Microfluidic‐Assisted Evolution of a Robust NAD + ‐Dependent Enzyme with Improved Isobutanol Tolerance at Elevated Temperatures

Prompted by the thermostability issue identified in recent work on enzyme discovery/engineering and its application, the directed evolution of an NAD + ‐dependent aldehyde dehydrogenase (ALDH) with improved thermostability and isobutanol tolerance at 50 °C, properties required for its successful implementation in cell‐free isobutanol biosynthesis, is described herein. ALDH not only plays an important role in multienzyme cascades for the production of platform chemicals but also represents a bottleneck due to its modest stability. Using a custom‐built absorbance‐activated droplet sorter, ultrahigh‐throughput microfluidic screening of a randomized library of 63,000 members is performed, leading to the discovery of a variant with a 250‐fold prolonged half‐life at 50 °C without significant loss of activity. Subsequently, the most promising mutations are distributed on designer templates in the combinatorial staggered extension process library to create a new generation of variants. One of these variants shows a threefold increase in k cat K m −1 . Another shows significantly higher stability in 3% v/v isobutanol, retaining ≈50% of its initial activity after 6 h of incubation at 50 °C. Finally, a cell‐free multienzymatic cascade using the ultimate variant demonstrates its superior stability in 4% v/v isobutanol at 50 °C, highlighting the success of engineering to overcome the cofactor‐related challenge of establishing cascade balance.