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Figure S1. Optical density at 540 m values of five selected butyraldehyde dehydrogenase (Bld) mutants mixed with Schiff's reagent.

Figure S2. Sequence alignment between Bld and two template proteins (aldehyde dehydrogenase of Listeria monocytogenes [Protein Data Bank ID: 3K9D] and alcohol dehydrogenase of Vibrio parahaemolyticus [Protein Data Bank ID: 3MY7]). Sequence alignment of Bld to 3K9D and 3MY7 sequences was performed for homology modeling. The results showed 15.0% sequence identity and 34.1% sequence similarity. Conserved catalytic cysteine residues (Cys275 in the case of Bld) are indicated with a red asterisk.

Table SI. Primers used for genome engineering.

Table SII. Cloning polymerase chain reaction primers for 1,4-butanediol pathway enzymes.

Table SIII. Primers used for site-directed mutagenesis.

Table SIV. Butyraldehyde dehydrogenase (Bld) L273X generated through site-directed mutagenesis.

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