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.