Refining the mutations based on manual curation of the structures and stability scores. From Model 2, we selected three ancestors with unique features: ancestor 3 closely mimicked the wild type with 98% similarity; ancestor 16 notably introduced a proline at position 75; and ancestor 23 was the last variant starting with a regular sequence pattern (MSEIGT), suggesting high solubility and expression potential based on its 88% similarity to the PDB sequence 4WCV. To increase our chances of producing soluble variants, we manually curated proposed mutations based on the structural predictions from AlphaFold and stability assessments using the MutCompute tool (Table ). Mutation manual curation led to the classification of VAE-proposed mutations into safe and risky categories, respectively (Section 2 Manual curation of designs in round 3 of SI). Starting from ancestor 3, we kept four non-risky mutations. We removed two structurally and statistically risky mutations, P34V and L238F, from the original six-point variant proposed by the VAE, producing the design AncDhaA14. As experimental validation of identified risky mutations, we selected ancestor 0 with eight substitutions (AncDhaA13). For the second manually curated variant (AncDhaA16), we selected ancestor 15 as the template, which included as the last VAE ancestor no insertions, and we removed the risky mutations P34V and L238F, leaving nine mutations compared to the DhaA wild type. To experimentally determine the effect of suggested proline insertion with risky mutations, we selected ancestor 16 as predicted by VAE (AncDhaA15) (Fig. ). As a template for the third manual curation target, we selected ancestor 23. We incorporated all mutations suggested by the VAE except the risky ones (P34V, L238F) and the proline insertions, resulting in a final design carrying 32 mutations (AncDhaA17).