A probable new pathway for the biosynthesis of putrescine in Escherichia coli.
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ABSTRACT:
SUBMITTER: Cataldi AA
PROVIDER: S-EPMC1146616 | biostudies-other | 1986 Mar
REPOSITORIES: biostudies-other
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Project description:BackgroundAvian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).ResultsFollowing whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.ConclusionsDeletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.
| S-EPMC6307189 | biostudies-literature
Project description:In the bio-based polymer industry, putrescine is in the spotlight for use as a material. We constructed strains of Escherichia coli to assess its putrescine production capabilities through the arginine decarboxylase pathway in batch fermentation. N-Acetylglutamate (ArgA) synthase is subjected to feedback inhibition by arginine. Therefore, the 19th amino acid residue, Tyr, of argA was substituted with Cys to desensitize the feedback inhibition of arginine, resulting in improved putrescine production. The inefficient initiation codon GTG of argA was substituted with the effective ATG codon, but its replacement did not affect putrescine production. The essential genes for the putrescine production pathway, speA and speB, were cloned into the same plasmid with argAATG Y19C to form an operon. These genes were introduced under different promoters; lacIp, lacIqp, lacIq1p, and T5p. Among these, the T5 promoter demonstrated the best putrescine production. In addition, disruption of the puuA gene encoding enzyme of the first step of putrescine degradation pathway increased the putrescine production. Of note, putrescine production was not affected by the disruption of patA, which encodes putrescine aminotransferase, the initial enzyme of another putrescine utilization pathway. We also report that the strain KT160, which has a genomic mutation of YifEQ100TAG, had the greatest putrescine production. At 48 h of batch fermentation, strain KT160 grown in terrific broth with 0.01 mM IPTG produced 19.8 mM of putrescine.
| S-EPMC8674398 | biostudies-literature
Project description:Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 μm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the culture supernatant. Additionally, the ΔsapBCDF strain did not facilitate uptake of putrescine from the culture supernatant. Quantification of stable isotope-labeled putrescine derived from stable isotope-labeled arginine supplemented in the medium revealed that SapBCDF exported putrescine from E. coli cells to the culture supernatant. It was previously reported that SapABCDF of Salmonella enterica sv. typhimurium and Haemophilus influenzae conferred resistance toantimicrobial peptides; however, the E. coli ΔsapBCDF strain did not affect resistance to antimicrobial peptide LL-37. These results strongly suggest that the natural function of the SapBCDF proteins is the export of putrescine.
| S-EPMC5159496 | biostudies-literature
Project description:Lipid A is a highly conserved component of lipopolysaccharide (LPS), itself a major component of the outer membrane of Gram-negative bacteria. Lipid A is essential to cells and elicits a strong immune response from humans and other animals. We developed a quantitative model of the nine enzyme-catalyzed steps of Escherichia coli lipid A biosynthesis, drawing parameters from the experimental literature. This model accounts for biosynthesis regulation, which occurs through regulated degradation of the LpxC and WaaA (also called KdtA) enzymes. The LpxC degradation signal appears to arise from the lipid A disaccharide concentration, which we deduced from prior results, model results, and new LpxK overexpression results. The model agrees reasonably well with many experimental findings, including the lipid A production rate, the behaviors of mutants with defective LpxA enzymes, correlations between LpxC half-lives and cell generation times, and the effects of LpxK overexpression on LpxC concentrations. Its predictions also differ from some experimental results, which suggest modifications to the current understanding of the lipid A pathway, such as the possibility that LpxD can replace LpxA and that there may be metabolic channeling between LpxH and LpxB. The model shows that WaaA regulation may serve to regulate the lipid A production rate when the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) concentration is low and/or to control the number of KDO residues that get attached to lipid A. Computation of flux control coefficients showed that LpxC is the rate-limiting enzyme if pathway regulation is ignored, but that LpxK is the rate-limiting enzyme if pathway regulation is present, as it is in real cells. Control also shifts to other enzymes if the pathway substrate concentrations are not in excess. Based on these results, we suggest that LpxK may be a much better drug target than LpxC, which has been pursued most often.
| S-EPMC4412817 | biostudies-literature
Project description:The lipid A moiety of Escherichia coli lipopolysaccharide is a hexa-acylated disaccharide of glucosamine that makes up the outer monolayer of the outer membrane. Arabidopsis thaliana contains nuclear genes encoding orthologs of key enzymes of bacterial lipid A biosynthesis, including LpxA, LpxC, LpxD, LpxB, LpxK and KdtA. Although structurally related lipid A molecules are found in most other gram-negative bacteria, lipid A and its precursors have not been directly detected in plants previously. However, homozygous insertional knockout mutations or RNAi knock-down constructs of Arabidopsis lpx and kdtA mutants revealed accumulation (or disappearance) of the expected monosaccharide or disaccharide lipid A precursors by mass spectrometry of total lipids extracted from 10-day old seedlings of these mutants. In addition, fluorescence microscopy of lpx-gfp fusions in transgenic Arabidopsis plants suggests that the Lpx and KdtA proteins are expressed and targeted to mitochondria. Although the structure of the lipid A end product generated by plants is still unknown, our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli.
| S-EPMC3136250 | biostudies-literature
Project description:The hydroxylation of tryptophan is an important reaction in the biosynthesis of natural products. 5-Hydroxytryptophan (5HTP) is not only an important compound for its pharmaceutical value but also because it is the precursor of other molecules, such as serotonin. In this study, we have extended the metabolism of an E. coli strain to produce 5HTP. Aromatic amino acid hydroxylase from Cupriavidus taiwanensis (CtAAAH) was selected using an in silico structure-based approach. We have predicted and selected several substrate-determining residues using sequence, phylogenetic and functional divergence analyses; we also did rational design on CtAAAH to shift the enzyme preference from phenylalanine to tryptophan. Whole cell bioconversion assays were used to show the effect of predicted sites. In general, all of them decreased the preference toward phenylalanine and increased the tryptophan synthesis activity. The best performer, CtAAAH-W192F, was transformed into a strain that had the tryptophanase gene disrupted and carried a human tetrahydrobiopterin (BH4) regeneration pathway. The resulting strain was capable of synthesizing 2.5 mM 5HTP after 24 hours. This work demonstrates the application of computational approaches for protein engineering and further coupling with the bacterial metabolism.
| S-EPMC6999490 | biostudies-literature
Project description:In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.
| S-EPMC2893496 | biostudies-literature
Project description:BackgroundThe shortage of food based feedstocks has been one of the stumbling blocks in industrial biomanufacturing. The acetone bioproduction from the traditional acetone-butanol-ethanol fermentation is limited by the non-specificity of products and competitive utilization of food-based substrates. Using genetically modified Escherichia coli to produce acetone as sole product from the cost-effective non-food based substrates showed great potential to overcome these problems.ResultsA novel acetone biosynthetic pathway were constructed based on genes from Clostridium acetobutylicum (thlA encoding for thiolase, adc encoding for acetoacetate decarboxylase, ctfAB encoding for coenzyme A transferase) and Escherichia coli MG1655 (atoB encoding acetyl-CoA acetyltransferase, atoDA encoding for acetyl-CoA: acetoacetyl-CoA transferase subunit ? and ?). Among these constructs, one recombinant MG1655 derivative containing the hybrid pathway consisting of thlA, atoDA, and adc, produced the highest level of acetone from acetate. Reducing the gluconeogenesis pathway had little effect on acetone production, while blocking the TCA cycle by knocking out the icdA gene enhanced the yield of acetone significantly. As a result, acetone concentration increased up to 113.18 mM in 24 h by the resting cell culture coupling with gas-stripping methods.ConclusionsAn engineered E. coli strain with optimized hybrid acetone biosynthetic pathway can utilize acetate as substrate efficiently to synthesize acetone without other non-gas byproducts. It provides a potential method for industrial biomanufacturing of acetone by engineered E. coli strains from non-food based substrate.
| S-EPMC6330746 | biostudies-literature
Project description:To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l(-1)). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters.
| S-EPMC4411949 | biostudies-literature
Project description:The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of D-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on D-malate as a carbon source, the D-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB(-) strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (L(+)-tartrate, D-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution.
| S-EPMC4200263 | biostudies-literature