scholarly journals L-Carnitine Production Through Biosensor-Guided Construction of the Neurospora crassa Biosynthesis Pathway in Escherichia coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Pierre Kugler ◽  
Marika Trumm ◽  
Marcel Frese ◽  
Volker F. Wendisch
Keyword(s):  
Escherichia Coli ◽  
Neurospora Crassa ◽  
De Novo ◽  
Nitrogen Sources ◽  
Bioactive Compound ◽  
Biosynthesis Pathway ◽  
One Pot ◽  
E Coli ◽  
Renewable Feedstocks ◽  
Eukaryotic Microorganisms

L-Carnitine is a bioactive compound derived from L-lysine and S-adenosyl-L-methionine, which is closely associated with the transport of long-chain fatty acids in the intermediary metabolism of eukaryotes and sought after in the pharmaceutical, food, and feed industries. The L-carnitine biosynthesis pathway has not been observed in prokaryotes, and the use of eukaryotic microorganisms as natural L-carnitine producers lacks economic viability due to complex cultivation and low titers. While biotransformation processes based on petrochemical achiral precursors have been described for bacterial hosts, fermentative de novo synthesis has not been established although it holds the potential for a sustainable and economical one-pot process using renewable feedstocks. This study describes the metabolic engineering of Escherichia coli for L-carnitine production. L-carnitine biosynthesis enzymes from the fungus Neurospora crassa that were functionally active in E. coli were identified and applied individually or in cascades to assemble and optimize a four-step L-carnitine biosynthesis pathway in this host. Pathway performance was monitored by a transcription factor-based L-carnitine biosensor. The engineered E. coli strain produced L-carnitine from supplemented L-Nε-trimethyllysine in a whole cell biotransformation, resulting in 15.9 μM carnitine found in the supernatant. Notably, this strain also produced 1.7 μM L-carnitine de novo from glycerol and ammonium as carbon and nitrogen sources through endogenous Nε-trimethyllysine. This work provides a proof of concept for the de novoL-carnitine production in E. coli, which does not depend on petrochemical synthesis of achiral precursors, but makes use of renewable feedstocks instead. To the best of our knowledge, this is the first description of L-carnitine de novo synthesis using an engineered bacterium.

scholarly journals Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

2012 ◽  
Vol 78 (20) ◽  
pp. 7407-7413 ◽  
Author(s):  
Qian Zhang ◽  
Tao Yan
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Potassium Acetate ◽  
Desiccation Stress ◽  
Desiccation Resistance ◽  
Content Type ◽  
Soil Desiccation ◽  
E Coli ◽  
Organic Solute ◽  
Trehalose Synthesis

ABSTRACTNaturalized soilEscherichia colipopulations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soilE. colistrains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day−1) than that of MG1655 (0.85 day−1). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among theE. colistrains. AllE. colistrains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman's ρ = −1.0;P= 0.02).De novotrehalose synthesis was further determined for 15E. colistrains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. MostE. colistrains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soilE. colistrains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).

scholarly journals Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenning Liu ◽  
Xue Zhang ◽  
Dengwei Lei ◽  
Bin Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Phosphopantetheinyl Transferase ◽  
Chromosome Engineering ◽  
Microbial Cell Factory ◽  
E Coli ◽  
Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Antibacterial activity of Escherichia coli from squid ink (Loligo sp.) n-Hexane extracts

2021 ◽  
Vol 869 (1) ◽  
pp. 012033
Author(s):  
D R Utami ◽  
I Irwan ◽  
S Agustina ◽  
S Karina ◽  
S Afriani
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Secondary Metabolite ◽  
Bioactive Compound ◽  
Inhibition Zone ◽  
Hexane Extract ◽  
Inhibition Zone Diameter ◽  
E Coli ◽  
Hexane Extracts ◽  
Intermediate Concentration

Abstract Squid is one of the export commodities in Indonesia. In general, the use of squid meat, while the ink is only as waste. In fact, Squid ink contain bioactive compound that potential as anti-inflammatory, antihypertensive, anti-diabetic,anti-microbial and anti-malaria agents. The purpose of the study is to determine the types of secondary metabolite compounds contained in n-hexane extract of Loligo sp. ink using maceration method to determine its antibacterial activity against Escherichia coli. The results of secondary metabolite compounds obtained from the n-hexane extract of Loligo sp. ink are alkaloid, saponins, glycosides and phenol. The results of antibacterial test against E. coli using the disc method obtained the average of inhibition zone diameter at the concentration of 4% is 6.3 mm (intermediate), concentration of 8% is 7.83 mm (intermediate), concentration of 16% is 14.5 mm (susceptible) and concentration of 32% is 10.83 mm (intermediate). The antibacterial activity in n-hexane extract of Loligo sp. ink is optimal at the concentration of 16% against E. coli bacteria.

scholarly journals PREPARASI DAN MODIFIKASI KIMIA STRUKTUR KITOOLIGOSAKARIDA-2,5-ANHIDRO-D-MANNOFURANOSA (KOSAMF) DARI KITOSAN SERTA UJI ANTIBAKTERI TERHADAP Staphylococcus aureus DAN Escherichia coli

2016 ◽  
Vol 9 (2) ◽  
pp. 15
Author(s):  
Emil Salim ◽  
Hasnirwan ◽  
Sanusi Ibrahim ◽  
Afrizal
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
One Pot ◽  
E Coli ◽  
Ft Ir

Kitooligosakarida-2,5-anhidro-D-mannofuranosa (KOSamf) dipersiapkan dengan derajat polimerisasi tertentu melalui reaksi nitrous deaminasi dari kitosan parsial N-deasetilasi. KOSamf kemudian diubah menjadi turunan KOSamf dengan mengkopling gugus aldehid yang terdapat pada unit 2,5-anhidro-D-mannofuranosa (amf). KOSamf dengan 3,5-dikloroanilin dan NaBH3CN dalam pelarut buffer ammonium asetat direaksikan melalui metoda one-pot aminasi reduktif untuk menghasilkan turunan KOSamf yang berpotensi sebagai senyawa antibakteri dan antijamur. Struktur kimia dari turunan KOSamf yang telah disintesis dikarakterisasi dengan Spektroskopi FT-IR dan NMR. Senyawa turunan KOSamf memiliki aktivitas sebagai antibakteri terhadap bakteri Staphylococcus aureus dengan zona inhibisi 12,5 mm dan bakteri E. coli dengan zona inhibisi 10,5 mm.

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

scholarly journals Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins.

1991 ◽  
Vol 174 (5) ◽  
pp. 1167-1177 ◽  
Author(s):  
J Vuopio-Varkila ◽  
G K Schoolnik
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
De Novo ◽  
Outer Membrane Proteins ◽  
Temporal Correlation ◽  
Enteropathogenic Escherichia Coli ◽  
E Coli

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

scholarly journals Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 774
Author(s):  
Virginio Cepas ◽  
Victoria Ballén ◽  
Yaiza Gabasa ◽  
Miriam Ramírez ◽  
Yuly López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
De Novo ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Qrt Pcr ◽  
Transposon Insertion ◽  
De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

scholarly journals Increase in Furfural Tolerance in Ethanologenic Escherichia coli LY180 by Plasmid-Based Expression ofthyA

2012 ◽  
Vol 78 (12) ◽  
pp. 4346-4352 ◽  
Author(s):  
Huabao Zheng ◽  
Xuan Wang ◽  
Lorraine P. Yomano ◽  
Keelnatham T. Shanmugam ◽  
Lonnie O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cellular Response ◽  
De Novo ◽  
Content Type ◽  
Genomic Libraries ◽  
Methyl Donor ◽  
E Coli ◽  
Furfural Tolerance ◽  
Small Benefit ◽  
Ethanologenic Escherichia Coli

ABSTRACTFurfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilisYB886,Escherichia coliNC3, andZymomonas mobilisCP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing thethyAgene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in thede novopathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression ofthyAwas no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA inE. coliand to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA inE. coliwould be expected to increase the cellular requirement for dTMP. Increased expression ofthyA(E. coli,B. subtilis, orZ. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

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