Optimal regulatory strategies for metabolic pathways in Escherichia coli depending on protein costs.
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ABSTRACT:
SUBMITTER: Wessely F
PROVIDER: S-EPMC3159982 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMelatonin has attracted substantial attention because of its excellent prospects for both medical applications and crop improvement. The microbial production of melatonin is a safer and more promising alternative to chemical synthesis approaches. Researchers have failed to produce high yields of melatonin in common heterologous hosts due to either the insolubility or low enzyme activity of proteins encoded by gene clusters related to melatonin biosynthesis.ResultsHere, a combinatorial gene pathway for melatonin production was successfully established in Escherichia coli by combining the physostigmine biosynthetic genes from Streptomyces albulus and gene encoding phenylalanine 4-hydroxylase (P4H) from Xanthomonas campestris and caffeic acid 3-O-methyltransferase (COMT) from Oryza sativa. A threefold improvement of melatonin production was achieved by balancing the expression of heterologous proteins and adding 3% glycerol. Further protein engineering and metabolic engineering were conducted to improve the conversion of N-acetylserotonin (NAS) to melatonin. Construction of COMT variant containing C303F and V321T mutations increased the production of melatonin by fivefold. Moreover, the deletion of speD gene increased the supply of S-adenosylmethionine (SAM), an indispensable cofactor of COMT, which doubled the yield of melatonin. In the final engineered strain EcMEL8, the production of NAS and melatonin reached 879.38 ± 71.42 mg/L and 136.17 ± 1.33 mg/L in a shake flask. Finally, in a 2-L bioreactor, EcMEL8 produced 1.06 ± 0.07 g/L NAS and 0.65 ± 0.11 g/L melatonin with tryptophan supplementation.ConclusionsThis study established a novel combinatorial pathway for melatonin biosynthesis in E. coli and provided alternative strategies for improvement of melatonin production.
| S-EPMC8403405 | biostudies-literature
Project description:In response to environmental changes, the connections ("arrows") in gene regulatory networks determine which genes modulate their expression, but the quantitative parameters of the network ("the numbers on the arrows") are equally important in determining the resulting phenotype. What are the objectives and constraints by which evolution determines these parameters? We explore these issues by analyzing gene expression changes in a number of yeast metabolic pathways in response to nutrient depletion. We find that a striking pattern emerges that couples the regulatory architecture of the pathway to the gene expression response. In particular, we find that pathways controlled by the intermediate metabolite activation (IMA) architecture, in which an intermediate metabolite activates transcription of pathway genes, exhibit the following response: the enzyme immediately downstream of the regulatory metabolite is under the strongest transcriptional control, whereas the induction of the enzymes upstream of the regulatory intermediate is relatively weak. This pattern of responses is absent in pathways not controlled by an IMA architecture. The observation can be explained by the constraint imposed by the fundamental feedback structure of the network, which places downstream enzymes under a negative feedback loop and upstream ones under a positive feedback loop. This general design principle for transcriptional control of a metabolic pathway can be derived from a simple cost/benefit model of gene expression, in which the observed pattern is an optimal solution. Our results suggest that the parameters regulating metabolic enzyme expression are optimized by evolution, under the strong constraint of the underlying regulatory architecture.
| S-EPMC3324031 | biostudies-other
Project description:MotivationThe effort to build a whole-cell model requires the development of new modeling approaches, and in particular, the integration of models for different types of processes, each of which may be best described using different representation. Flux-balance analysis (FBA) has been useful for large-scale analysis of metabolic networks, and methods have been developed to incorporate transcriptional regulation (regulatory FBA, or rFBA). Of current interest is the integration of these approaches with detailed models based on ordinary differential equations (ODEs).ResultsWe developed an approach to modeling the dynamic behavior of metabolic, regulatory and signaling networks by combining FBA with regulatory Boolean logic, and ordinary differential equations. We use this approach (called integrated FBA, or iFBA) to create an integrated model of Escherichia coli which combines a flux-balance-based, central carbon metabolic and transcriptional regulatory model with an ODE-based, detailed model of carbohydrate uptake control. We compare the predicted Escherichia coli wild-type and single gene perturbation phenotypes for diauxic growth on glucose/lactose and glucose/glucose-6-phosphate with that of the individual models. We find that iFBA encapsulates the dynamics of three internal metabolites and three transporters inadequately predicted by rFBA. Furthermore, we find that iFBA predicts different and more accurate phenotypes than the ODE model for 85 of 334 single gene perturbation simulations, as well for the wild-type simulations. We conclude that iFBA is a significant improvement over the individual rFBA and ODE modeling paradigms.AvailabilityAll MATLAB files used in this study are available at http://www.simtk.org/home/ifba/.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC6702764 | biostudies-literature
Project description:In order to understand how cellular metabolism has taken its modern form, the conservation and variations between metabolic pathways were evaluated by using a genetic algorithm (GA). The GA approach considered information on the complete metabolism of the bacterium Escherichia coli K-12, as deposited in the KEGG database, and the enzymes belonging to a particular pathway were transformed into enzymatic step sequences by using the breadth-first search algorithm. These sequences represent contiguous enzymes linked to each other, based on their catalytic activities as they are encoded in the Enzyme Commission numbers. In a posterior step, these sequences were compared using a GA in an all-against-all (pairwise comparisons) approach. Individual reactions were chosen based on their measure of fitness to act as parents of offspring, which constitute the new generation. The sequences compared were used to construct a similarity matrix (of fitness values) that was then considered to be clustered by using a k-medoids algorithm. A total of 34 clusters of conserved reactions were obtained, and their sequences were finally aligned with a multiple-sequence alignment GA optimized to align all the reaction sequences included in each group or cluster. From these comparisons, maps associated with the metabolism of similar compounds also contained similar enzymatic step sequences, reinforcing the Patchwork Model for the evolution of metabolism in E. coli K-12, an observation that can be expanded to other organisms, for which there is metabolism information. Finally, our mapping of these reactions is discussed, with illustrations from a particular case.
| S-EPMC4423528 | biostudies-literature
Project description:Antibiotic resistance is often associated with metabolic costs. To investigate the metabolic consequences of antibiotic resistance, the genomic and transcriptomic profiles of an amoxicillin-resistant Escherichia coli strain and the wild type it was derived from were compared. A total of 125 amino acid substitutions and 7 mutations that were located <1,000 bp upstream of differentially expressed genes were found in resistant cells. However, broad induction and suppression of genes were observed when comparing the expression profiles of resistant and wild-type cells. Expression of genes involved in cell wall maintenance, DNA metabolic processes, cellular stress response, and respiration was most affected in resistant cells regardless of the absence or presence of amoxicillin. The SOS response was downregulated in resistant cells. The physiological effect of the acquisition of amoxicillin resistance in cells grown in chemostat cultures consisted of an initial increase in glucose consumption that was followed by an adaptation process. Furthermore, no difference in maintenance energy was observed between resistant and sensitive cells. In accordance with the transcriptomic profile, exposure of resistant cells to amoxicillin resulted in reduced salt and pH tolerance. Taken together, the results demonstrate that the acquisition of antibiotic resistance in E. coli is accompanied by specifically reorganized metabolic networks in order to circumvent metabolic costs. The overall effect of the acquisition of resistance consists not so much of an extra energy requirement, but more a reduced ecological range.
| S-EPMC3719774 | biostudies-literature
Project description:Intracellular compartmentalization of cooperating enzymes is a strategy that is frequently used by cells. Segregation of enzymes that catalyze sequential reactions can alleviate challenges such as toxic pathway intermediates, competing metabolic reactions, and slow reaction rates. Inspired by nature, synthetic biologists also seek to encapsulate engineered metabolic pathways within vesicles or proteinaceous shells to enhance the yield of industrially and pharmaceutically useful products. Although enzymatic compartments have been extensively studied experimentally, a quantitative understanding of the underlying design principles is still lacking. Here, we study theoretically how the size and enzymatic composition of compartments should be chosen so as to maximize the productivity of a model metabolic pathway. We find that maximizing productivity requires compartments larger than a certain critical size. The enzyme density within each compartment should be tuned according to a power-law scaling in the compartment size. We explain these observations using an analytically solvable, well-mixed approximation. We also investigate the qualitatively different compartmentalization strategies that emerge in parameter regimes where this approximation breaks down. Our results suggest that the different sizes and enzyme packings of α- and β-carboxysomes each constitute an optimal compartmentalization strategy given the properties of their respective protein shells.
| S-EPMC5340097 | biostudies-literature
Project description:Transcription networks consist of hundreds of transcription factors with thousands of often overlapping target genes. While we can reliably measure gene expression changes, we still understand relatively little why expression changes the way it does. How does a coordinated response emerge in such complex networks and how many input signals are necessary to achieve it? Here, we unravel the regulatory program of gene expression in Escherichia coli central carbon metabolism with more than 30 known transcription factors. Using a library of fluorescent transcriptional reporters, we comprehensively quantify the activity of central metabolic promoters in 26 environmental conditions. The expression patterns were dominated by growth rate-dependent global regulation for most central metabolic promoters in concert with highly condition-specific activation for only few promoters. Using an approximate mathematical description of promoter activity, we dissect the contribution of global and specific transcriptional regulation. About 70% of the total variance in promoter activity across conditions was explained by global transcriptional regulation. Correlating the remaining specific transcriptional regulation of each promoter with the cell's metabolome response across the same conditions identified potential regulatory metabolites. Remarkably, cyclic AMP, fructose-1,6-bisphosphate, and fructose-1-phosphate alone explained most of the specific transcriptional regulation through their interaction with the two major transcription factors Crp and Cra. Thus, a surprisingly simple regulatory program that relies on global transcriptional regulation and input from few intracellular metabolites appears to be sufficient to coordinate E. coli central metabolism and explain about 90% of the experimentally observed transcription changes in 100 genes.
| S-EPMC5293157 | biostudies-literature
Project description:The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.
| S-EPMC1932724 | biostudies-literature
Project description:During metabolic evolution to improve succinate production in Escherichia coli strains, significant changes in cellular metabolism were acquired that increased energy efficiency in two respects. The energy-conserving phosphoenolpyruvate (PEP) carboxykinase (pck), which normally functions in the reverse direction (gluconeogenesis; glucose repressed) during the oxidative metabolism of organic acids, evolved to become the major carboxylation pathway for succinate production. Both PCK enzyme activity and gene expression levels increased significantly in two stages because of several mutations during the metabolic evolution process. High-level expression of this enzyme-dominated CO(2) fixation and increased ATP yield (1 ATP per oxaloacetate). In addition, the native PEP-dependent phosphotransferase system for glucose uptake was inactivated by a mutation in ptsI. This glucose transport function was replaced by increased expression of the GalP permease (galP) and glucokinase (glk). Results of deleting individual transport genes confirmed that GalP served as the dominant glucose transporter in evolved strains. Using this alternative transport system would increase the pool of PEP available for redox balance. This change would also increase energy efficiency by eliminating the need to produce additional PEP from pyruvate, a reaction that requires two ATP equivalents. Together, these changes converted the wild-type E. coli fermentation pathway for succinate into a functional equivalent of the native pathway that nature evolved in succinate-producing rumen bacteria.
| S-EPMC2777959 | biostudies-literature
Project description:Microorganisms adjust metabolic activity to cope with diverse environments. While many studies have provided insights into how individual pathways are regulated, the mechanisms that give rise to coordinated metabolic responses are poorly understood. Here, we identify the regulatory mechanisms that coordinate catabolism and anabolism in Escherichia coli. Integrating protein, metabolite, and flux changes in genetically implemented catabolic or anabolic limitations, we show that combined global and local mechanisms coordinate the response to metabolic limitations. To allocate proteomic resources between catabolism and anabolism, E. coli uses a simple global gene regulatory program. Surprisingly, this program is largely implemented by a single transcription factor, Crp, which directly activates the expression of catabolic enzymes and indirectly reduces the expression of anabolic enzymes by passively sequestering cellular resources needed for their synthesis. However, metabolic fluxes are not controlled by this regulatory program alone; instead, fluxes are adjusted mostly through passive changes in the local metabolite concentrations. These mechanisms constitute a simple but effective global regulatory program that coarsely partitions resources between different parts of metabolism while ensuring robust coordination of individual metabolic reactions.
| S-EPMC8045939 | biostudies-literature