Integrated laboratory evolution and rational engineering of GalP/Glk-dependent Escherichia coli for higher yield and productivity of L-tryptophan biosynthesis

ABSTRACT In comparison with intensive studies of genetic mechanisms related to biological evolutionary systems, much less analysis has been conducted on metabolic network responses to adaptive evolution that are directly associated with evolved metabolic phenotypes. Metabolic mechanisms involved in laboratory evolution of Escherichia coli on gluconeogenic carbon sources, such as lactate, were studied based on intracellular flux states determined from 13C tracer experiments and 13C-constrained flux analysis. At the end point of laboratory evolution, strains exhibited a more than doubling of the average growth rate and a 50% increase in the average biomass yield. Despite different evolutionary trajectories among parallel evolved populations, most improvements were obtained within the first 250 generations of evolution and were generally characterized by a significant increase in pathway capacity. Partitioning between gluconeogenic and pyruvate catabolic flux at the pyruvate node remained almost unchanged, while flux distributions around the key metabolites phosphoenolpyruvate, oxaloacetate, and acetyl-coenzyme A were relatively flexible over the course of evolution on lactate to meet energetic and anabolic demands during rapid growth on this gluconeogenic carbon substrate. There were no clear qualitative correlations between most transcriptional expression and metabolic flux changes, suggesting complex regulatory mechanisms at multiple levels of genetics and molecular biology. Moreover, higher fitness gains for cell growth on both evolutionary and alternative carbon sources were found for strains that adaptively evolved on gluconeogenic carbon sources compared to those that evolved on glucose. These results provide a novel systematic view of the mechanisms underlying microbial adaptation to growth on a gluconeogenic substrate.

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Amplification of the tryptophan operon gene in Escherichia coli chromosome to increase l-tryptophan biosynthesis

Applied Microbiology and Biotechnology ◽

10.1007/bf00170384 ◽

1993 ◽

Vol 40 (2-3) ◽

Cited By ~ 13

Author(s):

Err-Cheng Chan ◽

Hsiu-Lan Tsai ◽

Shih-Lin Chen ◽

Due-Gang Mou

Keyword(s):

Escherichia Coli ◽

Tryptophan Biosynthesis ◽

Tryptophan Operon ◽

Operon Gene

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Aromatic Acid Metabolites of Escherichia coli K-12 Can Induce the marRAB Operon

Journal of Bacteriology ◽

10.1128/jb.00371-10 ◽

2010 ◽

Vol 192 (18) ◽

pp. 4786-4789 ◽

Cited By ~ 29

Author(s):

Lon M. Chubiz ◽

Christopher V. Rao

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Stress Tolerance ◽

Aromatic Acid ◽

Tryptophan Biosynthesis ◽

Metabolic Intermediates ◽

Acid Metabolites ◽

K 12 ◽

Solvent Stress

ABSTRACT MarR is a key regulator of the marRAB operon involved in antibiotic resistance and solvent stress tolerance in Escherichia coli. We show that two metabolic intermediates, 2,3-dihydroxybenzoate and anthranilate, involved in enterobactin and tryptophan biosynthesis, respectively, can activate marRAB transcription. We also found that a third intermediate involved in ubiquinone biosynthesis, 4-hydroxybenzoate, activates marRAB transcription in the absence of TolC. Of the three, however, only 2,3-dihydroxybenzoate directly binds MarR and affects its activity.

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Use of Adaptive Laboratory Evolution To Discover Key Mutations Enabling Rapid Growth of Escherichia coli K-12 MG1655 on Glucose Minimal Medium

Applied and Environmental Microbiology ◽

10.1128/aem.02246-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 17-30 ◽

Cited By ~ 138

Author(s):

Ryan A. LaCroix ◽

Troy E. Sandberg ◽

Edward J. O'Brien ◽

Jose Utrilla ◽

Ali Ebrahim ◽

...

Keyword(s):

Escherichia Coli ◽

Rapid Growth ◽

Scientific Discovery ◽

Maximum Growth ◽

Time Frame ◽

Adaptive Laboratory Evolution ◽

Genetic Changes ◽

Laboratory Evolution ◽

Content Type ◽

K 12

ABSTRACTAdaptive laboratory evolution (ALE) has emerged as an effective tool for scientific discovery and addressing biotechnological needs. Much of ALE's utility is derived from reproducibly obtained fitness increases. Identifying causal genetic changes and their combinatorial effects is challenging and time-consuming. Understanding how these genetic changes enable increased fitness can be difficult. A series of approaches that address these challenges was developed and demonstrated usingEscherichia coliK-12 MG1655 on glucose minimal media at 37°C. By keepingE. coliin constant substrate excess and exponential growth, fitness increases up to 1.6-fold were obtained compared to the wild type. These increases are comparable to previously reported maximum growth rates in similar conditions but were obtained over a shorter time frame. Across the eight replicate ALE experiments performed, causal mutations were identified using three approaches: identifying mutations in the same gene/region across replicate experiments, sequencing strains before and after computationally determined fitness jumps, and allelic replacement coupled with targeted ALE of reconstructed strains. Three genetic regions were most often mutated: the global transcription generpoB, an 82-bp deletion between the metabolicpyrEgene andrph, and an IS element between the DNA structural genehnsandtdk. Model-derived classification of gene expression revealed a number of processes important for increased growth that were missed using a gene classification system alone. The methods described here represent a powerful combination of technologies to increase the speed and efficiency of ALE studies. The identified mutations can be examined as genetic parts for increasing growth rate in a desired strain and for understanding rapid growth phenotypes.

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Adaptive laboratory evolution for improved tolerance of isobutyl acetate in Escherichia coli

Metabolic Engineering ◽

10.1016/j.ymben.2021.11.002 ◽

2021 ◽

Author(s):

Morgan M. Matson ◽

Mateo M. Cepeda ◽

Angela Zhang ◽

Anna E. Case ◽

Erol S. Kavvas ◽

...

Keyword(s):

Escherichia Coli ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

Isobutyl Acetate

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Laboratory evolution of Escherichia coli enables life based on fluorinated amino acids

10.1101/665950 ◽

2019 ◽

Author(s):

Federica Agostini ◽

Ludwig Sinn ◽

Daniel Petras ◽

Christian J. Schipp ◽

Vladimir Kubyshkin ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Quality Control ◽

Protein Folding ◽

Regulatory Networks ◽

Membrane Integrity ◽

Genetic Mutations ◽

Laboratory Evolution ◽

Fluorinated Amino Acids ◽

Large Rearrangements

AbstractOrganofluorine compounds are toxic to various living beings in different habitats. On the other hand, fluorine incorporation into single proteins via related amino acid analogues has become common practice in protein engineering. Thus, an essential question remains: can fluorinated amino acids generally be used as xeno-nutrients to build up biomass, or do large amounts of fluorine in the cells render them nonviable? To gain information about the effect of long-term exposure of a cellular proteome to fluorinated organic compounds, we constructed an experiment based on bacterial adaptation in artificial fluorinated habitats. We propagated Escherichia coli (E. coli) in the presence of either 4- or 5-fluoroindole as essential precursors for the in situ synthesis of tryptophan (Trp) analogues. We found that full adaptation requires astonishingly few genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity and quality control of protein folding. These findings highlight the cellular mechanisms of the evolutionary adaption process to unnatural amino acids and provide the molecular foundation for novel and innovative bioengineering of microbial strains with potential for biotechnological applications.One Sentence SummaryLaboratory evolution enabled for the first time Escherichia coli to use fluorinated indoles as essential precursors for protein synthesis by introducing few genetic mutations but large rearrangements in regulatory networks, membrane integrity and quality control of protein folding.

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Multiple optimal phenotypes overcome redox and glycolytic intermediate metabolite imbalances inEscherichia coli pgiknockout evolutions

10.1101/342709 ◽

2018 ◽

Cited By ~ 1

Author(s):

Douglas McCloskey ◽

Sibei Xu ◽

Troy E. Sandberg ◽

Elizabeth Brunk ◽

Ying Hefner ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Product ◽

Major Gene ◽

Regulatory Function ◽

Second Step ◽

Valuable Insight ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

K 12 ◽

Mg1655 Strain

AbstractA mechanistic understanding of how new phenotypes develop to overcome the loss of a gene product provides valuable insight on both the metabolic and regulatory function of the lost gene. Thepgigene, whose product catalyzes the second step in glycolysis, was deleted in a growth optimizedEscherichia coliK-12 MG1655 strain. The knock-out (KO) strain exhibited an 80% drop in growth rate, that was largely recovered in eight replicate, but phenotypically distinct, cultures after undergoing adaptive laboratory evolution (ALE). Multi omic data sets showed that the loss ofpgisubstantially shifted pathway usage leading to a redox and sugar phosphate stress response. These stress responses were overcome by unique combinations of innovative mutations selected for by ALE. Thus, we show the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after loss of a major gene product.ImportanceA mechanistic understanding of how new phenotypes develop to overcome the loss of a gene product provides valuable insight on both the metabolic and regulatory function of the lost gene. Thepgigene, whose product catalyzes the second step in glycolysis, was deleted in a growth optimizedEscherichia coliK-12 MG1655 strain. Eight replicate adaptive laboratory evolution (ALE) resulted in eight phenotypically distinct endpoints that were able to overcome the gene loss. Utilizing multi-omics analysis, we show the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after loss of a major gene product.

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