Repression by glucose of acetohydroxy acid synthetase in Escherichia coli B.
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ABSTRACT:
SUBMITTER: Coukell MB
PROVIDER: S-EPMC1187508 | biostudies-other | 1969 Feb
REPOSITORIES: biostudies-other
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Project description:The expression of the lac operon of E. coli is subject to positive feedback during growth in the presence of gratuitous inducers, but its existence in the presence of lactose remains controversial. The key question in this debate is: Do the lactose enzymes, Lac permease and β-galactosidase, promote accumulation of allolactose? If so, positive feedback exists since allolactose does stimulate synthesis of the lactose enzymes. Here, we addressed the above question by developing methods for determining the intracellular allolactose concentration as well as the kinetics of enzyme induction and dilution. We show that, during lac induction in the presence of lactose, the intracellular allolactose concentration increases with the lactose enzyme level, which implies that lactose enzymes promote allolactose accumulation, and positive feedback exists. We also show that, during lac repression in the presence of lactose + glucose, the intracellular allolactose concentration decreases with the lactose enzyme levels, which suggests that, under these conditions, the positive feedback loop turns in the reverse direction. The induction and dilution rates derived from the transient data show that the positive feedback loop is reversed due to a radical shift of the steady-state induction level. This is formally identical to the mechanism driving catabolite repression in the presence of TMG + glucose.
| S-EPMC8943705 | biostudies-literature
Project description:As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation of the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutant of the gene encoding the cyclic AMP receptor protein, crp*, has been shown to disable CCR and improve the co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site-specific crp* chromosomal mutant in E. coli BL21 star (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomics further showed that glucose was routed to the C5 carbon utilization pathways to support both de novo nucleotide synthesis and energy production in the crp* mutant strain. Metabolite analyses further show that overflow metabolism contributes to the slower growth in the crp* mutant. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass.IMPORTANCEAs the need for renewable biofuel and biochemical production processes continues to grow, there is an associated need for microbial technology capable of utilizing cheap, widely available, and renewable carbon substrates. This work details the construction and characterization of the first B-lineage Escherichia coli strain with mutated cyclic AMP receptor protein, Crp*, which deregulates the carbon catabolite repression (CCR) system and enables the co-utilization of multiple sugar sources in the growth medium. In this study, we focus our analysis on glucose and xylose utilization as these two sugars are the primary components in lignocellulosic biomass hydrolysate, a promising renewable carbon feedstock for industrial bioprocesses. This strain is valuable to the field as it enables the use of mixed sugar sources in traditional fed-batch based approaches, whereas the wild-type carbon catabolite repression system leads to biphasic growth and possible buildup of non-preferential sugars, reducing process efficiency at scale.
| S-EPMC10880614 | biostudies-literature
Project description:Aminoacyl-tRNA synthetases (ARSs) join amino acids to their cognate tRNAs to initiate protein synthesis. Class II ARS possess a unique catalytic domain fold, possess active site signature sequences, and are dimers or tetramers. The dimeric class I enzymes, notably TyrRS, exhibit half-of-sites reactivity, but its mechanistic basis is unclear. In class II histidyl-tRNA synthetase (HisRS), amino acid activation occurs at different rates in the two active sites when tRNA is absent, but half-of-sites reactivity has not been observed. To investigate the mechanistic basis of the asymmetry, and explore the relationship between adenylate formation and conformational events in HisRS, a fluorescently labeled version of the enzyme was developed by conjugating 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) to a cysteine introduced at residue 212, located in the insertion domain. The binding of the substrates histidine, ATP, and 5'-O-[N-(l-histidyl)sulfamoyl]adenosine to MDCC-HisRS produced fluorescence quenches on the order of 6-15%, allowing equilibrium dissociation constants to be measured. The rates of adenylate formation measured by rapid quench and domain closure as measured by stopped-flow fluorescence were similar and asymmetric with respect to the two active sites of the dimer, indicating that conformational change may be rate-limiting for product formation. Fluorescence resonance energy transfer experiments employing differential labeling of the two monomers in the dimer suggested that rigid body rotation of the insertion domain accompanies adenylate formation. The results support an alternating site model for catalysis in HisRS that may prove to be common to other class II aminoacyl-tRNA synthetases.
| S-EPMC2743188 | biostudies-literature
Project description:The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to cAMP receptor protein-mediated inhibition of lac transcription and EIIAGlc-mediated inhibition of lactose transport (inducer exclusion). The growing evidence against the first mechanism has led to the postulate that the repression is driven by inducer exclusion. Although inducer exclusion reduces the permease activity only 2-fold in fully induced cells, it could be more potent in partially induced cells. Here, we show that even in partially induced cells, inducer exclusion reduces the permease activity no more than 6-fold. Moreover, the repression is so small because these experiments are performed in the presence of chloramphenicol. Indeed, when glucose is added to a culture growing on glycerol and TMG, but no chloramphenicol, lac expression is repressed 900-fold. This repression is primarily due to reversal of the positive feedback loop, i.e., the decline of the intracellular TMG level leads to a lower permease level, which reduces the intracellular TMG level even further. The repression in the absence of chloramphenicol is therefore primarily due to positive feedback, which does not exist during measurements of inducer exclusion.
| S-EPMC8943701 | biostudies-literature
Project description:Highlights • E. coli was engineered to produce fumaric acid from glucose under aerobic conditions.• Fermentation-deficient strain with modified glucose transport was used as a chassis.• Novel design for biosynthesis of the product via modified TCA cycle was implemented.• Artificial bypass of 2-ketoglutarate-succinate semialdehyde-succinate was constructed.• Yield of 0.86 mol/mol, amounting to 86% of theoretical maximum, was achieved. Escherichia coli was engineered for efficient aerobic conversion of glucose to fumaric acid. A novel design for biosynthesis of the target product through the modified TCA cycle rather than via glyoxylate shunt, implying oxaloacetate formation from pyruvate and artificial channelling of 2-ketoglutarate towards succinic acid via succinate semialdehyde formation, was implemented. The main fumarases were inactivated in the core strain MSG1.0 (∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PL-glk, Ptac-galP) by the deletion of the fumA, fumB, and fumC genes. The Bacillus subtilis pycA gene was expressed in the strain to ensure pyruvate to oxaloacetate conversion. The Mycobacterium tuberculosis kgd gene was expressed to enable succinate semialdehyde formation. The resulting strain was able to convert glucose to fumaric acid with a yield of 0.86 mol/mol, amounting to 86% of the theoretical maximum. The results demonstrated the high potential of the implemented strategy for development of efficient strains for bio-based fumaric acid production.
| S-EPMC8801760 | biostudies-literature
Project description:The enzyme AHAS (acetohydroxy acid synthase), which is involved in the biosynthesis of valine, leucine and isoleucine, is the target of several classes of herbicides. A model of tobacco AHAS was generated based on the X-ray structure of yeast AHAS. Well conserved residues at the herbicide-binding site were identified, and the roles of three of these residues (Phe-205, Val-570 and Phe-577) were determined by site-directed mutagenesis. The Phe-205 mutants F205A, F205H, F205W and F205Y showed markedly decreased levels of catalytic efficiency, and cross-resistance to two or three classes of herbicides, i.e. Londax (a sulphonylurea herbicide), Cadre (an imidazolinone herbicide) and TP (a triazolopyrimidine derivative). None of the mutations caused significant changes in the secondary or tertiary structure of the enzyme. Four mutants of Phe-577, i.e. F577D, F577E, F577K and F577R, showed unaltered V(max) values, but substantially decreased catalytic efficiency. However, these mutants were highly resistant to two or three of the tested herbicides. The three mutants F577D, F577E and F577R had a similar secondary structure to that of wild-type AHAS. Conservative mutations of Phe-577, i.e. F577W and F577Y, did not affect the kinetic properties of the enzyme or its inhibition by herbicides. The mutation Val-570 to Asn abolished the binding affinity of the enzyme for FAD as well as its activity, and also caused a change in the tertiary structure of AHAS. However, the mutant V570Q was active, but resistant to two classes of herbicides, i.e. Londax and TP. The conservative mutant V570I was substantially reduced in catalytic efficiency and moderately resistant to the three herbicides. The results of this study suggest that residues Phe-205, Val-570 and Phe-577 in tobacco AHAS are located at or near the binding site that is common for the three classes of herbicides. In addition, Phe-205 and Val-570 are probably located at the herbicide-binding site that may overlap partially with the active site. Selected mutants of Phe-577 are expected to be utilized to construct herbicide-resistant transgenic plants.
| S-EPMC1134043 | biostudies-literature
Project description:LC MS/MS analysis (Fred Hutchinson Cancer Research Center, Seattle, Washington)
2021-12-20 | MSV000088593 | MassIVE
Project description:Acetohydroxy acid isomeroreductase catalyses a two-step reaction, an alkyl migration and a NADPH-dependent reduction, in the assembly of the carbon skeletons of branched-chain amino acids. Detailed investigations of acetohydroxy acid isomeroreductase aimed at elucidating the biosynthetic pathway of branched-chain amino acids and at designing new inhibitors of the enzyme having herbicidal potency have so far been conducted with the enzymes isolated from bacteria. To gain more information on a plant system, the gene encoding the mature acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) leaf chloroplasts has been used to transform Escherichia coli cells and to overexpress the enzyme. A rapid protocol is described that allows the preparation of large quantities of pure spinach chloroplast acetohydroxy acid isomeroreductase. Kinetic and structural properties of the plant enzyme expressed in Escherichia coli are compared with those reported in our previous studies on the native enzymes purified from spinach chloroplasts and with those reported for the corresponding enzymes isolated from Escherichia coli and Salmonella typhimurium. Both the plant and the bacterial enzymes obey an ordered mechanism in which NADPH binds first, followed by substrate (either 2-acetolactate or 2-aceto-2-hydroxybutyrate). Inhibition studies employing an inactive substrate analogue, 2-hydroxy-2-methyl-3-oxopentanoate, showed, however, that the binding of 2-hydroxy-2-methyl-3-oxopentanoate and NADPH occurs randomly, suggestive of some flexibility of the plant enzyme active site. The observed preference of the enzyme for 2-aceto-2-hydroxybutyrate over 2-acetolactate is discussed with regard to the contribution of acetohydroxy acid isomeroreductase activity in the partitioning between isoleucine and valine biosyntheses. Moreover, the kinetic properties of the chloroplast enzyme support the notion that biosynthesis of branched-chain amino acids in plants is controlled by light. As judged by analytical-ultracentrifugation and gel-filtration analyses the overexpressed plant enzyme is a dimer of identical subunits.
| S-EPMC1131967 | biostudies-other
Project description:How microbes dynamically coordinate uptake and simultaneous utilization of nutrients in complex nutritional ecosystems is still an open question. Here, we develop a constraint-based modeling approach that exploits non-targeted exo-metabolomics data to unravel adaptive decision-making processes in dynamic nutritional environments. We thereby investigate metabolic adaptation of Escherichia coli to continuously changing conditions during batch growth in complex medium. Unexpectedly, model-based analysis of time resolved exo-metabolome data revealed that fastest growth coincides with preferred catabolism of amino acids, which, in turn, reduces glucose uptake and increases acetate overflow. We show that high intracellular levels of the amino acid degradation metabolites pyruvate and oxaloacetate can directly inhibit the phosphotransferase system (PTS), and reveal their functional role in mediating regulatory decisions for uptake and catabolism of alternative carbon sources. Overall, the proposed methodology expands the spectrum of possible applications of flux balance analysis to decipher metabolic adaptation mechanisms in naturally occurring habitats and diverse organisms.
| S-EPMC6659692 | biostudies-literature
Project description:Background4-Hydroxymandelic acid (4-HMA) is a valuable aromatic fine chemical and widely used for production of pharmaceuticals and food additives. 4-HMA is conventionally synthesized by chemical condensation of glyoxylic acid with excessive phenol, and the process is environmentally unfriendly. Microbial cell factory would be an attractive approach for 4-HMA production from renewable and sustainable resources.ResultsIn this study, a biosynthetic pathway for 4-HMA production was constructed by heterologously expressing the fully synthetic 4-hydroxymandelic acid synthase (shmaS) in our L-tyrosine-overproducing Escherichia coli BKT5. The expression level of shmaS was optimized to improve 4-HMA production by fine tuning of four promoters of different strength combined with three plasmids of different copy number. Furthermore, two genes aspC and tyrB in the competitive pathway were deleted to block the formation of byproduct to enhance 4-HMA biosynthesis. The final engineered E. coli strain HMA15 utilized glucose and xylose simultaneously and produced 15.8 g/L of 4-HMA by fed-batch fermentation in 60 h.ConclusionsMetabolically engineered E. coli strain for 4-HMA production was designed and constructed, and efficiently co-fermented glucose and xylose, the major components in the hydrolysate mixture of agricultural biomass. Our research provided a promising biomanufacturing route to produce 4-HMA from lignocellulosic biomass.
| S-EPMC4884394 | biostudies-literature