Excel 2013

Activity assays on the chromogenic 4-NPF substrate were performed in a reaction volume of 80 μl containing up to 200 μg of total protein of the cleared lysates. Dilution of the lysate was performed as needed to ensure that the signal of product formation was within the dynamic range of the assay for each enzyme. The reaction further contained 2.5 mM of 4-NPF in 50 mM buffer (MES at pH 5.5 or 6.5; or HEPES at pH 7.5) supplemented with protease inhibitors (cOmplete™ Protease Inhibitor Cocktail without EDTA, Roche). After incubation (30 minutes at 30°C), the reaction was stopped by adding 120 μl of 0.5 M of carbonate/bicarbonate pH 9.6, and the absorbance was measured at 415 nm on an iMark™ microplate reader (Bio-Rad). The obtained absorbance was corrected using a negative control lysate from an untransformed strain, which was applied in the same concentration as the sample. The amount of fucose that was released was inferred by linear regression by including a dilution series of 4-nitrophenol on each plate, with concentrations ranging from 0-1 mM (1 mM corresponds to 80 nmol per well). For selected enzymes, each with varying degrees of activity towards 4-NPF, the assay was repeated with up to four biological replicates. Data processing and visualization was done in Microsoft Excel 2013 and GraphPad Prism 7.

Data analyses were performed using Microsoft Excel 2013 and GraphPad Prism7. A two-tailed Student’s t test, a two-way ANOVA and a Mantel-Cox were used to determine significance. In all types of statistical analysis values of p < 0.05 were considered significant. Data are represented as the mean ± SEM (standard error of the mean) of individual data points of at least three independent samples. Number of independent samples and statistical method used in each experiment are reported in the figure legends. For all experiments similar variances between groups were observed. Normal distribution of samples was not determined. In the DAVID functional annotation analyses for the RNA-seq experiments EASE Score, a modified Fisher Exact p-values, were used to determine significance as recommended.