Accessible biocatalyst development by rapid in vitro semi-rational engineering (RISE) of enzymes

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzyme Engineering

Background:

• Enzyme engineering for biocatalysis is costly and time-consuming.
• Wider adoption of biocatalysis in chemical and pharmaceutical industries is limited.
• Existing methods like directed evolution are expensive and slow.

Purpose of the Study:

• To develop a rapid, accessible enzyme engineering workflow (RISE).
• To reduce costs and timelines associated with enzyme tailoring.
• To promote biocatalysis implementation in early chemical development.

Main Methods:

• RISE workflow integrates computational design, rapid DNA library generation (PCR), cell-free protein synthesis, and iterative mutagenesis.
• Focused variant libraries were computationally designed.
• Linear mutant DNA libraries were generated rapidly via PCR.
• Cell-free protein synthesis was used from linear template DNA.
• Iterative cycles of mutagenesis, expression, and testing were employed.

Main Results:

• Engineered a ketimine reductase (RnKIRED) with improved performance.
• Achieved stereoselectivity inversion in one reaction.
• Obtained a 400-fold activity improvement in another reaction.
• Enabled gram-scale synthesis of key intermediates for ACE2 inhibitor drugs.

Conclusions:

• RISE workflow makes enzyme engineering accessible to chemistry labs.
• RISE bridges the gap between wild-type enzymes and directed evolution.
• The developed workflow accelerates biocatalysis adoption in industry.