Fig. 6

Elucidating XYR genes in Y. lipolytica and enzyme characterization. a Screening for the missing XYR gene from Y. lipolytica by revisiting the E. coli xylose challenge. Cell densities of E. coli ΔxylA strains grown in 1 % xylose minimal media for 6 days expressing a candidate XYR (1–13, or SDR) and XDH (Plasmids 3–16) from Y. lipolytica (teal) were measured. The known S. stipitis XYR (black) and XDH (Y. lipolytica) (Plasmid 17) were used as a positive control. b Enzyme assay of XDH in BL21 cell lysates using NAD⁺ cofactor and xylitol as a substrate. One unit is defined as the reduction of 1 μmol NAD⁺ per min per mg protein. Enzyme assay of XYR1 and XYR2 in BL21 cell lysates using NADPH cofactor and xylose as a substrate. One unit is defined as the oxidation of 1 μmol NADPH per min per mg protein. Protein concentrations were calculated by densitometry from the coomassie image after SDS–PAGE electrophoresis. c Enzyme assay of XYR1 and XYR2 in Y. lipolytica cell lysates using NADPH cofactor and xylose as a substrate. XYR1 and XYR2 overexpressed using Plasmids 32 and 33. Control strain harboring plasmid 18 was also tested. One unit is defined as the oxidation of 1 μmol NADPH per min per mg protein. Protein concentrations are calculated by Bradford Assay for total cellular protein. All error bars represent SD. (n = 3)