Relative activity measurements of acetolactate synthase (ALS) enzymes

Full details of the ALS enzyme assays are described in Additional file 3: Method S6). Yeast cells were grown in SD selection medium and then the collected cells were disrupted with glass beads using a Shake Master Neo (Bio Medical Science, Tokyo, Japan) to obtain cell crude extracts. The ALS activity assay basically followed previously described procedures [33, 34]. Briefly, a pre-mixture containing thiamine pyrophosphate, FAD, MgCl₂ and crude yeast cell extract was pre-incubated, and then the reaction was started by adding pyruvate. The reaction was stopped by adding sulfonic acid and the reaction mixture was incubated to convert 2-acetolactate to acetoin. Acetoin was then further oxidized to diacetyl with creatine and α-naphthol. The relative ALS activity based on the colorimetric assay of acetoin (or diacetyl) was determined by measuring the absorbance (at 525 nm) of the developed red color using an EnVision multilabel plate reader (Perkin Elmer, Waltham, MA, USA).