Enhanced Protocatechuic Acid Production From Glucose Using Pseudomonas putida 3-Dehydroshikimate Dehydratase Expressed in a Phenylalanine-Overproducing Mutant of Escherichia coli.
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SUBMITTER: Orn OE
PROVIDER: S-EPMC8264583 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum encoded by the qsuB gene is related to the previously described QuiC1 protein (39.9% identity) from Pseudomonas putida. Both QuiC1 and QsuB are two-domain bacterial DSDs. The N-terminal domain provides dehydratase activity, while the C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate dioxygenase. Here, the QsuB protein and its N-terminal domain (N-QsuB) were expressed in the T7 system, purified and characterized. QsuB was present mainly in octameric form (60%), while N-QsuB had a predominantly monomeric structure (80%) in aqueous buffer. Both proteins possessed DSD activity with one of the following cofactors (listed in the order of decreasing activity): Co2+, Mg2+, Mn2+. The Km and kcat values for the QsuB enzyme (Km ~ 1 mM, kcat ~ 61 s-1) were two and three times higher than those for N-QsuB. 3,4-DHBA inhibited QsuB (Ki ~ 0.38 mM, Ki' ~ 0.96 mM) and N-QsuB (Ki ~ 0.69 mM) enzymes via mixed and noncompetitive inhibition mechanism, respectively. E. coli MG1655ΔaroEPlac‒qsuB strain produced three times more 3,4-DHBA from glucose in test tube fermentation than the MG1655ΔaroEPlac‒n-qsuB strain. The C-terminal domain activity towards 3,4-DHBA was not established in vitro. This domain was proposed to promote protein oligomerization for maintaining structural stability of the enzyme. The dimer formation of QsuB protein was more predictable (ΔG = ‒15.8 kcal/mol) than the dimerization of its truncated version N-QsuB (ΔG = ‒0.4 kcal/mol).
| S-EPMC7442255 | biostudies-literature
Project description:Alongside fermentable sugars, weak acids, and furan derivatives, lignocellulosic hydrolysates contain non-negligible amounts of lignin-derived aromatic compounds. The biological funnel of lignin offers a new strategy for the "natural" production of protocatechuic acid (PCA). Herein, Pseudomonas putida KT2440 was engineered to produce PCA from lignin-derived monomers in hydrolysates by knocking out protocatechuate 3,4-dioxygenase and overexpressing vanillate-O-demethylase endogenously, while acetic acid was used for cell growth. The sugar catabolism was further blocked to prevent the loss of fermentable sugar. Using the engineered strain, a total of 253.88 mg/L of PCA was obtained with a yield of 70.85% from corncob hydrolysate 1. The highest titer of 433.72 mg/L of PCA was achieved using corncob hydrolysate 2 without any additional nutrients. This study highlights the potential ability of engineered strains to address the challenges of PCA production from lignocellulosic hydrolysate, providing novel insights into the utilization of hydrolysates.
| S-EPMC11013400 | biostudies-literature
Project description:Decreased biomass growth in iron (Fe)-limited Pseudomonas is generally attributed to downregulated expression of Fe-requiring proteins accompanied by an increase in siderophore biosynthesis. Here, we applied a stable isotope-assisted metabolomics approach to explore the underlying carbon metabolism in glucose-grown Pseudomonas putida KT2440. Compared to Fe-replete cells, Fe-limited cells exhibited a sixfold reduction in growth rate but the glucose uptake rate was only halved, implying an imbalance between glucose uptake and biomass growth. This imbalance could not be explained by carbon loss via siderophore production, which accounted for only 10% of the carbon-equivalent glucose uptake. In lieu of the classic glycolytic pathway, the Entner-Doudoroff (ED) pathway in Pseudomonas is the principal route for glucose catabolism following glucose oxidation to gluconate. Remarkably, gluconate secretion represented 44% of the glucose uptake in Fe-limited cells but only 2% in Fe-replete cells. Metabolic (13) C flux analysis and intracellular metabolite levels under Fe limitation indicated a decrease in carbon fluxes through the ED pathway and through Fe-containing metabolic enzymes. The secreted siderophore was found to promote dissolution of Fe-bearing minerals to a greater extent than the high extracellular gluconate. In sum, bypasses in the Fe-limited glucose metabolism were achieved to promote Fe availability via siderophore secretion and to reroute excess carbon influx via enhanced gluconate secretion.
| S-EPMC4767421 | biostudies-literature
Project description:Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.
| S-EPMC6680230 | biostudies-literature
Project description:BackgroundEfficient conversion of plant biomass to commodity chemicals is an important challenge that needs to be solved to enable a sustainable bioeconomy. Deconstruction of biomass to sugars and lignin yields a wide variety of low molecular weight carbon substrates that need to be funneled to product. Pseudomonas putida KT2440 has emerged as a potential platform for bioconversion of lignin and the other components of plant biomass. However, P. putida is unable to natively utilize several of the common sugars in hydrolysate streams, including galactose.ResultsIn this work, we integrated a De Ley-Doudoroff catabolic pathway for galactose catabolism into the chromosome of P. putida KT2440, using genes from several different organisms. We found that the galactonate catabolic pathway alone (DgoKAD) supported slow growth of P. putida on galactose. Further integration of genes to convert galactose to galactonate and to optimize the transporter expression level resulted in a growth rate of 0.371 h-1. Additionally, the best-performing strain was demonstrated to co-utilize galactose with glucose.ConclusionsWe have engineered P. putida to catabolize galactose, which will allow future engineered strains to convert more plant biomass carbon to products of interest. Further, by demonstrating co-utilization of glucose and galactose, continuous bioconversion processes for mixed sugar streams are now possible.
| S-EPMC6927180 | biostudies-literature
Project description:Providing an anodic potential in a bio-electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro-fermentation technology. Growth and carbon turnover start with carbon uptake into the periplasm and cytosol. P. putida KT2440 has three native transporting systems for glucose, each differing in energy and redox demand. This architecture previously led to the hypothesis that internal redox and energy constraints ultimately limit cytoplasmic carbon utilization in a bio-electrochemical system. However, it remains largely unclear which uptake route is predominantly used by P. putida under electro-fermentative conditions. To elucidate this, we created three gene deletion mutants of P. putida KT2440, forcing the cells to exclusively utilize one of the routes. When grown in a bio-electrochemical system, the pathway mutants were heavily affected in terms of sugar consumption, current output and product formation. Surprisingly, however, we found that about half of the acetate formed in the cytoplasm originated from carbon that was put into the system via the inoculation biomass, while the other half came from the consumption of substrate. The deletion of individual sugar uptake routes did not alter significantly the secreted acetate concentrations among different strains even with different carbon sources. This means that the stoichiometry of the sugar uptake routes is not a limiting factor during electro-fermentation and that the low rates might be caused by other reasons, for example energy limitations or a yet-to-be-identified oxygen-dependent regulatory mechanism.
| S-EPMC10832537 | biostudies-literature
Project description:In vivo expression technology (IVET) was employed to study colonization of Phytophthora parasitica by a biological control bacterium, Pseudomonas putida 06909, based on a new selection marker. The pyrB gene, which encodes aspartate transcarbamoylase, an enzyme used for pyrimidine biosynthesis, was cloned from P. putida 06909. A pyrB-disrupted mutant did not grow in pyrimidine-deficient media unless it was complemented with pyrBC' behind an active promoter. Thirty clones obtained from P. putida 06909 that were expressed on fungal hyphae but not on culture media were isolated by IVET based on the promoterless transcriptional fusion between pyrBC' and lacZ. Nineteen of these clones were induced during late-stage bacterial growth in vitro, while 11 of the clones were expressed only when they were inoculated onto fungal hyphae. Restriction analysis of these 11 clones revealed that there were five unique clones. Sequence analyses of three of the five unique clones showed that the 3' ends of the clones fused to pyrB were similar to genes encoding diacylglycerol kinase (DAGK), bacterial ABC transporters, and outer membrane porins. The sequences of the two other clones were not similar to the sequences of any of the genes in the database used. A LuxR family response regulator was found upstream of DAGK, and a LysR family response regulator was found upstream of the ABC transporter. The location of the inducible promoter of two clones suggested that DAGK and the ABC transporter are induced and may play a role in colonization of the fungus P. parasitica by P. putida 06909.
| S-EPMC92071 | biostudies-literature
Project description:Pseudomonas is an efficient plant growth-promoting rhizobacteria (PGPR); however, intolerance to drought and high temperature limit its application in agriculture as a bioinoculant. Transposon 5 (Tn5) mutagenesis was used to generate a stress tolerant mutant from a PGPR Pseudomonas putida NBRI1108 isolated from chickpea rhizosphere. A mutant NBRI1108T, selected after screening of nearly 10,000 transconjugants, exhibited significant tolerance towards high temperature and drought. Southern hybridization analysis of EcoRI and XhoI restricted genomic DNA of NBRI1108T confirmed that it had a single Tn5 insertion. The metabolic changes in the polar and non-polar extracts of NBRI1108 and NBRI1108T were examined using 1H, 31P nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Thirty six chemically diverse metabolites consisting of amino acids, fatty acids and phospholipids were identified and quantified. Insertion of Tn5 influenced amino acid and phospholipid metabolism and resulted in significantly higher concentration of aspartic acid, glutamic acid, glycinebetaine, glycerophosphatidylcholine (GPC) and putrescine in NBRI1108T as compared to that in NBRI1108. The concentration of glutamic acid, glycinebetaine and GPC increased by 34%, 95% and 100%, respectively in the NBRI1108T as compared to that in NBRI1108. High concentration of glycerophosphatidylethanolamine (GPE) and undetected GPC in NBRI1108 indicates that biosynthesis of GPE may have taken place via the methylation pathway of phospholipid biosynthesis. However, high GPC and low GPE concentration in NBRI1108T suggest that methylation pathway and phosphatidylcholine synthase (PCS) pathway of phospholipid biosynthesis are being followed in the NBRI1108T. Application of multivariate principal component analysis (PCA) on the quantified metabolites revealed clear variations in NBRI1108 and NBRI1108T in polar and non-polar metabolites. Identification of abiotic stress tolerant metabolites from the NBRI1108T suggest that Tn5 mutagenesis enhanced tolerance towards high temperature and drought. Tolerance to drought was further confirmed in greenhouse experiments with maize as host plant, where NBRI1108T showed relatively high biomass under drought conditions.
| S-EPMC4309533 | biostudies-literature
Project description:Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent K(D) (equilibrium dissociation constant) of 15 muM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1 degrees C and 57.6 degrees C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 muM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5'-TGAAACCGGTTTCA-3'. This palindrome overlaps with the RNA polymerase binding site.
| S-EPMC2937388 | biostudies-literature
Project description:BackgroundRhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.ResultsHere, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C(10):C(10)). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/g(glucose) corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.ConclusionsA functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.
| S-EPMC3258213 | biostudies-literature