Clayton Dilks, Rhiannon LaVine, Claire Buchanan, Daniel Russo, and Elizabete Carmo-Silva

Adaptation to increasing environmental temperatures is essential to plant survival and human food production. Thermal tolerance is controlled by a complex network of factors in plants including but not limited to genetic variation and environmental context. Rubisco activase (Rca) is a key photosynthetic enzyme with low thermal tolerance. Here, we report a large machine learning-directed screen of >1,400 synthetic cassava Rca enzymes which identified mutations that convey increased thermal stability while minimizing introduced mutations. We demonstrate multiple synthetic proteins that maintain activity at 8°C higher than wildtype cassava Rca including a single mutation that retains most activity post heat-shock.