Kohout_2026_Brief.Bioinform_27_

Evolution-guided protein design remains one of the most effective strategies for engineering proteins with enhanced stability, activity, or specificity. To make these approaches more accessible, we previously developed FireProtASR-a fully automated pipeline for ancestral sequence reconstruction (ASR). Here, we present FireProtASR 2.0, a significantly enhanced version that extends the design space beyond ancestral inference by integrating a successor sequence predictor (SSP) and a generative model based on variational autoencoders (VAEs). These new modules enable both 'prospective' and 'retrospective' evolutionary design strategies. The SSP module predicts likely future mutations based on site-wise evolutionary trends, and the method was previously validated through in silico benchmarks, demonstrating improvements in thermostability and activity. The VAE module captures global evolutionary constraints in a low-dimensional latent space, from which novel functional ancestral-like variants can be sampled. The VAE-based design strategy was previously validated experimentally on the haloalkane dehalogenase family, yielding variants with enhanced thermostability while maintaining catalytic activity. Both these modules are newly available in FireProtASR in a fully automated pipeline, guiding the users via an interactive graphical user interface. With expanded functionality, modernized user interface, and a more robust backend, FireProtASR 2.0 provides a comprehensive, accessible, and fully automated platform for evolutionary-based protein engineering (https://loschmidt.chemi.muni.cz/fireprotasr/).