Ancestral Sequence Reconstruction to Accelerate Non-heme Iron-dependent Biocatalyst Engineering

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.