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

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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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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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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Escherichia coli Data-Driven Strain Design Using Aggregated Adaptive Laboratory Evolution Mutational Data

ACS Synthetic Biology ◽

10.1021/acssynbio.1c00337 ◽

2021 ◽

Author(s):

Patrick V. Phaneuf ◽

Daniel C. Zielinski ◽

James T. Yurkovich ◽

Josefin Johnsen ◽

Richard Szubin ◽

...

Keyword(s):

Escherichia Coli ◽

Data Driven ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution ◽

Strain Design

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Adaptive laboratory evolution of methylotrophic Escherichia coli enables synthesis of all amino acids from methanol-derived carbon

Applied Microbiology and Biotechnology ◽

10.1007/s00253-020-11058-0 ◽

2021 ◽

Author(s):

Jie Ren Gerald Har ◽

Alec Agee ◽

R. Kyle Bennett ◽

Eleftherios T. Papoutsakis ◽

Maciek R. Antoniewicz

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution

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Benefits of a Recombination-Proficient Escherichia coli System for Adaptive Laboratory Evolution

Applied and Environmental Microbiology ◽

10.1128/aem.01850-16 ◽

2016 ◽

Vol 82 (22) ◽

pp. 6736-6747 ◽

Cited By ~ 10

Author(s):

George Peabody ◽

James Winkler ◽

Weston Fountain ◽

David A. Castro ◽

Enzo Leiva-Aravena ◽

...

Keyword(s):

Escherichia Coli ◽

Strong Support ◽

Mutational Load ◽

Adaptive Laboratory Evolution ◽

Beneficial Mutations ◽

Laboratory Evolution ◽

Content Type ◽

Sexual Recombination ◽

Simple Kinetic Model ◽

Neutral Mutations

ABSTRACTAdaptive laboratory evolution typically involves the propagation of organisms asexually to select for mutants with the desired phenotypes. However, asexual evolution is prone to competition among beneficial mutations (clonal interference) and the accumulation of hitchhiking and neutral mutations. The benefits of horizontal gene transfer toward overcoming these known disadvantages of asexual evolution were characterized in a strain ofEscherichia coliengineered for superior sexual recombination (genderless). Specifically, we experimentally validated the capacity of the genderless strain to reduce the mutational load and recombine beneficial mutations. We also confirmed that inclusion of multiple origins of transfer influences both the frequency of genetic exchange throughout the chromosome and the linkage of donor DNA. We built a simple kinetic model to estimate recombination frequency as a function of transfer size and relative genotype enrichment in batch transfers; the model output correlated well with the experimental data. Our results provide strong support for the advantages of utilizing the genderless strain over its asexual counterpart during adaptive laboratory evolution for generating beneficial mutants with reduced mutational load.IMPORTANCEOver 80 years ago Fisher and Muller began a debate on the origins of sexual recombination. Although many aspects of sexual recombination have been examined at length, experimental evidence behind the behaviors of recombination in many systems and the means to harness it remain elusive. In this study, we sought to experimentally validate some advantages of recombination in typically asexualEscherichia coliand determine if a sexual strain ofE. colican become an effective tool for strain development.

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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 Escherichia coli W enhances gamma-aminobutyric acid production using glycerol as the carbon source

Metabolic Engineering ◽

10.1016/j.ymben.2021.11.004 ◽

2021 ◽

Author(s):

Kangsan Kim ◽

Chen Yuan Hou ◽

Donghui Choe ◽

Minjeong Kang ◽

Suhyung Cho ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Acid Production ◽

Aminobutyric Acid ◽

Gamma Aminobutyric Acid ◽

Adaptive Laboratory Evolution ◽

Laboratory Evolution

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Laboratory Evolution to Alternating Substrate Environments Yields Distinct Phenotypic and Genetic Adaptive Strategies

Applied and Environmental Microbiology ◽

10.1128/aem.00410-17 ◽

2017 ◽

Vol 83 (13) ◽

Cited By ~ 39

Author(s):

Troy E. Sandberg ◽

Colton J. Lloyd ◽

Bernhard O. Palsson ◽

Adam M. Feist

Keyword(s):

Escherichia Coli ◽

Adaptive Strategies ◽

Carbon Substrate ◽

Natural Occurrence ◽

Natural Setting ◽

Data Types ◽

Adaptive Laboratory Evolution ◽

Genetic Changes ◽

Laboratory Evolution ◽

Content Type

ABSTRACT Adaptive laboratory evolution (ALE) experiments are often designed to maintain a static culturing environment to minimize confounding variables that could influence the adaptive process, but dynamic nutrient conditions occur frequently in natural and bioprocessing settings. To study the nature of carbon substrate fitness tradeoffs, we evolved batch cultures of Escherichia coli via serial propagation into tubes alternating between glucose and either xylose, glycerol, or acetate. Genome sequencing of evolved cultures revealed several genetic changes preferentially selected for under dynamic conditions and different adaptation strategies depending on the substrates being switched between; in some environments, a persistent “generalist” strain developed, while in another, two “specialist” subpopulations arose that alternated dominance. Diauxic lag phenotype varied across the generalists and specialists, in one case being completely abolished, while gene expression data distinguished the transcriptional strategies implemented by strains in pursuit of growth optimality. Genome-scale metabolic modeling techniques were then used to help explain the inherent substrate differences giving rise to the observed distinct adaptive strategies. This study gives insight into the population dynamics of adaptation in an alternating environment and into the underlying metabolic and genetic mechanisms. Furthermore, ALE-generated optimized strains have phenotypes with potential industrial bioprocessing applications. IMPORTANCE Evolution and natural selection inexorably lead to an organism's improved fitness in a given environment, whether in a laboratory or natural setting. However, despite the frequent natural occurrence of complex and dynamic growth environments, laboratory evolution experiments typically maintain simple, static culturing environments so as to reduce selection pressure complexity. In this study, we investigated the adaptive strategies underlying evolution to fluctuating environments by evolving Escherichia coli to conditions of frequently switching growth substrate. Characterization of evolved strains via a number of different data types revealed the various genetic and phenotypic changes implemented in pursuit of growth optimality and how these differed across the different growth substrates and switching protocols. This work not only helps to establish general principles of adaptation to complex environments but also suggests strategies for experimental design to achieve desired evolutionary outcomes.

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