Variant production. Protein overexpression was carried out in three rounds (Table ) to assess the solubility and whole-cell activity of DhaA variants. In the first round, the solubility of DhaA ancestor variants was analyzed after their overproduction in E. coli and purification, where only AncDhaA1 was purified as a soluble protein in sufficient yield (Fig. ). This variant was also the only active one within the screening of the dehalogenase activity in whole cells using a halide oxidation (HOX) assay (Fig. ) . The variants produced in the first round showed overall low solubility, consistent with outcomes from several previous machine learning-based pipelines for protein design, achieving a solubility success rate of around 20% . In the second round, the ancestors 5, 7, and 8 (AncDhaA10-12) were selected for production to stay closer to the template. Among these variants, AncDhaA10 showed the highest expression, followed by AncDhaA11, while AncDhaA12 exhibited the lowest expression and solubility (Fig. ). AncDhaA10 was also shown to be active by HOX assay (Fig. ). Based on the results from both rounds, AncDhaA1, AncDhaA10, and AncDhaA11 were chosen for further biochemical characterization. From the third round of designs, six variants showed average or high solubility and only AncDhaA17 and AncDhaA19 were poorly soluble in the tested buffer. The HOX assay revealed two highly active variants AncDhaA13 and AncDhaA14 with activities comparable to templates and positive controls (Fig. ). Variants AncDhaA16 and AncDhaA20 exhibited lower activity, comparable to that of AncHLD-RLuc (Fig. ), while AncDhaA15 and AncDhaA18 showed low activities (Fig. ).