Figure 5.

Combinatorial OR-type libraries for CASTing. (A) OR-type mutations at two separate positions in a sequence generates combinatorial OR-type mutations, where all different combinations of mutations from set 1 and set 2 are created, such that optimal paired mutations can be discovered. Simultaneous mutagenesis using the NNK codon using conventional AND-type mutations produces over 10¹⁵ genetic combinations, while the corresponding combinatorial OR-type library encoded 25600 combinations, a 4.4 × 10¹⁰-fold reduction in library size. (B) Sets 1 (residues [209]–[213]) and 2 ([241]–[245]) were selected as they sit on either side of the MAO-N active site channel. Both sets contained five mutagenic primers, each randomizing one amino acid. The library created every mutation combination between sets 1 and 2, that is, {[MP1] AND [MP1]} OR {[MP1] AND [MP2]}, etc. (C) The “hit” combination, exhibiting novel activity to non-native substrates, encoded mutations at the [1] (A209S) and [5] (L245C) positions.