Activating silent glycolysis bypasses in Escherichia coli

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal, the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal immediately operated in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary for implementing a functional methylglyoxal pathway. Furthermore, we engineered the serine shunt which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection we performed an adaptive laboratory evolution study using an enolase deletion strain. The evolved mutants were shown to use the serine shunt. Our study reveals the flexible redesignation of metabolic pathways to create new metabolite links and rewire central metabolism.

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Adaptive laboratory evolution ofEscherichia coliunder acid stress

10.1101/659706 ◽

2019 ◽

Cited By ~ 1

Author(s):

Bin Du ◽

Connor A. Olson ◽

Anand V. Sastry ◽

Xin Fang ◽

Patrick V. Phaneuf ◽

...

Keyword(s):

Growth Rates ◽

Expression Profiles ◽

Acid Stress ◽

Gene Expression Profiles ◽

Laboratory Strain ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

E Coli ◽

A Genome ◽

K 12

AbstractThe ability ofEscherichia colito tolerate acid stress is important for its survival and colonization in the human digestive tract. Here, we performed adaptive laboratory evolution of the laboratory strainE. coliK-12 MG1655 at pH 5.5 in glucose minimal medium. By 800 generations, six independent populations under evolution reached 18.0% higher growth rates than their starting strain at pH 5.5, while maintaining comparable growth rates to the starting strain at pH 7. We characterized the evolved strains to find that: (1) whole genome sequencing of isolated clones from each evolved population revealed mutations inrpoCappearing in 5 of 6 sequenced clones; (2) gene expression profiles revealed different strategies to mitigate acid stress, that are related to amino acid metabolism and energy production and conversion. Thus, a combination of adaptive laboratory evolution, genome resequencing, and expression profiling reveals, on a genome-scale, the strategies thatE. colideploys to mitigate acid stress.

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EColiCore2: a reference network model of the central metabolism of Escherichia coli and relationships to its genome-scale parent model

Scientific Reports ◽

10.1038/srep39647 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 18

Author(s):

Oliver Hädicke ◽

Steffen Klamt

Keyword(s):

Escherichia Coli ◽

Reference Model ◽

Model Organism ◽

Solution Space ◽

Scale Model ◽

Reference Network ◽

Central Metabolism ◽

E Coli ◽

A Genome ◽

Genome Scale

Abstract Genome-scale metabolic modeling has become an invaluable tool to analyze properties and capabilities of metabolic networks and has been particularly successful for the model organism Escherichia coli. However, for several applications, smaller metabolic (core) models are needed. Using a recently introduced reduction algorithm and the latest E. coli genome-scale reconstruction iJO1366, we derived EColiCore2, a model of the central metabolism of E. coli. EColiCore2 is a subnetwork of iJO1366 and preserves predefined phenotypes including optimal growth on different substrates. The network comprises 486 metabolites and 499 reactions, is accessible for elementary-modes analysis and can, if required, be further compressed to a network with 82 reactions and 54 metabolites having an identical solution space as EColiCore2. A systematic comparison of EColiCore2 with its genome-scale parent model iJO1366 reveals that several key properties (flux ranges, reaction essentialities, production envelopes) of the central metabolism are preserved in EColiCore2 while it neglects redundancies along biosynthetic routes. We also compare calculated metabolic engineering strategies in both models and demonstrate, as a general result, how intervention strategies found in a core model allow the identification of valid strategies in a genome-scale model. Overall, EColiCore2 holds promise to become a reference model of E. coli’s central metabolism.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.638426 ◽

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.

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Adaptive evolution reveals a tradeoff between growth rate and oxidative stress during naphthoquinone-based aerobic respiration

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909987116 ◽

2019 ◽

Vol 116 (50) ◽

pp. 25287-25292 ◽

Cited By ~ 15

Author(s):

Amitesh Anand ◽

Ke Chen ◽

Laurence Yang ◽

Anand V. Sastry ◽

Connor A. Olson ◽

...

Keyword(s):

Growth Rate ◽

Resource Allocation ◽

Bacterial Species ◽

Scale Model ◽

Adaptive Laboratory Evolution ◽

Lower Growth ◽

Respiratory Quinone ◽

A Genome ◽

Clear Shift ◽

Genome Scale

Evolution fine-tunes biological pathways to achieve a robust cellular physiology. Two and a half billion years ago, rapidly rising levels of oxygen as a byproduct of blooming cyanobacterial photosynthesis resulted in a redox upshift in microbial energetics. The appearance of higher-redox-potential respiratory quinone, ubiquinone (UQ), is believed to be an adaptive response to this environmental transition. However, the majority of bacterial species are still dependent on the ancient respiratory quinone, naphthoquinone (NQ). Gammaproteobacteria can biosynthesize both of these respiratory quinones, where UQ has been associated with aerobic lifestyle and NQ with anaerobic lifestyle. We engineered an obligate NQ-dependent γ-proteobacterium, Escherichia coli ΔubiC, and performed adaptive laboratory evolution to understand the selection against the use of NQ in an oxic environment and also the adaptation required to support the NQ-driven aerobic electron transport chain. A comparative systems-level analysis of pre- and postevolved NQ-dependent strains revealed a clear shift from fermentative to oxidative metabolism enabled by higher periplasmic superoxide defense. This metabolic shift was driven by the concerted activity of 3 transcriptional regulators (PdhR, RpoS, and Fur). Analysis of these findings using a genome-scale model suggested that resource allocation to reactive oxygen species (ROS) mitigation results in lower growth rates. These results provide a direct elucidation of a resource allocation tradeoff between growth rate and ROS mitigation costs associated with NQ usage under oxygen-replete condition.

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Novel Mode Engineering for β-Alanine Production in Escherichia coli with the Guide of Adaptive Laboratory Evolution

Microorganisms ◽

10.3390/microorganisms9030600 ◽

2021 ◽

Vol 9 (3) ◽

pp. 600

Author(s):

Jian Xu ◽

Li Zhou ◽

Meng Yin ◽

Zhemin Zhou

Keyword(s):

Escherichia Coli ◽

Anaerobic Condition ◽

Energy Production ◽

Phosphoenolpyruvate Carboxykinase ◽

Production Efficiency ◽

Genetic Mutations ◽

Cell Factory ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

E Coli

The strategy of anaerobic biosynthesis of β-alanine by Escherichia coli (E. coli) has been reported. However, the low energy production under anaerobic condition limited cell growth and then affected the production efficiency of β-alanine. Here, the adaptive laboratory evolution was carried out to improve energy production of E. coli lacking phosphoenolpyruvate carboxylase under anaerobic condition. Five mutants were isolated and analyzed. Sequence analysis showed that most of the consistent genetic mutations among the mutants were related with pyruvate accumulation, indicating that pyruvate accumulation enabled the growth of the lethal parent. It is possible that the accumulated pyruvate provides sufficient precursors for energy generation and CO2 fixing reaction catalyzed by phosphoenolpyruvate carboxykinase. B0016-100BB (B0016-090BB, recE::FRT, mhpF::FRT, ykgF::FRT, mhpB:: mhpB *, mhpD:: mhpD *, rcsA:: rcsA *) was engineered based on the analysis of the genetic mutations among the mutants for the biosynthesis of β-alanine. Along with the recruitment of glycerol as the sole carbon source, 1.07 g/L β-alanine was generated by B0016-200BB (B0016-100BB, aspA::FRT) harboring pET24a-panD-AspDH, which was used for overexpression of two key enzymes in β-alanine fermentation process. Compared with the starting strain, which can hardly generate β-alanine under anaerobic condition, the production efficiency of β-alanine of the engineered cell factory was significantly improved.

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Adaptive laboratory evolution of a genome-reduced Escherichia coli

Nature Communications ◽

10.1038/s41467-019-08888-6 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 38

Author(s):

Donghui Choe ◽

Jun Hyoung Lee ◽

Minseob Yoo ◽

Soonkyu Hwang ◽

Bong Hyun Sung ◽

...

Keyword(s):

Escherichia Coli ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

A Genome

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Mutant Variants of the Substrate-Binding Protein DppA fromEscherichia coliEnhance Growth on Nonstandard γ-Glutamyl Amide-Containing Peptides

Applied and Environmental Microbiology ◽

10.1128/aem.00340-18 ◽

2018 ◽

Vol 84 (13) ◽

Cited By ~ 2

Author(s):

Tilmann Kuenzl ◽

Xiaochun Li-Blatter ◽

Puneet Srivastava ◽

Piet Herdewijn ◽

Timothy Sharpe ◽

...

Keyword(s):

Amino Acid ◽

Binding Protein ◽

Substrate Binding ◽

Building Blocks ◽

Side Chains ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

Wide Range ◽

Amino Acid Side Chains ◽

Substrate Binding Protein

ABSTRACTThe import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) ofEscherichia coliis an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA.In vitrocharacterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides.IMPORTANCEFundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.

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Adaptive laboratory evolution triggers pathogen-dependent broad-spectrum antimicrobial potency in Streptomyces

10.1101/2021.01.20.427378 ◽

2021 ◽

Author(s):

Dharmesh Harwani ◽

Jyotsna Begani ◽

Sweta Barupal ◽

Jyoti Lakhani

Keyword(s):

Antimicrobial Activity ◽

Antibiotic Production ◽

Multidrug Resistant ◽

Molecular Evidence ◽

Evolutionary Engineering ◽

Wild Type ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

E Coli ◽

Mutant Phenotypes

AbstractIn the present study, adaptive laboratory evolution was used to stimulate antibiotic production in a weak antibiotic-producing Streptomyces strain JB140. The seven different competition experiments utilized three serial passages (three cycles of adaptation-selection of 15 days each) of a weak antibiotic-producing Streptomyces strain (wild-type) against one (biculture) or two (triculture) or three (quadriculture) target pathogens. This resulted in the evolution of a weak antibiotic-producing strain into the seven unique mutant phenotypes that acquired the ability to constitutively exhibit increased antimicrobial activity against bacterial pathogens. The mutant not only effectively inhibited the growth of the tested pathogens but also observed to produce antimicrobial against multidrug-resistant (MDR) E. coli. Intriguingly, the highest antimicrobial activity was registered with the Streptomyces mutants that were adaptively evolved against the three pathogens (quadriculture competition). In contrast to the adaptively evolved mutants, a weak antimicrobial activity was detected in the un-evolved, wild-type Streptomyces. To get molecular evidence of evolution, RAPD profiles of the wild-type Streptomyces and its evolved mutants were compared that revealed significant polymorphism among them. These results demonstrated that competition-based adaptive laboratory evolution method can constitute a platform for evolutionary engineering to select improved phenotypes (mutants) with increased production of antibiotics against targeted pathogens.

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Generation of an E. coli platform strain for improved sucrose utilization using adaptive laboratory evolution

Microbial Cell Factories ◽

10.1186/s12934-019-1165-2 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 5

Author(s):

Elsayed T. Mohamed ◽

Hemanshu Mundhada ◽

Jenny Landberg ◽

Isaac Cann ◽

Roderick I. Mackie ◽

...

Keyword(s):

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

E Coli ◽

Sucrose Utilization

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Improving Designer Glycan Production inEscherichia colithrough Model-Guided Metabolic Engineering

10.1101/160853 ◽

2017 ◽

Cited By ~ 2

Author(s):

Joseph A. Wayman ◽

Cameron Glasscock ◽

Thomas J. Mansell ◽

Matthew P. DeLisa ◽

Jeffrey D. Varner

Keyword(s):

Protein Modification ◽

Gene Knockout ◽

Optimal Growth ◽

Fluorescent Labeling ◽

Protein Glycosylation ◽

Scale Model ◽

E Coli ◽

A Genome ◽

Glycosylation Pathway ◽

Genome Scale

AbstractAsparagine-linked (N-linked) glycosylation is the most common protein modification in eukaryotes, affecting over two-thirds of the proteome. Glycosylation is also critical to the pharmacokinetic activity and immunogenicity of many therapeutic proteins currently produced in complex eukaryotic hosts. The discovery of a protein glycosylation pathway in the pathogenCampylobacter jejuniand its subsequent transfer into laboratory strains ofEscherichia colihas spurred great interest in glycoprotein production in prokaryotes. However, prokaryotic glycoprotein production has several drawbacks, including insufficient availability of non-native glycan precursors. To address this limitation, we used a constraint-based model ofE. colimetabolism in combination with heuristic optimization to design gene knockout strains that overproduced glycan precursors. First, we incorporated reactions associated withC. jejuniglycan assembly into a genome-scale model ofE. colimetabolism. We then identified gene knockout strains that coupled optimal growth to glycan synthesis. Simulations suggested that these growth-coupled glycan overproducing strains had metabolic imbalances that rerouted flux toward glycan precursor synthesis. We then validated the model-identified knockout strains experimentally by measuring glycan expression using a flow cytometric-based assay involving fluorescent labeling of cell surface-displayed glycans. Overall, this study demonstrates the promising role that metabolic modeling can play in optimizing the performance of a next-generation microbial glycosylation platform.

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