Altering the regioselectivity of cytochrome P450 BM-3 by saturation mutagenesis for the biosynthesis of indirubin

Hu, Sheng; Huang, Jun; Mei, Lehe; Yu, Qing; Yao, Shan‐Jing; Jin, Zhihua

doi:10.1016/j.molcatb.2010.07.001

Cited by 28 publications

(11 citation statements)

References 57 publications

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“…Saturation mutagenesis techniques provide full access to all amino acids by using degenerate codons that cover a comprehensive collection of amino acids ( 15 – 17 ). Such approaches frequently identify multiple nucleotide replacements (MNRs) within a codon to be superior to SNRs in a variety of contexts ( 16 , 18 – 26 ). Recent technological advances in DNA synthesis and sequencing have enabled more detailed surveys, using complete systematic saturation mutagenesis of a gene and permitting the interrogation of the full mutational landscape at single-amino-acid resolution ( 27 – 30 ).…”

Section: Introductionmentioning

confidence: 99%

Refactoring the Genetic Code for Increased Evolvability

Pines

Winkler

Pines³

et al. 2017

mBio

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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.

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Section: Introductionmentioning

confidence: 99%

Refactoring the Genetic Code for Increased Evolvability

Pines

Winkler

Pines³

et al. 2017

mBio

View full text Add to dashboard Cite

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“…Saturation mutagenesis techniques provide full access to all amino acids by using degenerate codons covering a comprehensive collection of amino acids (15)(16)(17). Such approaches frequently identify multiple nucleotide replacements (MNRs) within a codon to be superior to SNRs in a variety of contexts (16,(18)(19)(20)(21)(22)(23)(24)(25)(26). Recent technological advances in DNA synthesis and sequencing have enabled more detailed surveys of a complete systematic saturation mutagenesis of a gene, permitting the interrogation of the full mutational landscape at single amino acid resolution (27)(28)(29).…”

Section: Introductionmentioning

confidence: 99%

Refactoring the Genetic Code for Increased Evolvability

Pines

Winkler

Pines³

et al. 2017

Preprint

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The 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. Keywords: genetic code, evolution, saturation mutagenesis, genome synthesis 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.

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“…Recent experimental data on various mesophilic P450s by several groups, including ours, indicate that the protein dynamics is not only restricted to the active site regions but also involves regions remote from the catalytic site ( 27 , 30 – 34 ). For example, in P450-BM3, mutations done at remote sites affected ligand binding, likely due to change in active site dynamics ( 35 ). Also, in CYP101A1, collective dynamic modes on the picosecond to nanosecond timescales were detected to be prevalent throughout the protein, and not just the active site, that create access to the active site for the substrate ( 26 , 36 ).…”

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confidence: 99%

Entropic contribution to enhanced thermal stability in the thermostable P450 CYP119

Liu

Lemmonds

Huang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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SignificanceUnderstanding the thermodynamic factors responsible for enhanced stability of thermophilic cytochrome P450 enzymes is significant in their development as efficient catalysts for high-temperature monooxygenation reactions. These factors are usually inferred from structural comparison with mesophilic P450s and invoke rigidifying enthalpic interactions as primary thermodynamic determinants for thermostability. Using the thermophilic P450 CYP119 as an example, the present work, however, questions this enthalpy-driven notion of P450 thermostability and provides strong experimental evidence that their thermostability may actually be entropy-driven due to increased flexibility in the folded state relative to mesophilic P450s and that this flexibility is partitioned effectively between functional activity and thermal stability. This represents a major paradigm shift in understanding the basis of thermostability in thermophilic P450s.

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Altering the regioselectivity of cytochrome P450 BM-3 by saturation mutagenesis for the biosynthesis of indirubin

Cited by 28 publications

References 57 publications

Refactoring the Genetic Code for Increased Evolvability

Refactoring the Genetic Code for Increased Evolvability

Refactoring the Genetic Code for Increased Evolvability

Entropic contribution to enhanced thermal stability in the thermostable P450 CYP119

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