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Ancestral sequence reconstruction (ASR) uncovers thermostable enzymes for biocatalyst engineering. Engineering these ancestral proteins yields enhanced stability, activity, and broader substrate scope for synthetic chemistry applications.

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Area of Science:

  • Biocatalysis and synthetic chemistry
  • Enzyme engineering and directed evolution
  • Biotechnology and protein science

Background:

  • Non-heme iron-dependent (NHI) enzymes are valuable biocatalysts for selective oxidation reactions.
  • Protein engineering often faces limitations due to the innate stability of modern enzymes.
  • Ancestral sequence reconstruction (ASR) offers a strategy to identify evolutionarily stable enzyme variants.

Purpose of the Study:

  • To explore the utility of ASR in identifying thermostable NHI enzymes for protein engineering.
  • To compare the evolvability of a modern NHI enzyme with its reconstructed ancestor.
  • To develop a biocatalytic route to tropolones using engineered ancestral NHI enzymes.

Main Methods:

  • Utilized ASR to reconstruct ancestral NHI enzyme sequences.
  • Performed comparative in-lab evolution of modern and ancestral NHI enzymes.
  • Engineered the ancestral NHI enzyme for improved thermostability, expression, and activity.
  • Assessed substrate scope and catalytic efficiency of engineered variants.

Main Results:

  • The ancestral NHI enzyme exhibited superior stability and evolvability compared to its modern counterpart.
  • Engineered ancestral variants showed enhanced thermostability and expression levels.
  • Variants demonstrated increased catalytic rates and a broader substrate scope.
  • Successful biocatalytic synthesis of tropolones was achieved using engineered enzymes.

Conclusions:

  • ASR is an effective strategy for mining robust enzyme backbones for biocatalyst engineering.
  • Engineering ancestral NHI enzymes accelerates the development of more stable and synthetically useful biocatalysts.
  • This approach overcomes limitations of innate enzyme stability, enabling broader applications in synthetic chemistry.