scholarly journals Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

2020 ◽  
Author(s):  
Anna Christina Bohnenkamp ◽  
Aleksander J. Kruis ◽  
Astrid E. Mars ◽  
Rene H. Wijffels ◽  
John van der Oost ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Production Efficiency ◽  
Yeast Species ◽  
Anaerobic Conditions ◽  
E Coli ◽  
Alcohol Acetyltransferase ◽  
Enhanced Production ◽  
Key Enzyme ◽  
Encoding Genes ◽  
Multilevel Optimisation

Abstract BackgroundEthyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. ResultsWe engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/T7 and XylS/Pm promoters allowed optimisation of their expression levels. ConclusionEngineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced ethyl acetate from glucose at an unprecedented level, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

scholarly journals Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

2020 ◽  
Author(s):  
Anna Christina Bohnenkamp ◽  
Aleksander J. Kruis ◽  
Astrid E. Mars ◽  
Rene H. Wijffels ◽  
John van der Oost ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Production Efficiency ◽  
Yeast Species ◽  
Coli Strain ◽  
E Coli ◽  
Alcohol Acetyltransferase ◽  
Enhanced Production ◽  
Key Enzyme ◽  
Encoding Genes ◽  
Multilevel Optimisation

Abstract Background Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/ T7 and XylS/ Pm promoters allowed optimisation of their expression levels. Conclusion Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced ethyl acetate from glucose at an unprecedented level, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

Deoxyribonucleotide Synthesis in an Escherichia coli Mutant (H 1491) which Lacks Ribonucleotide Reductase Subunit B2

1989 ◽  
Vol 44 (7-8) ◽  
pp. 715-718 ◽  
Author(s):  
Jens Harder ◽  
Hartmut Follmann ◽  
Klaus Hantke
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Strictly Anaerobic ◽  
Anaerobic Conditions ◽  
Sensitive Mutant ◽  
E Coli ◽  
Key Enzyme ◽  
Minimal Media

An iron-sensitive mutant of E. coli with a Mudl phage insertion in the nrdB gene lacks subunit B2 of the key enzyme of DNA synthesis, ribonucleotide reductase. Nevertheless, these cells are capable of growing in minimal media under anaerobic conditions, indicating a second enzyme or pathway for deoxyribonucleotide synthesis. We here show that ribonucleotide reduction cannot be unambiguously measured in bacterial extracts whereas phosphorylase- catalyzed deoxyribosyl transfer does occur; however these salvage reactions could not function in vivo in the absence of deoxyribosides. It is suggested that the cells possess a specific, anaerobic ribonucleotide reductase which escapes detection under aerobic standard conditions, similar to the situation found in strictly anaerobic methanogens.

scholarly journals Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

2021 ◽  
Author(s):  
Fatma Ben Abid ◽  
Clement K. M. Tsui ◽  
Yohei Doi ◽  
Anand Deshmukh ◽  
Christi L. McElheny ◽  
...  
Keyword(s):  
Common Species ◽  
Clinical Samples ◽  
Whole Genome ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Sequence Types ◽  
Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

scholarly journals Foreign investment in emerging legal medicinal cannabis markets: the Jamaica case study

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Marta Rychert ◽  
Machel Anthony Emanuel ◽  
Chris Wilkins
Keyword(s):  
Civil Society ◽  
Foreign Investment ◽  
Production Efficiency ◽  
Production Quality ◽  
Small Scale ◽  
Organic Cultivation ◽  
Enhanced Production ◽  
Political Interference ◽  
Medicinal Cannabis ◽  
The Impact

Abstract Introduction The establishment of a legal market for medicinal cannabis under the Dangerous Drugs Amendment Act 2015 has positioned Jamaica at the forefront of cannabis law reform in the developing world. Many local cannabis businesses have attracted investment from overseas, including from Canada, US and Europe. Aim To explore the opportunities and risks of foreign investment in an emerging domestic legal cannabis market in a developing country. Methods Thematic analysis of semi-structured face-to-face interviews with 22 key informants (KIs) from the Jamaican government, local cannabis industry, academia and civil society, and field observations of legal and illegal cannabis cultivators. Results KIs from the Jamaican public agencies and domestic cannabis entrepreneurs saw foreign investment as an essential source of capital to finance the start-up costs of legal cannabis businesses. Local cannabis entrepreneurs prioritised investors with the greatest financial resources, brand reputation and export networks. They also considered how allied an investor was with their business vision (e.g., organic cultivation, medical vs. recreational). The key benefits of partnering with a foreign investor included transfer of technical knowledge and financial capital, which enhanced production, quality assurance and seed-to-sale tracking. Some KIs expressed concern over investors’ focus on increasing production efficiency and scale at the expense of funding research and development (R&D) and clinical trials. KIs from the local industry, government agencies and civil society highlighted the risks of ‘predatory’ shareholder agreements and domestic political interference. Concerns were raised about the impact of foreign investment on the diversity of the domestic cannabis sector in Jamaica, including the commitment to transition traditional illegal small-scale cannabis cultivators to the legal sector. Conclusion While foreign investment has facilitated the commercialisation of the cannabis sector in Jamaica, regulatory measures are also needed to protect the domestic industry and support the transition of small-scale illegal cultivators to the legal regime. Foreign investments may alter the economic, social and political determinants of health in transitioning from illegal to legal cannabis market economy.

Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum

2012 ◽  
Vol 58 (3) ◽  
pp. 278-286 ◽  
Author(s):  
Jae-Hyung Jo ◽  
Hye-Young Seol ◽  
Yun-Bom Lee ◽  
Min-Hong Kim ◽  
Hyung-Hwan Hyun ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Biosynthetic Pathway ◽  
Isocitrate Lyase ◽  
Glutamate Synthase ◽  
Flask Culture ◽  
Control Strain ◽  
Enhanced Production ◽  
Key Enzyme ◽  
Microbial Strains ◽  
Glyoxylate Pathway

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.

scholarly journals 166. Activity of a Novel β-lactamase Inhibitor QPX7728 Combined With β-lactams Against st258 klebsiella Pneumoniae and st131 escherchia Coli Isolates Producing β-lactamases

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S212-S213
Author(s):  
Mariana Castanheira ◽  
Jill Lindley ◽  
Timothy B Doyle ◽  
Andrew P Davis ◽  
Olga Lomovskaya
Keyword(s):  
Multidrug Resistant ◽  
Chemical Diversity ◽  
Sequencing Analysis ◽  
E Coli ◽  
Escherchia Coli ◽  
Global Spread ◽  
Encoding Genes ◽  
Center Research ◽  
Lactamase Inhibitor ◽  
Research Grant

Abstract Background ST258 K. pneumoniae and ST131 E. coli clones are considered vectors for the global spread of multidrug resistance. We evaluated the activity of β-lactams in combination with QPX7728, a novel β-lactamase inhibitor active against all β-lactamase classes, against a collection of 210 isolates belonging to these clones collected from a worldwide surveillance study. Methods A total of 118 ST258 K. pneumoniae and 92 ST131 E. coli (single loci variant also included) were susceptibility tested by reference broth microdilution against various β-lactams ± QPX7728 and comparator agents. All isolates were screened for β-lactamases using whole genome sequencing analysis. Results All β-lactam agents had limited activity against 118 ST258 K. pneumoniae (1.7–7.6% susceptible). Among these, 104 carried carbapenemase-encoding genes: 66 KPC variants, 20 NDM and 17 OXA-48-like. One isolate carried 2 carbapenemases. The addition of QPX7728 at 4 mg/L or 8 mg/L lowered the MICs for cefepime (MIC50/90, 0.25/1 mg/L and MIC50/90, 0.12/0.5 mg/L), ceftolozane (MIC50/90, 0.5/ > 32 mg/L and MIC50/90, 0.25/16 mg/L), ertapenem (MIC50/90, 0.12/2 mg/L and MIC50/90, 0.06/0.5 mg/L), and meropenem (MIC50/90, 0.06/0.5 mg/L and MIC50/90, 0.03/0.12 mg/L; Table). QPX7728 at 4 mg/L reduced the ceftibuten (MIC50/90, 0.25/8 mg/L) or tebipenem (MIC50/90, 0.12/2 mg/L) MICs for ST258 isolates. E. coli ST131 carried mainly CTX-M variant (85 isolates), but 6 isolates harbored carbapenemases. Carbapenems were the only β-lactams displaying > 80.0% activity against ST131 E. coli, followed by piperacillin-tazobactam (79.3% susceptible). Only 5.4%and 41.3% ST131 isolates were susceptible to cefepime and ceftibuten, respectively. MIC50/MIC90 values for these agents with QPX7728 were ≤ 0.015/≤ 0.015 mg/L for cefepime and ≤ 0.015/0.06 mg/L for ceftolozane with the inhibitor at 8 mg/L and ≤ 0.015/0.03 mg/L for ceftibuten with the inhibitor at 4 mg/L. Conclusion QPX7728 lowered the MICs for all agents tested to clinically achievable levels when tested against isolates multidrug resistant belonging to important clones responsible to the dissemination of KPC, CTX variants, and metallo-β-lactamases. The development of this broad β-lactamase inhibitor should be pursued. Table 1 Disclosures Mariana Castanheira, PhD, 1928 Diagnostics (Research Grant or Support)A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Amplyx Pharmaceuticals (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Jill Lindley, Allergan (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Timothy B. Doyle, Allergan (Research Grant or Support)Allergan (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Olga Lomovskaya, PhD, Qpex Biopharma (Employee)

scholarly journals Autotransporter Protein-Encoding Genes of Diarrheagenic Escherichia coli Are Found in both Typical and Atypical Enteropathogenic E. coli Strains

2012 ◽  
Vol 79 (1) ◽  
pp. 411-414 ◽  
Author(s):  
Afonso G. Abreu ◽  
Vanessa Bueris ◽  
Tatiane M. Porangaba ◽  
Marcelo P. Sircili ◽  
Fernando Navarro-Garcia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Virulence Markers ◽  
Protein Encoding ◽  
Encoding Genes ◽  
Specific Virulence

ABSTRACTAutotransporter (AT) protein-encoding genes of diarrheagenicEscherichia coli(DEC) pathotypes (cah,eatA,ehaABCDJ,espC,espI,espP,pet,pic,sat, andtibA) were detected in typical and atypical enteropathogenicE. coli(EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.

scholarly journals High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

2013 ◽  
Vol 97 (9) ◽  
pp. 3893-3900 ◽  
Author(s):  
Odile Francesca Restaino ◽  
Ujjwal Bhaskar ◽  
Priscilla Paul ◽  
Lingyun Li ◽  
Mario De Rosa ◽  
...  
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Coli Strain ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
E Coli ◽  
Bioengineered Heparin ◽  
Key Enzyme

scholarly journals Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation

2005 ◽  
Vol 71 (12) ◽  
pp. 7880-7887 ◽  
Author(s):  
Sang Jun Lee ◽  
Dong-Yup Lee ◽  
Tae Yong Kim ◽  
Byung Hun Kim ◽  
Jinwon Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Succinic Acid ◽  
In Silico ◽  
Acid Production ◽  
Gene Knockout ◽  
Fermentation Products ◽  
E Coli ◽  
Metabolic Analysis ◽  
Enhanced Production ◽  
Succinic Acid Production

ABSTRACT Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 81-88
Author(s):  
E H Berglin ◽  
M B Edlund ◽  
G K Nyberg ◽  
J Carlsson
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Metal Ion ◽  
Chelating Agent ◽  
Fold Increase ◽  
Bactericidal Effect ◽  
Anaerobic Conditions ◽  
Dna Breakage ◽  
E Coli ◽  
K 12

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.

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