Systematic molecular evolution enables robust biomolecule discovery

DeBenedictis, Erik P.; Chory, Emma J.; Gretton, Dana W; Wang, Brian; Golas, Stefan; Esvelt, Kevin M.

doi:10.1038/s41592-021-01348-4

Cited by 43 publications

(54 citation statements)

References 51 publications

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“…Antibiotic selection markers have previously been used to identify functional qtRNAs ( Magliery et al, 2001 ). We applied an equivalent approach based on the use of an M13 bacteriophage tail fiber pIII as a selection marker ( DeBenedictis et al, 2022 ). In this selection scheme, a qtRNA is encoded on the genome of a ΔpIII M13 bacteriophage.…”

Section: Resultsmentioning

confidence: 99%

“…Many qtRNAs were quite inefficient in translation: the presence of a single quadruplet codon in an mRNA transcript can reduce total protein yield to less than 3% relative to an all-triplet mRNA ( Figure 2 ). Mutations at the anticodon loop sides of the qtRNAs have been observed to improve translation efficiency for TAGA-qtRNAs ( DeBenedictis et al, 2021 ; DeBenedictis et al, 2022 ; Niu et al, 2013 ). Triplet tRNAs are known to exhibit patterns that relate the bases in the anticodon loop sides to the bases in the anticodon itself ( Yarus, 1982 ), and similarly benefit from anticodon loop side mutations after anticodon replacement ( Kleina et al, 1990 ; Raftery and Yarus, 1987 ; Cervettini et al, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

Section: Evolution Of Qtrnas Through Point Insertionsmentioning

confidence: 99%

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Measuring the tolerance of the genetic code to altered codon size

DeBenedictis

Söll

Esvelt

2022

eLife

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Translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the Escherichia coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs). Many of these selectively incorporate a single amino acid in response to a specified four-base codon, as confirmed with mass spectrometry. However, efficient quadruplet codon translation often requires multiple tRNA mutations. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These may constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results suggest that the triplet codon code was selected because it is simpler and sufficient, not because a quadruplet codon code is unachievable. These data provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Evolution Of Qtrnas Through Point Insertionsmentioning

confidence: 99%

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Measuring the tolerance of the genetic code to altered codon size

DeBenedictis

Söll

Esvelt

2022

eLife

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“…Among these methods, eMAGE was derived from MAGE, which was originally implemented in E. coli [ 74 ]. In this context, procaryotic organisms derived methods, such as phage-assisted continuous evolution (PACE) [ 75 ], phage- and robotics-assisted near-continuous evolution (PRANCE) [ 76 ], and automated continuous evolution (ACE) [ 77 ] may also have the potential to be ported over to yeast and expand the toolkit for the genome evolution of yeast in the future.…”

Section: Discussionmentioning

confidence: 99%

Recent Advances in Directed Yeast Genome Evolution

Yao

Wang

Dai

2022

JoF

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Saccharomyces cerevisiae, as a Generally Recognized as Safe (GRAS) fungus, has become one of the most widely used chassis cells for industrial applications and basic research. However, owing to its complex genetic background and intertwined metabolic networks, there are still many obstacles that need to be overcome in order to improve desired traits and to successfully link genotypes to phenotypes. In this context, genome editing and evolutionary technology have rapidly progressed over the last few decades to facilitate the rapid generation of tailor-made properties as well as for the precise determination of relevant gene targets that regulate physiological functions, including stress resistance, metabolic-pathway optimization and organismal adaptation. Directed genome evolution has emerged as a versatile tool to enable researchers to access desired traits and to study increasingly complicated phenomena. Here, the development of directed genome evolutions in S. cerevisiae is reviewed, with a focus on different techniques driving evolutionary engineering.

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“…In some sense, these selection-based methods passively harness artificial selection, as individuals with novel or enhanced functions of interest will tend to outcompete other conspecifics without requiring intervention beyond initial environmental manipulations. In combination with continuous culture devices, this approach to directing evolution can be used to achieve high throughput microbial directed evolution, “automatically” evaluating many variants without manual analysis (DeBenedictis et al, 2021; Toprak et al, 2012; Wang et al, 2021). For example, to study mechanisms of antibiotic resistance, researchers have employed morbidostats that continuously monitor the growth of evolving microbial populations and dynamically adjust antibiotic concentrations to maintain constant selection on further resistance (Toprak et al, 2012).…”

Section: Directed Evolutionmentioning

confidence: 99%

Artificial selection methods from evolutionary computing show promise for directed evolution of microbes

Lalejini

Dolson

Vostinar

et al. 2022

Preprint

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Directed microbial evolution harnesses evolutionary processes in the laboratory to construct microorganisms with enhanced or novel functional traits. Attempting to direct evolutionary processes for applied goals is fundamental to evolutionary computation, which harnesses the principles of Darwinian evolution as a general purpose search engine for solutions to challenging computational problems. Despite their overlapping approaches, artificial selection methods from evolutionary computing are not commonly applied to living systems in the laboratory. In this work, we ask if parent selection algorithms---procedures for choosing promising progenitors---from evolutionary computation might be useful for directing the evolution of microbial populations when selecting for multiple functional traits. To do so, we introduce an agent-based model of directed microbial evolution, which we used to evaluate how well three selection algorithms from evolutionary computing (tournament selection, lexicase selection, and non-dominated elite selection) performed relative to methods commonly used in the laboratory (elite and top-10% selection). We found that multi-objective selection techniques from evolutionary computing (lexicase and non-dominated elite) generally outperformed the commonly used directed evolution approaches when selecting for multiple traits of interest. Our results motivate ongoing work transferring these multi-objective selection procedures into the laboratory. Additionally, our findings suggest that more sophisticated artificial selection methods from evolutionary computation should also be evaluated for use in directed microbial evolution.

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Systematic molecular evolution enables robust biomolecule discovery

Cited by 43 publications

References 51 publications

Measuring the tolerance of the genetic code to altered codon size

Measuring the tolerance of the genetic code to altered codon size

Recent Advances in Directed Yeast Genome Evolution

Artificial selection methods from evolutionary computing show promise for directed evolution of microbes

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