Thermostable Enzyme Variants in the Lower Mevalonate Pathway Improve Isoprenoid Production by Cell-Free Biocatalysis

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• Cell-free biocatalysis offers sustainable chemical production but faces challenges with enzyme stability and reaction longevity.
• Instability of enzymes hinders the efficiency and scalability of cell-free systems.

Purpose of the Study:

• To enhance the productivity of the lower mevalonate pathway for isoprenoid production using thermophilic enzymes.
• To address enzyme instability challenges in cell-free biocatalysis.

Main Methods:

• Assembled and compared a thermophilic Archaea I mevalonate pathway against a classical mesophilic pathway.
• Evaluated enzyme thermostability up to 60 °C and operating lifetime at 22 °C.
• Assessed product yield (limonene) and enzyme stability under solvent exposure.

Main Results:

• Thermophilic enzymes exhibited a 6× longer operating lifetime at 22 °C compared to mesophilic enzymes.
• The thermophilic pathway achieved a 1.7× higher yield of limonene.
• Thermostable enzymes demonstrated improved resilience to solvent exposure (ethanol, isoprenol).

Conclusions:

• Employing thermophilic enzymes significantly improves the stability and productivity of cell-free mevalonate pathways.
• Sourcing biocatalysts from thermophiles enhances cell-free system robustness for isoprenoid biosynthesis.
• This strategy enables more efficient and resilient cell-free production of valuable chemicals.