scholarly journals Biosynthesis of ethyl caffeate via caffeoyl-CoA acyltransferase expression in Escherichia coli

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Shin-Won Lee ◽  
Han Kim ◽  
Joong-Hoon Ahn
Keyword(s):  
Escherichia Coli ◽  
Caffeic Acid ◽  
Shikimate Pathway ◽  
Acid Synthesis ◽  
Biological Properties ◽  
E Coli ◽  
Pathway Gene ◽  
Coa Ligase ◽  
Prunus Yedoensis ◽  
Tyrosine Ammonia Lyase

AbstractHydroxycinnamic acids (HCs) are natural compounds that form conjugates with diverse compounds in nature. Ethyl caffeate (EC) is a conjugate of caffeic acid (an HC) and ethanol. It has been found in several plants, including Prunus yedoensis, Polygonum amplexicaule, and Ligularia fischeri. Although it exhibits anticancer, anti-inflammatory, and antifibrotic activities, its biosynthetic pathway in plants still remains unknown. This study aimed to design an EC synthesis pathway and clone genes relevant to the same. Genes involved in the caffeic acid synthesis pathway (tyrosine ammonia-lyase (TAL) and p-coumaric acid hydroxylase (HpaBC)) were introduced into Escherichia coli along with 4-coumaroyl CoA ligase (4CL) and acyltransferases (AtCAT) cloned from Arabidopsis thaliana. In presence of ethanol, E. coli harboring the above genes successfully synthesized EC. Providing more tyrosine through the overexpression of shikimate-pathway gene-module construct and using E. coli mutant enhanced EC yield; approximately 116.7 mg/L EC could be synthesized in the process. Synthesis of four more alkyl caffeates was confirmed in this study; these might potentially possess novel biological properties, which would require further investigation.

scholarly journals Biosynthesis of resveratrol using metabolically engineered Escherichia coli

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Jin Yeong Park ◽  
Jeong-Hyeon Lim ◽  
Joong-Hoon Ahn ◽  
Bong-Gyu Kim
Keyword(s):  
Escherichia Coli ◽  
Carbon Sources ◽  
Shikimate Pathway ◽  
Stilbene Synthase ◽  
Culture Conditions ◽  
E Coli ◽  
The Past ◽  
Coa Ligase ◽  
Trans Stilbene ◽  
Tyrosine Ammonia Lyase

AbstractResveratrol (3,5,4′-trihydroxy-trans-stilbene) is a phenolic compound widely used in pharmaceutics and nutraceutics. Although resveratrol is produced by some plant species, including grapes, peanuts, and berries, the content of resveratrol and its derivatives are very low. Therefore, an alternative biosynthetic method using microorganisms, such as Escherichia coli, has been developed over the past two decades. In the present study, a resveratrol-over-producing E. coli strain was developed using three strategies. First, we increased the synthesis of p-coumaric acid, a precursor of resveratrol, by manipulating genes in the shikimate pathway of E. coli. Second, three genes involved in resveratrol biosynthesis, such as tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS), were cloned from diverse sources, such as plants and microorganisms, and the best combination was selected to maximize resveratrol production in E. coli. Finally, culture conditions, such as cell concentration, culture temperature, and carbon sources, were established for optimal resveratrol production. Through these strategies, approximately 80.4 mg/L of resveratrol was biosynthesized after 48 h of culture using glycerol as a carbon source.

scholarly journals Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450

2019 ◽  
Author(s):  
Kristina Haslinger ◽  
Kristala L.J. Prather
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Caffeic Acid ◽  
De Novo ◽  
Redox System ◽  
Chemical Extraction ◽  
E Coli ◽  
System Combination ◽  
Cell Factories ◽  
Tyrosine Ammonia Lyase

AbstractBackgroundCaffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far.ResultsThe herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 μM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even an initially unproductive Cytochrome P450/ redox system combination productive.ConclusionsThe caffeic acid titer achieved in this study is about 25% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.

scholarly journals Induction and control of the autolytic system of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 26-34
Author(s):  
M Leduc ◽  
R Kasra ◽  
J van Heijenoort
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Acid Synthesis ◽  
Induction Method ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Carbonyl Cyanide ◽  
And Control ◽  
First Time ◽  
Induced Autolysis

Various methods of inducing autolysis of Escherichia coli cells were investigated, some being described here for the first time. For the autolysis of growing cells only induction methods interfering with the biosynthesis of peptidoglycan were taken into consideration, whereas with harvested cells autolysis was induced by rapid osmotic or EDTA shock treatments. The highest rates of autolysis were observed after induction by moenomycin, EDTA, or cephaloridine. The different autolyses examined shared certain common properties. In particular, regardless of the induction method used, more or less extensive peptidoglycan degradation was observed, and 10(-2) M Mg2+ efficiently inhibited the autolytic process. However, for other properties a distinction was made between methods used for growing cells and those used for harvested cells. Autolysis of growing cells required RNA, protein, and fatty acid synthesis. No such requirements were observed with shock-induced autolysis performed with harvested cells. Thus, the effects of Mg2+, rifampicin, chloramphenicol, and cerulenin clearly suggest that distinct factors are involved in the control of the autolytic system of E. Coli. Uncoupling agents such as sodium azide, 2,4-dinitrophenol, and carbonyl-cyanide-m-chlorophenyl hydrazone used at their usual inhibiting concentration had no effect on the cephaloridine or shock-induced autolysis.

scholarly journals Anaerobic Cysteine Degradation and Potential Metabolic Coordination in Salmonella enterica and Escherichia coli

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Melissa Loddeke ◽  
Barbara Schneider ◽  
Tamiko Oguri ◽  
Iti Mehta ◽  
Zhenyu Xuan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Salmonella Enterica ◽  
Acid Synthesis ◽  
Lower Growth ◽  
Amino Acid Synthesis ◽  
Content Type ◽  
E Coli ◽  
Sulfur Containing ◽  
Sulfur Containing Compounds

ABSTRACT Salmonella enterica has two CyuR-activated enzymes that degrade cysteine, i.e., the aerobic CdsH and an unidentified anaerobic enzyme; Escherichia coli has only the latter. To identify the anaerobic enzyme, transcript profiling was performed for E. coli without cyuR and with overexpressed cyuR. Thirty-seven genes showed at least 5-fold changes in expression, and the cyuPA (formerly yhaOM) operon showed the greatest difference. Homology suggested that CyuP and CyuA represent a cysteine transporter and an iron-sulfur-containing cysteine desulfidase, respectively. E. coli and S. enterica ΔcyuA mutants grown with cysteine generated substantially less sulfide and had lower growth yields. Oxygen affected the CyuR-dependent genes reciprocally; cyuP-lacZ expression was greater anaerobically, whereas cdsH-lacZ expression was greater aerobically. In E. coli and S. enterica, anaerobic cyuP expression required cyuR and cysteine and was induced by l-cysteine, d-cysteine, and a few sulfur-containing compounds. Loss of either CyuA or RidA, both of which contribute to cysteine degradation to pyruvate, increased cyuP-lacZ expression, which suggests that CyuA modulates intracellular cysteine concentrations. Phylogenetic analysis showed that CyuA homologs are present in obligate and facultative anaerobes, confirming an anaerobic function, and in archaeal methanogens and bacterial acetogens, suggesting an ancient origin. Our results show that CyuA is the major anaerobic cysteine-catabolizing enzyme in both E. coli and S. enterica, and it is proposed that anaerobic cysteine catabolism can contribute to coordination of sulfur assimilation and amino acid synthesis. IMPORTANCE Sulfur-containing compounds such as cysteine and sulfide are essential and reactive metabolites. Exogenous sulfur-containing compounds can alter the thiol landscape and intracellular redox reactions and are known to affect several cellular processes, including swarming motility, antibiotic sensitivity, and biofilm formation. Cysteine inhibits several enzymes of amino acid synthesis; therefore, increasing cysteine concentrations could increase the levels of the inhibited enzymes. This inhibition implies that control of intracellular cysteine levels, which is the immediate product of sulfide assimilation, can affect several pathways and coordinate metabolism. For these and other reasons, cysteine and sulfide concentrations must be controlled, and this work shows that cysteine catabolism contributes to this control.

scholarly journals Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

2021 ◽  
Vol 12 ◽  
Author(s):  
R. Kyle Bennett ◽  
Gwendolyn J. Gregory ◽  
Jacqueline E. Gonzalez ◽  
Jie Ren Gerald Har ◽  
Maciek R. Antoniewicz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Subunit ◽  
Biological Properties ◽  
Methanol Tolerance ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
E Coli ◽  
Translational Accuracy ◽  
Methanol Utilization ◽  
Carbon Compounds

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

scholarly journals Ferulic Acid production by metabolically engineered Escherichia coli

2021 ◽  
Author(s):  
Huajun Lv ◽  
Ying Zhang ◽  
Jie Shao ◽  
Haili Liu ◽  
Yong Wang
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Caffeic Acid ◽  
Pyridine Nucleotide ◽  
Promoter Strength ◽  
T7 Promoter ◽  
Nadph Regeneration ◽  
E Coli ◽  
Ferulic Acid Production ◽  
K 12

Abstract Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics areas. Production of FA by eco-friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding Tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p - coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as Caffeic acid O-methytransferase. Fermentation results revealed JM109(DE3) as more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 ori or p15a ori), and the combination p15a-T5 works best. To further improve FA production, E.coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also over expressed. Based on the flask fermentation data which shows that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E.coli K-12 strains reported to the best of our knowledge.

scholarly journals Biological Properties of Structurally Related α-Helical Cationic Antimicrobial Peptides

1999 ◽  
Vol 67 (4) ◽  
pp. 2005-2009 ◽  
Author(s):  
Monisha G. Scott ◽  
Hong Yan ◽  
Robert E. W. Hancock
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Tumor Necrosis ◽  
Biological Properties ◽  
Cationic Antimicrobial Peptide ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Resistant Strains ◽  
Conventional Antibiotics ◽  
Necrosis Factor

ABSTRACT A series of α-helical cationic antimicrobial peptide variants with small amino acid changes was designed. Alterations in the charge, hydrophobicity, or length of the variant peptides did not improve the antimicrobial activity, and there was no statistically significant correlation between any of these factors and the MIC forPseudomonas aeruginosa, Escherichia coli, orSalmonella typhimurium. Individual peptides demonstrated synergy with conventional antibiotics against antibiotic-resistant strains of P. aeruginosa. The peptides varied considerably in the ability to bind E. coli O111:B4 lipopolysaccharide (LPS), and this correlated significantly with their antimicrobial activity and ability to block LPS-stimulated tumor necrosis factor and interleukin-6 production. In general, the peptides studied here demonstrated a broad range of activities, including antimicrobial, antiendotoxin, and enhancer activities.

scholarly journals Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene

2002 ◽  
Vol 364 (3) ◽  
pp. 795-805 ◽  
Author(s):  
Adrian P. BROWN ◽  
Simon CARNABY ◽  
Clare BROUGH ◽  
Melissa BRAZIER ◽  
Antoni R. SLABAS
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Lysophosphatidic Acid ◽  
Leaf Tissue ◽  
Acid Synthesis ◽  
Protein Antigens ◽  
Maximal Level ◽  
E Coli ◽  
Developing Seeds ◽  
Membrane Bound

Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27pg/μg of membrane protein in leaf tissue and ≤ 12.5pg/μg of membrane protein in developing embryos. The amount of LAT2 reaches a peak at 305pg/μg of membrane protein 25daf and is not expressed 20daf or before. This is the first study to quantify these membrane-bound proteins in a plant tissue. The maximal level of LAT2 protein coincides with the maximal level of erucic acid synthesis in the seeds. Both full-length proteins were expressed in the Escherichia coli LPAAT mutant JC201, and membranes from these strains were used to investigate the substrate selectivity of these two enzymes, demonstrating that they are different. Finally, we report that LAT2 and a maize LPAAT enzyme (MAT1) can functionally replace the E. coli plsC gene after its deletion in the chromosome, whereas LAT1 and a coconut LPAAT (Coco1) cannot. This is probably due to differences in substrate utilization.

ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

2015 ◽  
Vol 25 (6) ◽  
pp. 394-402 ◽  
Author(s):  
Taylor L. Fischer ◽  
Robert J. White ◽  
Katherine F.K. Mares ◽  
Devin E. Molnau ◽  
Justin J. Donato
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
E Coli ◽  
Enoyl Acp Reductase ◽  
Selection For

<b><i>Background/Aims:</i></b> We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in <i>Escherichia coli</i>. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. <b><i>Methods:</i></b> ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into <i>E. coli</i>, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. <b><i>Results:</i></b> Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into <i>E. coli</i>, whereas the mutant remained susceptible to triclosan<i>. </i>Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. <b><i>Conclusion:</i></b> ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

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