scholarly journals Refactoring the Genetic Code for Increased Evolvability

2017 ◽  
Author(s):  
Gur Pines ◽  
James D. Winkler ◽  
Assaf Pines ◽  
Ryan T. Gill
Keyword(s):  
Genetic Code ◽  
Directed Evolution ◽  
Single Gene ◽  
Point Mutations ◽  
Saturation Mutagenesis ◽  
Mutagenic Potential ◽  
Single Nucleotide ◽  
Common Error ◽  
Mutational Landscape ◽  
Genetic Codes

AbstractThe standard genetic code is robust to mutations and base-pairing errors during transcription and translation. Point mutations are most likely to be synonymous or preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best performing mutant requires a replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyper-evolvable organism serving as an ideal platform for directed evolution experiments. We then conclude by evaluating potential applications for recoded organisms within the synthetic biology field.Significance StatementThe conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene using common error-prone methods. Here we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transition to a larger pool of amino acids and with a greater degree of chemical differences, using a single nucleotide replacement within the codon, thus increasing evolvability both at the single gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation and fitness maximization.

scholarly journals Refactoring the Genetic Code for Increased Evolvability

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Gur Pines ◽  
James D. Winkler ◽  
Assaf Pines ◽  
Ryan T. Gill
Keyword(s):  
Genetic Code ◽  
Directed Evolution ◽  
Evolutionary Biology ◽  
Single Gene ◽  
Single Nucleotide ◽  
Common Error ◽  
Computational Approaches ◽  
Mutational Landscape ◽  
Genetic Codes ◽  
New Codes

ABSTRACT The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyperevolvable organism that could serve as an ideal platform for directed evolution experiments. We then conclude by evaluating the challenges of constructing such recoded organisms and their potential applications within the field of synthetic biology. IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization. IMPORTANCE The conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene via common error-prone methods. Here, we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transitions to a larger pool of amino acids and with a greater extent of chemical differences, based on a single nucleotide replacement within the codon, thus increasing evolvability both at the single-gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement, along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation, and fitness maximization.

scholarly journals Basic principles of the genetic code extension

2019 ◽  
Author(s):  
Paweł Błażej ◽  
Małgorzata Wnetrzak ◽  
Dorota Mackiewicz ◽  
Paweł Mackiewicz
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Point Mutations ◽  
Coding System ◽  
Base Pairs ◽  
Induced Subgraphs ◽  
Single Nucleotide ◽  
Basic Principles ◽  
Code Extension ◽  
Incremental Addition

AbstractCompounds including non-canonical amino acids or other artificially designed molecules can find a lot of applications in medicine, industry and biotechnology. They can be produced thanks to the modification or extension of the standard genetic code (SGC). Such peptides or proteins including the non-canonical amino acids can be constantly delivered in a stable way by organisms with the customized genetic code. Among several methods of engineering the code, using non-canonical base pairs is especially promising, because it enables generating many new codons, which can be used to encode any new amino acid. Since even one pair of new bases can extend the SGC up to 216 codons generated by six-letter nucleotide alphabet, the extension of the SGC can be achieved in many ways. Here, we proposed a stepwise procedure of the SGC extension with one pair of non-canonical bases to minimize the consequences of point mutations. We reported relationships between codons in the framework of graph theory. All 216 codons were represented as nodes of the graph, whereas its edges were induced by all possible single nucleotide mutations occurring between codons. Therefore, every set of canonical and newly added codons induces a specific subgraph. We characterized the properties of the induced subgraphs generated by selected sets of codons. Thanks to that, we were able to describe a procedure for incremental addition of the set of meaningful codons up to the full coding system consisting of three pairs of bases. The procedure of gradual extension of the SGC makes the whole system robust to changing genetic information due to mutations and is compatible with the views assuming that codons and amino acids were added successively to the primordial SGC, which evolved to minimize harmful consequences of mutations or mistranslations of encoded proteins.

scholarly journals Computational Analysis of Genetic Code Variations Optimized for the Robustness against Point Mutations with Wobble-like Effects

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1338
Author(s):  
Elena Fimmel ◽  
Markus Gumbel ◽  
Martin Starman ◽  
Lutz Strüngmann
Keyword(s):  
Genetic Code ◽  
Computational Analysis ◽  
Point Mutations ◽  
Weighted Graph ◽  
Single Nucleotide Variants ◽  
Negative Effects ◽  
Standard Genetic Code ◽  
Single Nucleotide ◽  
Random Code ◽  
Optimal Weights

It is believed that the codon–amino acid assignments of the standard genetic code (SGC) help to minimize the negative effects caused by point mutations. All possible point mutations of the genetic code can be represented as a weighted graph with weights that correspond to the probabilities of these mutations. The robustness of a code against point mutations can be described then by means of the so-called conductance measure. This paper quantifies the wobble effect, which was investigated previously by applying the weighted graph approach, and seeks optimal weights using an evolutionary optimization algorithm to maximize the code’s robustness. One result of our study is that the robustness of the genetic code is least influenced by mutations in the third position—like with the wobble effect. Moreover, the results clearly demonstrate that point mutations in the first, and even more importantly, in the second base of a codon have a very large influence on the robustness of the genetic code. These results were compared to single nucleotide variants (SNV) in coding sequences which support our findings. Additionally, it was analyzed which structure of a genetic code evolves from random code tables when the robustness is maximized. Our calculations show that the resulting code tables are very close to the standard genetic code. In conclusion, the results illustrate that the robustness against point mutations seems to be an important factor in the evolution of the standard genetic code.

scholarly journals Visualizing Amino Acid Substitutions in a Physicochemical Vector Space

2021 ◽  
Author(s):  
Louis R Nemzer
Keyword(s):  
Amino Acid ◽  
Genetic Code ◽  
Three Dimensional ◽  
Point Mutations ◽  
Amino Acid Substitutions ◽  
Standard Genetic Code ◽  
Single Nucleotide ◽  
Single Nucleotide Mutation ◽  
Nucleotide Mutation ◽  
Hereditary Disorders

A three-dimensional representation of the twenty proteinogenic amino acids in a physicochemical space is presented. Vectors corresponding to amino acid substitutions are classified based on whether they are accessible via a single-nucleotide mutation. It is shown that the standard genetic code establishes a "choice architecture" that permits nearly independent tuning of the properties related with size and those related with hydrophobicity. This work sheds light on the metarules of evolvability that may have shaped the standard genetic code to increase the probability that adaptive point mutations will be generated. An illustration of the usefulness of visualizing amino acid substitutions in a 3D physicochemical space is shown using data collected from the SARS-CoV-2 receptor binding domain. The substitutions most responsible for antibody escape are almost always inaccessible via single nucleotide mutation, and also change multiple properties concurrently. The results of this research can extend our understanding of certain hereditary disorders caused by point mutations, as well as guide the development of rational protein and vaccine design.

scholarly journals Basic principles of the genetic code extension

2020 ◽  
Vol 7 (2) ◽  
pp. 191384
Author(s):  
Paweł Błażej ◽  
Małgorzata Wnetrzak ◽  
Dorota Mackiewicz ◽  
Paweł Mackiewicz
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Point Mutations ◽  
Coding System ◽  
Base Pairs ◽  
Induced Subgraphs ◽  
Single Nucleotide ◽  
Basic Principles ◽  
Code Extension ◽  
Incremental Addition

Compounds including non-canonical amino acids (ncAAs) or other artificially designed molecules can find a lot of applications in medicine, industry and biotechnology. They can be produced thanks to the modification or extension of the standard genetic code (SGC). Such peptides or proteins including the ncAAs can be constantly delivered in a stable way by organisms with the customized genetic code. Among several methods of engineering the code, using non-canonical base pairs is especially promising, because it enables generating many new codons, which can be used to encode any new amino acid. Since even one pair of new bases can extend the SGC up to 216 codons generated by a six-letter nucleotide alphabet, the extension of the SGC can be achieved in many ways. Here, we proposed a stepwise procedure of the SGC extension with one pair of non-canonical bases to minimize the consequences of point mutations. We reported relationships between codons in the framework of graph theory. All 216 codons were represented as nodes of the graph, whereas its edges were induced by all possible single nucleotide mutations occurring between codons. Therefore, every set of canonical and newly added codons induces a specific subgraph. We characterized the properties of the induced subgraphs generated by selected sets of codons. Thanks to that, we were able to describe a procedure for incremental addition of the set of meaningful codons up to the full coding system consisting of three pairs of bases. The procedure of gradual extension of the SGC makes the whole system robust to changing genetic information due to mutations and is compatible with the views assuming that codons and amino acids were added successively to the primordial SGC, which evolved minimizing harmful consequences of mutations or mistranslations of encoded proteins.

scholarly journals Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition

2021 ◽  
Author(s):  
Gavin Kurgan ◽  
Moses Onyeabor ◽  
Steven C Holland ◽  
Eric Taylor ◽  
Aidan Schneider ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Zymomonas Mobilis ◽  
Sugar Transport ◽  
Point Mutations ◽  
Saturation Mutagenesis ◽  
Transport Systems ◽  
Xylose Utilization ◽  
Hydroxymethyl Group ◽  
Xylose Transport ◽  
Glucose Inhibition

Abstract Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process in bacterial biocatalysts. The sugar facilitator of Zymomonas mobilis, Glf, is capable of importing xylose at high rates without extra energy input, but is inhibited by D-glucose (the primary biomass sugar), potentially limiting the utility of this transporter for fermentation of sugar mixtures derived from lignocellulose. In this work we developed an Escherichia coli platform strain deficient in glucose and xylose transport to facilitate directed evolution of Glf to overcome glucose inhibition. Using this platform, we isolated nine Glf variants created by both random and site-saturation mutagenesis with increased xylose utilization rates ranging from 4.8-fold to 13-fold relative to wild-type Glf when fermenting 100 g l–1 glucose–xylose mixtures. Diverse point mutations such as A165M and L445I were discovered leading to released glucose inhibition. Most of these mutations likely alter sugar coordinating pocket for the 6-hydroxymethyl group of D-glucose. These discovered glucose-resistant Glf variants can be potentially used as energy-conservative alternatives to the native sugar transport systems of bacterial biocatalysts for fermentation of lignocellulose-derived sugars.

Directed Evolution of an Enantioselective Enoate-Reductase: Testing the Utility of Iterative Saturation Mutagenesis

2009 ◽  
Vol 351 (18) ◽  
pp. 3287-3305 ◽  
Author(s):  
Despina J. Bougioukou ◽  
Sabrina Kille ◽  
Andreas Taglieber ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Enoate Reductase ◽  
Iterative Saturation Mutagenesis

Translational bypassing: a new reading alternative of the genetic code

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1055-1059 ◽  
Author(s):  
Irina Groisman ◽  
Hanna Engelberg-Kulka
Keyword(s):  
16S Rrna ◽  
Genetic Code ◽  
Untranslated Region ◽  
Mrna Sequence ◽  
Cellular Gene ◽  
Single Nucleotide ◽  
Frameshift Event ◽  
Lac Z ◽  
Bypass Mechanism ◽  
Alternative Reading

The translation of the genetic code, once thought to be rigid, has been found to be quite flexible, and several alternatives in its reading have been described. An unusual alternative is translational bypassing, a frameshift event where the transition from frame 0 to another frame occurs by translational bypassing of an extended region of the mRNA sequence rather than by slippage past a single nucleotide, as has been described for most examples of frameshifting. Translational bypassing has been characterized in two cases, T4 gene 60 coding for a topoisomerase subunit and in a trpR–lac′Z fusion. The latter was discovered in our laboratory, and the unique bypass mechanism is investigated further in this study. Using a trpR+1–lac′Z fusion system, we show that the Gln codon at the beginning of lacZ end at the 3′ side of the gap is required for bypassing to occur. The Gln codon is part of an mRNA segment that can (potentially) base pair with a segment at the 5′ and of Escherichia coli 16S rRNA. A model of trpR+1–lac′Z bypassing is suggested in which the untranslated region of the mRNA is looped out through base pairing between a segment in the 5′ end of the 16S rRNA and two sites in the mRNA. Translational bypassing is a newly discovered mechanism of gene expression, and trpR is the first cellular gene identified in which such a mechanism could operate. The understanding of this mechanism and its associated signals may be considered a paradigm for the expression of other genes by this alternative reading of the genetic code.Key words: genetic code, translation, frameshifting, trpR.

scholarly journals Sensitive quantitative assay for point mutations in the rat H-ras gene based on single nucleotide primer extension

2010 ◽  
Vol 1 (4) ◽  
pp. 657-661 ◽  
Author(s):  
YOSHIHISA YANO ◽  
TOMOHIRO YANO ◽  
ANNA KINOSHITA ◽  
AI MATOBA ◽  
TADAYOSHI HASUMA ◽  
...  
Keyword(s):  
Point Mutations ◽  
Primer Extension ◽  
Quantitative Assay ◽  
Ras Gene ◽  
Single Nucleotide ◽  
Single Nucleotide Primer Extension

scholarly journals Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5658-5664 ◽  
Author(s):  
Soo-Jin Yang ◽  
Nagendra N. Mishra ◽  
Aileen Rubio ◽  
Arnold S. Bayer
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Reading Frame ◽  
A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

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