Optimizing enzyme thermostability by combining multiple mutations using protein language model

Optimizing enzyme thermostability is essential for advancements in protein science and industrial applications. Currently, (semi-)rational design and random mutagenesis methods can accurately identify single-point mutations that enhance enzyme thermostability. However, complex epistatic interactions often arise when multiple mutation sites are combined, leading to the complete inactivation of combinatorial mutants. As a result, constructing an optimized enzyme often requires repeated rounds of design to incrementally incorporate single mutation sites, which is highly time-consuming. In this study, we developed an AI-aided strategy for enzyme thermostability engineering that efficiently facilitates the recombination of beneficial single-point mutations. We utilized thermostability data from creatinase, including 18 single-point mutants, 22 double-point mutants, 21 triple-point mutants, and 12 quadruple-point mutants. Using these data as inputs, we used a temperature-guided protein language model, Pro-PRIME, to learn epistatic features and design combinatorial mutants. After two rounds of design, we obtained 50 combinatorial mutants with superior thermostability, achieving a success rate of 100%. The best mutant, 13M4, contained 13 mutation sites and maintained nearly full catalytic activity compared to the wild-type. It showed a 10.19°C increase in the melting temperature and an ~655-fold increase in the half-life at 58°C. Additionally, the model successfully captured epistasis in high-order combinatorial mutants, including sign epistasis (K351E) and synergistic epistasis (D17V/I149V). We elucidated the mechanism of long-range epistasis in detail using a dynamics cross-correlation matrix method. Our work provides an efficient framework for designing enzyme thermostability and studying high-order epistatic effects in protein-directed evolution.