Quantifying the thermostability landscape separating plant sesquiterpene synthase orthologs using Maximum Entropy
As global temperatures rise due to climate change, crops are becoming increasingly vulnerable to failure. We tested the accessibility of thermodynamic phenotypic diversity within limited positional changes using 29=512 mutants of a terpene synthase (TPS), Tobacco 5-epi-Aristolchene synthase. First, we measured the thermal unfolding curves of each mutant and found that mutations shifted the Tms both higher and lower, including a cohort of mutants that failed to fold. The low correlation coefficient between the Tms of these mutants and the proportions of each terpene product output by the enzymes revealed that thermostability and product output are independent traits. Maximum Noise Entropy analyses were used to analyze the impact of the 9 mutational positions on thermostability, revealing that three of these positions were mainly responsible for the trait of increased Tm. These positions form a functional network as measured by the nonlinearity of their combined effects on thermostability. Unexpectedly, the strongly destabilizing positions combine nonlinearly to ameliorate each others deleterious effects resulting in a synergistic dampening. Taken together, our study shows the high potential for specialized metabolic enzyme engineering but also reveals a complex interconnected system of amino acids that will continue to evade perfect predictability.