Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: a Model of Ssal- KRED binding 1a (blue) and NADPH (yellow). The targeted keto moiety in 1a and the hydride-donor C4 of the nicotinamide ring of the cofactor are circled in red. Catalytic residues are depicted as purple sticks. The six positions targeted in SSM libraries L2 are indicated in bold letters: F97, L174, A238, L241, M242, Q245. b Schematic representation of hot spot grouping for CSM libraries. While keeping mutation F97W fixed, five selected residues were grouped into four 3-site CSM libraries designated as L3 (174-242-245), L4 (241-242-245), L5 (238-241-242) and L6 (174-238-241). In addition, all five residues were simultaneously saturated in a 5-site CSM library (L7). c Surface representation depicting neutral to beneficial mutations (color-coded according to their FIOWT values) in the context of the wild-type enzyme. Linearly combining these mutations would have led to a theoretical library size of 29,400 variants. d Sequence-activity data of L3–L7 were used to train a ML algorithm. Predicted variants were combined in a small library of 75 variants (L10). Substitutions, which had not been found beneficial in context of the wild-type enzyme (L2) but were predicted to perform well in the ML-filtered library L10, are highlighted in red.

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Binding Assay, Mutagenesis, Sequencing, Activity Assay

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: Ssal -KRED hit variants were obtained via hot spot engineering (HSE), machine learning (ML) and iterative site mutagenesis (ISM). Positions are color-shadowed in accordance with the library they were investigated in (for reference, see Fig. , ). The fold-improvement over the wild type (FIOWT) was calculated from fold-improvement over the parent (FIOP) values. AA amino acid, − not applicable.

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Mutagenesis

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: a Binding model of 1a (blue) in the active pocket of NADPH-bound (yellow) Ssal -KRED (PDB ID: 1Y1P). Catalytic residues are indicated in purple. Targeted positions (23) are color-coded in accordance with the library they were investigated in (for reference, see c ). Amino acid positions covered in L1 (mutational scanning library) are not depicted. b Overview of the relative activity of the Ssal -KRED hit variants M1 – M6 compared to the wild-type enzyme. The fold-improvement over the wild type (FIOWT) was determined using an UV assay (Supplementary Fig. ). c Libraries (L1–L13) on protein surface, substrate entrance tunnel, substrate cavity and cofactor environment positions were built using single-site saturation mutagenesis (SSM), combinatorial saturation mutagenesis (CSM) or focused combinatorial (FC) mutagenesis, integrating hit combinations and combinatorial libraries. L10 is a filtered ML-library, which was designed by combining knowledge from previous rounds of engineering with ML predictions.

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Binding Assay, Activity Assay, UV Assay, Mutagenesis

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: Comparison of KRED-mediated syntheses of 2a

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Comparison

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: Kinetic characterization of selected Ssal -KRED variants using ketone 1a for the synthesis of 2a

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Variant Assay

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: Homology models of wild-type Ssal -KRED (light gray) and variants M1 (smudge), M3 (wheat) and M6 (slate) with binding modes of 1a . The cofactor is shown in pale yellow, sand, yellow and orange, respectively. Catalytic residues are indicated in red letters. a Superimposed WT and M1 displaying the aromatic cage-like binding site formed by F97W, M242W and W226 in the latter (only one variant is shown for clarity). b Close-up view on key positions 241, 242 and 245 in the WT, M3 and M6 . Coordination of 1a by catalytic S133 and Y177 is indicated in blue and red dashes for the WT and M6 , respectively. c Polar interactions of key position 134 with other residues in the WT (blue dashes) and M6 (red dashes). Interactions with P206 and N207 are lost in variant M6 . d Secondary structure differences on positions 97, 238 and 241 between WT and all evolved variants. Mutation A238K in M3 and M6 might act as a “tunnel gate” residue.

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Binding Assay, Variant Assay, Mutagenesis, Residue

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: a Model of Ssal- KRED binding 1a (blue) and NADPH (yellow). The targeted keto moiety in 1a and the hydride-donor C4 of the nicotinamide ring of the cofactor are circled in red. Catalytic residues are depicted as purple sticks. The six positions targeted in SSM libraries L2 are indicated in bold letters: F97, L174, A238, L241, M242, Q245. b Schematic representation of hot spot grouping for CSM libraries. While keeping mutation F97W fixed, five selected residues were grouped into four 3-site CSM libraries designated as L3 (174-242-245), L4 (241-242-245), L5 (238-241-242) and L6 (174-238-241). In addition, all five residues were simultaneously saturated in a 5-site CSM library (L7). c Surface representation depicting neutral to beneficial mutations (color-coded according to their FIOWT values) in the context of the wild-type enzyme. Linearly combining these mutations would have led to a theoretical library size of 29,400 variants. d Sequence-activity data of L3–L7 were used to train a ML algorithm. Predicted variants were combined in a small library of 75 variants (L10). Substitutions, which had not been found beneficial in context of the wild-type enzyme (L2) but were predicted to perform well in the ML-filtered library L10, are highlighted in red.

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Binding Assay, Mutagenesis, Sequencing, Activity Assay

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: Ssal -KRED hit variants were obtained via hot spot engineering (HSE), machine learning (ML) and iterative site mutagenesis (ISM). Positions are color-shadowed in accordance with the library they were investigated in (for reference, see Fig. , ). The fold-improvement over the wild type (FIOWT) was calculated from fold-improvement over the parent (FIOP) values. AA amino acid, − not applicable.

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Mutagenesis

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: a Binding model of 1a (blue) in the active pocket of NADPH-bound (yellow) Ssal -KRED (PDB ID: 1Y1P). Catalytic residues are indicated in purple. Targeted positions (23) are color-coded in accordance with the library they were investigated in (for reference, see c ). Amino acid positions covered in L1 (mutational scanning library) are not depicted. b Overview of the relative activity of the Ssal -KRED hit variants M1 – M6 compared to the wild-type enzyme. The fold-improvement over the wild type (FIOWT) was determined using an UV assay (Supplementary Fig. ). c Libraries (L1–L13) on protein surface, substrate entrance tunnel, substrate cavity and cofactor environment positions were built using single-site saturation mutagenesis (SSM), combinatorial saturation mutagenesis (CSM) or focused combinatorial (FC) mutagenesis, integrating hit combinations and combinatorial libraries. L10 is a filtered ML-library, which was designed by combining knowledge from previous rounds of engineering with ML predictions.

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Binding Assay, Activity Assay, UV Assay, Mutagenesis

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: Comparison of KRED-mediated syntheses of 2a

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Comparison

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: Kinetic characterization of selected Ssal -KRED variants using ketone 1a for the synthesis of 2a

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Variant Assay

Journal: Communications Chemistry

Article Title: Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

doi: 10.1038/s42004-024-01130-5

Figure Lengend Snippet: Homology models of wild-type Ssal -KRED (light gray) and variants M1 (smudge), M3 (wheat) and M6 (slate) with binding modes of 1a . The cofactor is shown in pale yellow, sand, yellow and orange, respectively. Catalytic residues are indicated in red letters. a Superimposed WT and M1 displaying the aromatic cage-like binding site formed by F97W, M242W and W226 in the latter (only one variant is shown for clarity). b Close-up view on key positions 241, 242 and 245 in the WT, M3 and M6 . Coordination of 1a by catalytic S133 and Y177 is indicated in blue and red dashes for the WT and M6 , respectively. c Polar interactions of key position 134 with other residues in the WT (blue dashes) and M6 (red dashes). Interactions with P206 and N207 are lost in variant M6 . d Secondary structure differences on positions 97, 238 and 241 between WT and all evolved variants. Mutation A238K in M3 and M6 might act as a “tunnel gate” residue.

Article Snippet: Genes from the in-house KRED collection as well as Ssal -KRED libraries L1 and L7 were provided by Twist Bioscience (San Francisco, CA, USA).

Techniques: Binding Assay, Variant Assay, Mutagenesis, Residue