0333.v1 ◽

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.

Download Full-text

Stability of plasmid pBR322 within Escherichia coli cells

MOJ Biology and Medicine ◽

10.15406/mojbm.2021.06.00123 ◽

2021 ◽

Vol 6 (1) ◽

pp. 17-19

Author(s):

Tahsin Tabassum ◽

Tasmin Tabassum ◽

Nafisa Tabassum ◽

Syeda Muntaka Maniha ◽

Rashed Noor

Keyword(s):

Escherichia Coli ◽

Drug Resistance ◽

Heat Shock ◽

Plasmid Stability ◽

Bacterial Cells ◽

Plasmid Pbr322 ◽

E Coli ◽

Replica Plating ◽

Escherichia Coli Cells ◽

Pbr322 Plasmid

nsertion of plasmids into the bacterial cells is of great significance especially in course of the transfer of drug resistance, virulence and other traits. Retention of plasmids within the host bacteria is therefore an important factor for bacterial homeostasis. Current study inferred the pBR322 plasmid stability within the Escherichia coli competent cells. The calcium chloride heat shock method was used for the transformation purpose. The plasmid retention phenomenon was assessed through the replica plating. The results positively showed the plasmid retention within E. coli.

Download Full-text

Exogenous Histidine as a Co-factor which Exacerbates the Toxicity of Hydrogen Peroxide in Cultured Mammalian and Bacterial Cells

Alternatives to Laboratory Animals ◽

10.1177/026119299101900113 ◽

1991 ◽

Vol 19 (1) ◽

pp. 68-70

Author(s):

Giorgio Brandi ◽

Piero Sestili ◽

Andrea Guidarelli ◽

Giuditta Fiorella Schiavano ◽

Amedeo Albano ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Hydrogen Peroxide ◽

Mammalian Cells ◽

Culture Medium ◽

Chinese Hamster ◽

Bacterial Cells ◽

E Coli ◽

Escherichia Coli Cells ◽

Casamino Acids

The killing of Escherichia coli cells by H2O2 is higher when exposure to the oxidant is performed in a complete culture medium, as compared to saline. Whereas MgSO4, CaCl2, thiamine or glucose, added separately or in combination with the saline, had no effect on the cytotoxic response to H2O2, the cytotoxicity appeared highly dependent upon the presence of the casamino acids in the incubation medium. One of these amino acids, histidine, was found to greatly augment the toxicity of H2O2 in E. coli. This effect of histidine was also observed in mammalian cells. In fact, both the cytoxicity and the DNA damage produced by H2O2 in Chinese hamster ovary (CHO) cells were significantly increased by this amino acid.

Download Full-text

Production and bioactivity test of recombinant protein common carp growth hormone

Jurnal Akuakultur Indonesia ◽

10.19027/jai.10.44-50 ◽

2011 ◽

Vol 10 (1) ◽

pp. 44

Author(s):

Deny Sapto Chondro Utomo ◽

. Alimuddin ◽

Agus Oman Sudrajat ◽

Irvan Faizal

Keyword(s):

Escherichia Coli ◽

Growth Hormone ◽

Body Weight ◽

Recombinant Protein ◽

Common Carp ◽

Cyprinus Carpio ◽

Bacterial Cells ◽

Recombinant Growth Hormone ◽

E Coli ◽

Sds Page

This study aimed to produce recombinant growth hormone (rGH) protein of common carp (Cyprinus carpio) and evaluate its bioactivity. DNA fragment encoding mature GH protein of common carp (mCcGH) was amplified by polymerase chain reaction (PCR) method and PCR products were then ligated into pCold I to generate pCold I-mCcGH protein expression vector. Escherichia coli BL21 (DE3) harboring pCold I-mCcGH was cultured in the 2xYT medium at 15 °C for 24 hours and protein production was induced by isopropyl-beta-thio galactopyranoside (IPTG). The inclusion bodies containing rGH protein from E. coli transformants were isolated by sonication method and analyzed by SDS-PAGE. The result showed that rGH with molecular weight of about 25 kDa was obtained. Common carp juveniles with average body weight of 5.2±0.4 g were intramuscularly injected once a week for 4 weeks with rGH protein solution from 1 μg bacterial cells per gram fish body weight. The result showed that juveniles fish injected with rGH grew 106.56% higher than control. This result indicated that rGH produced in E. coli BL21 possessed biological activity and it may be useful to improve growth of aquaculture species. Key words: growth hormone, recombinant protein, common carp ABSTRAK Penelitian ini bertujuan menghasilkan protein rekombinan hormon pertumbuhan (growth hormone, GH) dari ikan mas (Cyprinus carpio) dan menguji bioaktivitasnya. Fragmen DNA penyandi protein matang (mature) GH ikan mas (mCcGH) diamplifikasi dengan menggunakan metode PCR dan hasilnya kemudian diligasi ke dalam pCold-I untuk menghasilkan konstruksi vektor ekspresi pCold-I-mCcGH. Plasmid pCold-I-mCcGH ditransformasi ke bakteri Escherichia coli BL21 (DE3), dikultur dalam media 2xYT cair pada suhu 15°C selama 24 jam dan produksi protein diinduksi dengan menggunakan isopropyl-beta-thio galactopyranoside (IPTG). Badan inklusi yang mengandung protein rekombinan GH (rGH) dari bakteri E. coli transforman diisolasi menggunakan metode sonikasi dan dianalisis dengan menggunakan SDS-PAGE. Hasil penelitian menunjukkan bahwa rGH dengan bobot molekul sekitar 25 kDa berhasil diproduksi. Benih ikan mas dengan bobot rata-rata 5,15±0,4 g diinjeksi secara intramuskular satu kali per minggu selama 4 minggu dengan larutan rGH hasil ekstraksi dari 1 µg pelet bakteri/g bobot ikan. Benih yang disuntik dengan rGH tumbuh sekitar 100% lebih cepat bila dibandingkan dengan kontrol yang tidak diinjeksi rGH. Hasil ini mengindikasikan bahwa rGH yang diproduksi dalam bakteri E. coli memiliki bioaktivitas dan dapat bermanfaat untuk memacu pertumbuhan spesies ikan-ikan budidaya. Kata kunci: hormon pertumbuhan, protein rekombinan, ikan mas

Download Full-text