scholarly journals Long-term adaptive evolution of genomically recodedEscherichia coli

2017 ◽  
Author(s):  
Timothy M. Wannier ◽  
Aditya M. Kunjapur ◽  
Daniel P. Rice ◽  
Michael J. McDonald ◽  
Michael M. Desai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genetic Code ◽  
Adaptive Evolution ◽  
Genome Engineering ◽  
Translation Termination ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Defined Media ◽  
Standard Amino Acid ◽  
Translation Apparatus

AbstractEfforts are underway to construct several recoded genomes anticipated to exhibit multi-virus resistance, enhanced non-standard amino acid (NSAA) incorporation, and capability for synthetic biocontainment. Though we succeeded in pioneering the first genomically recoded organism (Escherichia colistrain C321.ΔA), its fitness is far lower than that of its non-recoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for NSAA-linked applications requiring defined media such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.ΔA for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceed the growth rates of both the ancestral C321.ΔA and non-recoded strains, permitting use of the recoded chassis in several new contexts. We use next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstruct individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occur only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared to application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.ΔA strain.Significance StatementAfter demonstrating construction of an organism with an altered genetic code, we sought to evolve this organism for many generations to improve its fitness and learn what unique changes natural selection would bestow upon it. Although this organism initially had impaired fitness, we observed that adaptive laboratory evolution resulted in several selective mutations that corrected for insufficient translation termination and for unintended mutations that occurred when originally altering the genetic code. This work further bolsters our understanding of the pliability of the genetic code, it will help guide ongoing and future efforts seeking to recode genomes, and it results in a useful strain for non-standard amino acid incorporation in numerous contexts relevant for research and industry.

scholarly journals Adaptive evolution of genomically recodedEscherichia coli

2018 ◽  
Vol 115 (12) ◽  
pp. 3090-3095 ◽  
Author(s):  
Timothy M. Wannier ◽  
Aditya M. Kunjapur ◽  
Daniel P. Rice ◽  
Michael J. McDonald ◽  
Michael M. Desai ◽  
...  
Keyword(s):  
Adaptive Evolution ◽  
Live Cell Imaging ◽  
Protein Production ◽  
Genome Engineering ◽  
Cell Imaging ◽  
Laboratory Evolution ◽  
Defined Media ◽  
Translation Apparatus ◽  
Initial Genome ◽  
Generation Sequencing

Efforts are underway to construct several recoded genomes anticipated to exhibit multivirus resistance, enhanced nonstandard amino acid (nsAA) incorporation, and capability for synthetic biocontainment. Although our laboratory pioneered the first genomically recoded organism (Escherichia colistrain C321.∆A), its fitness is far lower than that of its nonrecoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for nsAA-linked applications requiring defined media, such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.∆A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceeded the growth rates of both the ancestral C321.∆A and nonrecoded strains. We used next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstructed individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occurred only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared with application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.∆A strain.

scholarly journals Evolving a mitigation of the stress response pathway to change the basic chemistry of life

2021 ◽  
Author(s):  
Isabella Tolle ◽  
Stefan Oehm ◽  
Michael Georg Hoesl ◽  
Christin Treiber-Kleinke ◽  
Lauri Peil ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Stress Response ◽  
Genetic Code ◽  
Protein Translation ◽  
Synthetic Analog ◽  
E Coli ◽  
Regulatory Constraints ◽  
Translation Apparatus ◽  
Canonical Amino Acid

ABSTRACTBillions of years of evolution have produced only slight variations in the standard genetic code, and the number and identity of proteinogenic amino acids have remained mostly consistent throughout all three domains of life. These observations suggest a certain rigidity of the genetic code and prompt musings as to the origin and evolution of the code. Here we conducted an adaptive laboratory evolution (ALE) to push the limits of the code restriction, by evolving Escherichia coli to fully replace tryptophan, thought to be the latest addition to the genetic code, with the analog L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). We identified an overshooting of the stress response system to be the main inhibiting factor for limiting ancestral growth upon exposure to β-(thieno[3,2-b]pyrrole ([3,2]Tp), a metabolic precursor of [3,2]Tpa, and Trp limitation. During the ALE, E. coli was able to “calm down” its stress response machinery, thereby restoring growth. In particular, the inactivation of RpoS itself, the master regulon of the general stress response, was a key event during the adaptation. Knocking out the rpoS gene in the ancestral background independent of other changes conferred growth on [3,2]Tp. Our results add additional evidence that frozen regulatory constraints rather than a rigid protein translation apparatus are Life’s gatekeepers of the canonical amino acid repertoire. This information will not only enable us to design enhanced synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo.SIGNIFICANCE STATEMENTThe (apparent) rigidity of the genetic code, as well as its universality, have long since ushered explorations into expanding the code with synthetic, new-to-nature building blocks and testing its boundaries. While nowadays even proteome-wide incorporation of synthetic amino acids has been reported on several occasions1–3, little is known about the underlying mechanisms.We here report ALE with auxotrophic E. coli that yielded successful proteome-wide replacement of Trp by its synthetic analog [3,2]Tpa accompanied with the selection for loss of RpoS4 function. Such laboratory domestication of bacteria by the acquisition of rpoS mitigation mutations is beneficial not only to overcome the stress of nutrient (Trp) starvation but also to evolve the paths to use environmental xenobiotics (e.g. [3,2]Tp) as essential nutrients for growth.We pose that regulatory constraints rather than a rigid and conserved protein translation apparatus are Life’s gatekeepers of the canonical amino acid repertoire (at least where close structural analogs are concerned). Our findings contribute a step towards understanding possible environmental causes of genetic changes and their relationship to evolution.Our evolved strain affords a platform for homogenous protein labeling with [3,2]Tpa as well as for the production of biomolecules5, which are challenging to synthesize chemically. Top-down synthetic biology will also benefit greatly from breaking through the boundaries of the frozen bacterial genetic code, as this will enable us to begin creating synthetic cells capable to utilize an expanded range of substrates essential for life.

Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code

Nature ◽  
1992 ◽  
Vol 356 (6369) ◽  
pp. 537-539 ◽  
Author(s):  
J. D. Bain ◽  
Christopher Switzer ◽  
Richard Chamberlin ◽  
Steven A. Benner
Keyword(s):  
Amino Acid ◽  
Genetic Code ◽  
Standard Amino Acid

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

2018 ◽  
Vol 84 (13) ◽  
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.

scholarly journals Engineering improved ethylene production: Leveraging systems Biology and adaptive laboratory evolution

2021 ◽  
Author(s):  
Sophie Vaud ◽  
Nicole Pearcy ◽  
Marko Hanževački ◽  
Alexander M.W. Van Hagen ◽  
Salah Abdelrazig ◽  
...  
Keyword(s):  
Systems Biology ◽  
Ethylene Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution

scholarly journals Searching for signatures of positive selection in cytochrome b gene associated with subterranean lifestyle in fast-evolving arvicolines (Arvicolinae, Cricetidae, Rodentia)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Olga V. Bondareva ◽  
Nadezhda A. Potapova ◽  
Kirill A. Konovalov ◽  
Tatyana V. Petrova ◽  
Natalia I. Abramson
Keyword(s):  
Amino Acid ◽  
Oxidative Phosphorylation ◽  
Adaptive Evolution ◽  
Complex Structure ◽  
Subterranean Rodents ◽  
Amino Acid Sequence Variation ◽  
Tertiary Protein Structure ◽  
Metabolic Performance ◽  
The Impact ◽  
Subterranean Species

Abstract Background Mitochondrial genes encode proteins involved in oxidative phosphorylation. Variations in lifestyle and ecological niche can be directly reflected in metabolic performance. Subterranean rodents represent a good model for testing hypotheses on adaptive evolution driven by important ecological shifts. Voles and lemmings of the subfamily Arvicolinae (Rodentia: Cricetidae) provide a good example for studies of adaptive radiation. This is the youngest group within the order Rodentia showing the fastest rates of diversification, including the transition to the subterranean lifestyle in several phylogenetically independent lineages. Results We evaluated the signatures of selection in the mitochondrial cytochrome b (cytB) gene in 62 Arvicolinae species characterized by either subterranean or surface-dwelling lifestyle by assessing amino acid sequence variation, exploring the functional consequences of the observed variation in the tertiary protein structure, and estimating selection pressure. Our analysis revealed that: (1) three of the convergent amino acid substitutions were found among phylogenetically distant subterranean species and (2) these substitutions may have an influence on the protein complex structure, (3) cytB showed an increased ω and evidence of relaxed selection in subterranean lineages, relative to non-subterranean, and (4) eight protein domains possess increased nonsynonymous substitutions ratio in subterranean species. Conclusions Our study provides insights into the adaptive evolution of the cytochrome b gene in the Arvicolinae subfamily and its potential implications in the molecular mechanism of adaptation. We present a framework for future characterizations of the impact of specific mutations on the function, physiology, and interactions of the mtDNA-encoded proteins involved in oxidative phosphorylation.

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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