E pure water

MRP crude samples were re-dissolved in E-Pure water and supernatants were adjusted to 50% acetonitrile (Thermo Fisher Scientific, Waltham, MA, USA) for sugar analysis, analyzed by high-performance liquid chromatography (HPLC), using an Agilent 1100 series (Santa Clara, California, USA), equipped with a Agilent, 5 µm, 4.6 × 250 mm column and a refractive index detector [21 (link)]. The mobile phase contained 75% acetonitrile and the flow rate was 1.0 mL/min. Standard calibration curves of xylose, glucose, fructose, and sucrose were built for quantitative analysis with a concentration range of 0.625–20 mg/mL. Ribose was used as an internal standard.

The OD₆₀₀ of cultures was measured using a spectrophotometer (Biomate 5, Thermo, NY) and dried cell weight (DCW) was obtained from an experimentally determined conversion factor of 0.454 g DCW/OD₆₀₀. Extracellular metabolites such as lactose, galactose, galactitol, glycerol, acetate, and ethanol were measured by HPLC (Agilent Technologies 1200 Series, Santa Clara, CA) with a Rezex^TM ROA-Organic Acid H + (8%) column (Phenomenex Inc., Torrance, CA) and a refractive index detector (RID). The column was eluted with 0.005 N H₂SO₄ at a flow rate of 0.6 mL/min at 50 °C. Galactose and tagatose could not be separated by this ROA-organic acid H + (8%) column (Supplementary Figure 3). Galactose and tagatose concentrations were measured using an Agilent Technologies 1200 Series HPLC equipped with a Rezex^TM RCM-Monosaccharide Ca + 2 (8%) column (Phenomenex Inc. Torrance, CA) and RID detector. The mobile phase was E-Pure™ Water (Barnstead E-Pure™ Water Purification Systems, Thermo Scientific) and was eluted at a flow rate of 0.6 mL/min at 80 °C. The maxium theoretical tagatose yield (0.526 g tagatose/g lactose) by engineered yeast was calculated based on the molecular weights of tagatose (180.16 g/mol) and lactose (342.3 g/mol) with an assumption that glucose is not converted into tagatose.