Additional file 2 of Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate

Additional file 2: Fig. S1. Adaptive evolution of the xylose consuming strain XylC2 V1 in the SSL, parallel lines. Time course of each parallel evolution line. Color bars indicate increased concentration of the SSL. a MM SSL evolution lines. b YE SSL evolution lines. Asterisk indicates the EV3 evolution line that was terminated for poor tolerance in the 70% SSL. Fig. S2. Evolution of the xylose consuming strain, single isolates. Growth profiles of the evolved strains cultivated in YPX medium. Fig. S3. Evolution of the xylose consuming strain. Growth of the selected SSL-tolerant strains XylC2 EV6_4 and EV9_5 in the a YE SSL and b MM SSL at pH = 4.5. The experiment was performed in duplicates, error bars represent standard deviation (N = 2). Fig. S4. Growth of the tolerant XylC2 EV6_4 and its dicarboxylic acid producing derivative XylC2 EV6_4 MA in 70% and 60% YE SSL. The experiments were performed in two parallel shake flasks, error bars represent standard deviation (N = 2). Fig. S5. Production of dicarboxylic acids by fermentation of the SSL in bioreactors, second parallel bioreactor experiment. Consumption of the carbon sources present in the SSL and biomass formation by the engineered XylC2 6_4 MA strain (on the left) and production of dicarboxylic acids from the SSL at pH = 4.5 (on the right). Fig. S6. Mapping of the mutations to the respective loci on the reference genome of S288c. Panel A: overview of the distribution across the whole genome (Chr I to XVI, top) of the total mutations (blue, bottom) and the aminoacid-changing mutations (red, middle) found across all strains. Panels B–R: Distribution of the variants, mapped to each individual chromosome, in each of the strains (V1 parent and EV1–EV12, indicated on the left). Mutations are highlighted in orange if heterozygous or in red if homozygous, and referred to the S288c genomic sequence (annotation is shown on top, genes are represented by green boxes. Only few gene names are visible due to limited space). Dashed blue boxes highlight chromosomal segments involved in LOH events. Fig. S7. Highlights of mutated genes. Distribution and position of the variants in each strain (V1 parent and EV1–EV12, indicated on the left). Mutations are highlighted in orange if heterozygous or in red if homozygous. Corresponding outcomes of each mutation on the protein are indicated. a SNG1 / YGR197C. b FIT3 / YOR383C. c FZF1 / YGL254W. Fig. S8. Results of the mutations found in the engineered xylose pathway genes. a