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Supplementary materials

RNA-Seq of the xylose-fermenting yeast Scheffersomyces stipitis cultivated in glucose or xylose

Tiezheng Yuan1, Yan Ren2,3, Kun Meng1, Yun Feng2,3, Peilong Yang 1, Shaojing Wang2,3, Pengjun

Shi1, Lei Wang2,3*, Daoxin Xie4*, Bin Yao1*

1 Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.

2 TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin 300457, China.

3 Tianjin Research Center for Functional Genomics and Biochip, Tianjin 300457, China.

4 School of Biological Sciences, Tsinghua University, Beijing 100084, China

*Corresponding authors：

[email protected] (B. Yao), Tel: 86-10-82106053, Fax: 86-10-82106055

[email protected] (D. Xie)

[email protected] (L. Wang)

1 Index

Supplementary methods

Adjustment of yeast cell mass grown in glucose or xylose on a dry weight basis...... 3

Supplementary figures

Fig. S1 Fold changes of RPKM in the glucose and xylose samples versus sequence length of

novel transcripts defined by RNA-Seq. X/G: Ratio of the values obtained for the xylose and

glucose samples...... 5

Fig. S2 GO analysis of proteins encoded by transcribed genes in the glucose sample (a), in the

xylose sample (b), through the whole genome (c) and differentially transcribed genes (d)....6

Fig. S3 Transcriptional control of gluconeogenic genes in S. cerevisiae (a) and predicted PPI

involved in gluconeogenesis in S. stipitis (b). The regulator that interacts with the enzymes

involved in gluconeogenesis is labeled with question mark because the shaded nodes are not

defined by the predicted PPIs...... 7

Supplementary tables

Table S1 Novel transcripts defined by RNA-Seq...... 8

Table S2 Summary of the expressed genes and DEGs with GO annotationa...... 12

Table S3 Summary of DEGs (B–H adjusted p < 0.05) involved in the KEGG metabolic pathways

...... 14

Table S4 Quantification of certain genes determined by RNA-Seq and qPCRa...... 16

Table S5 Transcription of the sugar transporter genes in S. stipitis a...... 18

Table S6 Transcription of genes involved in peroxisomes of S. stipitis...... 20

Table S7 Transcription of genes of iron transporters in S. stipitis and in S. cerevisiae...... 24

2 Supplementary methods

Adjustment of yeast cell mass grown in glucose or xylose on a dry weight basis

Based on the growth curves of S. stipitis in the presence of glucose or xylose, we collected yeast cells at the exponential growth phase after 6–8 h in YPG (glucose) or YPX (xylose).

glucose flask xylose flask

m 1.5 n 0 0 6

0 0 2 4 6 8 10 12 14 16 18 20 22 24 growth time (h)

Fig. Growth curves of S. stipitis in the presence of glucose or xylose

Metabolites were quantified in each yeast pellet (from the glucose or xylose culture) used for RNA-

W Seq. The cell weight factor (F) of the glucose or xylose samples was defined as F= , in which W A*V

(gram) is the dry weight of the pellets from the glucose or xylose samples, A is absorbance at 600 nm, and V (liter) is volume of the yeast culture.

The pellets from the glucose or xylose samples were freeze-dried for 2 days, weighed, and used to construct a correlation between absorbance at 600 nm and cell dry weight. The cell weight factor of the glucose and xylose samples was 0.22, and the absorbance and the volume of the culture could be used to calculate the yeast cell weight. The table below summarizes data for yeast cultured for 6–8 h.

Cultivation time (h) The glucose sample The xylose sample

A600 Total volume (ml) A600 Total volume (ml) 6 1.118 2300 1.129 2360 6 1.316 2360 1.275 2320

3 7 1.449 3200 1.337 3250 7 1.390 4030 1.431 4090 7 1.640 2280 1.322 2370 8 2.133 2720 2.082 2680

4 Supplementary figures

Fig. S1 Fold changes of RPKM in the glucose and xylose samples versus sequence length of novel transcripts defined by RNA-Seq. X/G: Ratio of the values obtained for the xylose and glucose samples

5 Fig. S2 GO analysis of proteins encoded by transcribed genes in the glucose sample (a), in the xylose sample (b), through the whole genome (c) and differentially transcribed genes (d)

6 Fig. S3 Transcriptional control of gluconeogenic genes in S. cerevisiae (a) and predicted PPI involved in gluconeogenesis in S. stipitis (b). The regulator that interacts with the enzymes involved in gluconeogenesis is labeled with question mark because the shaded nodes are not defined by the predicted PPIs

7 Supplementary tables

Table S1 Novel transcripts defined by RNA-Seq

Chromosome Transcript start Transcript end Direction CDSa CDS start CDS end Function NC_009042.1 837797 839344 - CDS 837797 838006 Hypothetical protein NC_009042.1 940356 941141 + CDS 940690 940944 Hypothetical protein NC_009042.1 1144267 1144318 + NC_009042.1 1145508 1145788 + NC_009042.1 1301717 1302936 - CDS 1301798 1302556 Hypothetical protein NC_009042.1 1311776 1311962 + NC_009042.1 1312023 1312465 - CDS 1312046 1312225 Hypothetical protein NC_009042.1 1654578 1656091 - CDS 1655635 1655910 Hypothetical protein NC_009042.1 2312999 2315072 + CDS 2313258 2314970 Hypothetical protein NC_009042.1 2401451 2402308 - CDS 2401956 2402219 Hypothetical protein NC_009042.1 2595350 2598681 - CDS 2595352 2597079 Hypothetical protein NC_009042.1 2717338 2717405 + NC_009043.1 194361 194547 - CDS 194392 194496 Hypothetical protein NC_009043.1 323055 324444 - CDS 323368 324420 Hypothetical protein NC_009043.1 523416 524638 - CDS 523483 523914 Hypothetical protein NC_009043.1 1195952 1196401 - CDS 1196202 1196387 Hypothetical protein NC_009044.1 68966 69607 + CDS 68974 69276 Hypothetical protein NC_009044.1 306859 307712 - CDS 306942 307622 Imidazoleglycerol-phosphate dehydratase NC_009044.1 1083282 1083637 - CDS 1083477 1083587 Dolichyl-diphosphooligosaccharide-protein

8 Chromosome Transcript start Transcript end Direction CDSa CDS start CDS end Function glycosyltransferase 4 kDa subunit NC_009044.1 1785957 1786035 + NC_009044.1 1793552 1794531 - CDS 1793835 1794077 Hypothetical protein NC_009045.1 445456 445538 + NC_009046.1 855793 855981 - CDS 855811 855912 Hypothetical protein NC_009046.1 1701282 1702281 - CDS 1701414 1701593 Hypothetical protein NC_009047.1 83476 84798 - CDS 84610 84789 Hypothetical protein NC_009047.1 505968 508614 - CDS 506571 508562 Hypothetical protein NC_009047.1 827598 831420 - CDS 828026 831088 Hypothetical protein NC_009047.1 977133 977183 + NC_009047.1 1003352 1003504 - CDS 1003354 1003461 Hypothetical protein NC_009047.1 1005353 1005491 - CDS 1005361 1005474 Hypothetical protein NC_009048.1 400 521 + NC_009048.1 479151 480387 + CDS 479794 480015 Hypothetical protein NC_009048.1 919403 920543 - CDS 919637 919810 Hypothetical protein NC_009068.1 129183 129705 - CDS 129399 129614 Hypothetical protein NC_009068.1 287748 287827 + NC_009068.1 305802 306278 - CDS 306085 306222 Hypothetical protein NC_009068.1 580401 580572 + NC_009068.1 984932 986840 + CDS 985991 986446 Hypothetical protein NC_009068.1 1504382 1505438 + CDS 1505172 1505363 Hypothetical protein NC_009068.1 1577322 1580736 + CDS 1578119 1578370 Hypothetical protein NC_009068.1 1824062 1824112 + NC_009068.1 1889413 1890395 - CDS 1889983 1890372 Hypothetical protein

9 Chromosome Transcript start Transcript end Direction CDSa CDS start CDS end Function NC_009068.1 2239683 2240369 + CDS 2239916 2240368 Hypothetical protein NC_009068.1 2600201 2603061 + CDS 2600222 2602924 Hypothetical protein NC_009068.1 3119722 3120419 + CDS 3119777 3119980 Hypothetical protein NC_009068.1 3297104 3297968 - CDS 3297509 3297910 Hypothetical protein NC_009068.1 3411864 3412037 + CDS 3411867 3412016 Hypothetical protein aCDS: coding sequence

10 Table S2 Summary of the expressed genes and DEGs with GO annotationa

GO ID p value AN EX DE GO terms Upregulated biological process GO:0005975* 8.91E-8 75 47 11 Carbohydrate metabolic process GO:0006006 4.38E-2 5 3 1 Glucose metabolic process GO:0006012 5.49E-4 3 3 3 Galactose metabolic process GO:0006108 4.39E-2 4 3 1 Malate metabolic process GO:0006631 1.33E-2 2 1 1 Fatty acid metabolic process GO:0006635 1.33E-2 2 1 1 Fatty acid β-oxidation GO:0008152* 5.85E-5 398 217 30 Metabolic process GO:0009228 4.35E-2 3 3 2 Thiamin biosynthetic process GO:0018346 2.95E-2 2 2 1 Protein amino acid prenylation GO:0044237 1.04E-2 24 11 2 Cellular metabolic process GO:0046835 1.47E-2 1 1 1 Carbohydrate phosphorylation GO:0055085 1.99E-3 316 142 15 Transmembrane transport GO:0055114* 1.09E-5 262 142 22 Oxidation reduction Upregulated molecular function GO:0003824 9.52E-3 489 282 28 Catalytic activity GO:0003978 1.19E-2 1 1 1 UDP-glucose-epimerase activity GO:0003995 2.79E-2 3 2 1 Acyl-CoA dehydrogenase activity GO:0003997 1.33E-2 2 1 1 Acyl-CoA oxidase activity GO:0004335 1.47E-2 1 1 1 Galactokinase activity GO:0004345 1.48E-2 1 1 1 Glucose-phosphate dehydrogenase activity GO:0004553 1.17E-3 34 17 3 Hydrolase activity, hydrolyzing O-glycosyl

compounds GO:0005215 1.15E-2 83 36 7 Transporter activity GO:0005488 4.73E-4 317 204 26 Binding GO:0008318 2.95E-2 2 2 1 Protein prenyltransferase activity GO:0016491* 4.79E-6 238 128 21 Oxidoreductase activity GO:0016773 6.28E-4 20 16 3 Phosphotransferase activity, alcohol group as

acceptor GO:0022891 5.63E-4 37 13 5 Substrate-specific transmembrane transporter

activity GO:0030060 4.39E-2 3 3 1 L-Malate dehydrogenase activity GO:0030246 4.32E-2 5 4 1 Carbohydrate binding GO:0042578 1.48E-2 1 1 1 Phosphoric ester hydrolase activity GO:0050660 3.90E-2 46 23 3 FAD binding GO:0050662 1.02E-2 26 12 3 CoA binding Downregulated cellular component GO:0005622 4.36E-2 308 217 3 Intracellular

11 aGOs (B–H adjusted p < 0.05) are marked with asterisks; AN, the number of genes with GO annotation; EX, the number of expressed genes with GO annotation; DE, the number of DEGs with GO annotation (p < 0.05).

12 Table S3 Summary of DEGs (B–H adjusted p < 0.05) involved in the KEGG metabolic pathways

Locus_tag RPKM RPKM p value Function KEGG metabolic pathway ID

(glucose) (xylose) PICST_29300 1 21 1.36E-5 β-Mannosidase 00511 PICST_33726 10 50 3.59E-4 Glucan-1,4-α-glucosidase 00052, 01100, 00500 PICST_34123 1 145 4.85E-14 β-Glucosidase 00460, 00500 PICST_39160 1 276 1.28E-17 Endo-1,4-β-glucanase 01100, 00500 PICST_49223 8 89 8.58E-7 Protein induced by osmotic stress 01100 PICST_51227 1 26 1.85E-5 β-Glucosidase 00460, 00500 PICST_51462 37 162 4.22E-4 Fructose-1,6-bisphosphatase 00010, 00030, 00051, 01100 PICST_56703 2 19 2.55E-4 Glucan-1,4-α-glucosidase 00052, 01100, 00500 PICST_59568 4 39 9.20E-6 Putative aldo-keto reductase 01100, 00650, 00051 PICST_65767 2 248 4.27E-17 β-Glucosidase 00460, 00500 PICST_66312 4 1881 3.58E-21 Endo-1,4-β-glucanase 01100, 00500 PICST_67803 163 942 1.39E-4 Secondary alcohol dehydrogenase 01100, 00010, 00980, 00071, 00350 PICST_68734 11 2156 3.63E-21 D-Xylulokinase 00040, 01100 PICST_75424 11 63 1.20E-4 Acyl-CoA oxidase 4 01100, 01040, 00592, 00071 PICST_76750 11 147 8.18E-9 UDP-glucose-4-epimerase 00520, 00052, 01100, 00010 PICST_78343 12 60 3.20E-4 Malate dehydrogenase 00020, 00620 00630 PICST_80169 17 100 8.51E-5 Vacuolar acid trehalase 00500 PICST_82625 81 355 3.63E-4 Galactokinase 00520, 00052, 01100 PICST_85065 250 1773 8.65E-5 Glucose-6-phosphate 1-dehydrogenase 01100, 00030, 00480 PICST_86346 20 198 1.41E-7 Peroxisomal 2,4-dienoyl-CoA reductase 01100, 00590 PICST_86603 12 87 2.00E-5 Putative ribitol kinase or glycerol kinase 01100

13 Locus_tag RPKM RPKM p value Function KEGG metabolic pathway ID

(glucose) (xylose) PICST_86924 40 2555 5.16E-20 D-Xylulose reductase (xylitol dehydrogenase) 00040, 01100 PICST_89614 67 11175 7.21E-20 D-Xylose reductase 00040, 01100

14 Table S4 Quantification of certain genes determined by RNA-Seq and qPCRa

Gene RPKM RPKM Ratio by RNA-Seq Reference gene Ratio by qPCR

(glucose) (xylose) ADH1 4116 6521 1.6 RPL13 1.54 ± 0.60 ADH4 4 15 3.8 ACT1 4.95 ± 3.49 ARO10 31 136 4.4 ACT1 0.73 ± 0.07 BGL5 1 145 145 ACT1 293 ± 238.44 BGL7 2 248 124 ACT1 14.37 ± 10.11 CGR1 27 92 3.4 ACT1 1.98 ± 0.18 CIT1 116 127 1.1 ACT1 0.66 ± 0.29 EGC2 4 1881 470.3 ACT1 1330 ± 1193 ENO1 2545 3222 1.3 RPL13 3.48 ± 0.12 FBA1 1851 1932 1.0 RPL13 1.47 ± 0.40 FBP1 37 162 4.4 ACT1 2.88 ± 1.09 FBP26 71 136 1.9 ACT1 0.68 ± 0.15 GAL1 81 355 4.4 ACT1 1.66 ± 0.56 GLK1 7773 6363 0.8 RPL13 1.06 ± 0.11 GLK2 19 65 3.4 ACT1 2.37 ± 0.07 GND1 730 1658 2.3 RPL13 1.82 ± 0.15 GPM1. 790 1082 1.4 RPL13 1.43 ± 0.12

1 GPM1. 190 315 1.7 ACT1 0.55 ± 0.15

2 GPM1. 56 45 0.8 ACT1 0.52 ± 0.13

3 GPM2 81 73 0.9 ACT1 0.67 ± 0.12 HXK1 82 88 1.1 ACT1 0.99 ± 0.30 IDH1 71 61 0.9 ACT1 0.94 ± 0.06 KGD1 50 44 0.9 ACT1 1.47 ± 0.33 PDC1 3033 3271 1.1 RPL13 1.82 ± 0.09 PDC2 2479 1001 0.4 RPL13 0.65 ± 0.06 PFK1 169 180 1.1 ACT1 1.08 ± 0.25 PGI1 402 1081 2.7 RPL13 2.93 ± 0.23 PYC1 43 36 0.8 ACT1 2.26 ± 0.93 PYC2 22 20 0.9 ACT1 1.44 ± 0.46

15 Gene RPKM RPKM Ratio by RNA-Seq Reference gene Ratio by qPCR

(glucose) (xylose) PYK1 755 694 0.9 ACT1 0.62 ± 0.16 RPE1 145 196 1.4 ACT1 1.13 ± 0.06 SOL1 66 69 1.0 ACT1 0.89 ± 0.10 SOL2 233 341 1.5 ACT1 0.59 ± 0.14 TAL1 440 2060 4.7 RPL13 3.15 ± 0.06 XKS1 11 2156 196.0 RPL13 6E+7 ± 1.2E+7 XYL1 67 11175 166.8 RPL13 34.44 ± 6.12 XYL2 40 2555 63.9 RPL13 39.03 ± 3.44 ZWF1 250 1773 7.1 RPL13 6.91 ± 1.09 a The RPKM ratio of xylose versus glucose samples is defined by RNA-Seq or qPCR. Transcription values that differ significantly between the RNA-Seq and qPCR analyses are shaded.

16 Table S5 Transcription of the sugar transporter genes in S. stipitis a

Locus_Tag RPK RPKM p Value Gene Function

M (xylose)

(gluco

se) PICST_87108 1 3 0.626 AUT1 Arabinose-proton symporter (arabinose transporter) PICST_58479 19 19 1 FUC1 L-Fucose permease (galactose transporter) PICST_53880 0 0 NULL HGT1 High-affinity glucose transporter PICST_55800 105 3501 4.487E-10 HGT2 Putative high-affinity glucose transporter PICST_29041 1 4 0.376 HXT2.1 Hexose transporter (tentative) PICST_54022 0 0 NULL HXT2.3 Possible hexose transporter PICST_80517 1 126 2.530E-13 HXT2.4 Possible hexose transporter PICST_11314 0 0 NULL HXT2.5 Possible hexose transporter PICST_31830 0 0 NULL HXT2.6 Hexose transporter PICST_48420 1 2 1 HXT4 Fructose symporter PICST_30035 0 0 NULL LAC1 Lactose permease PICST_29297 0 0 NULL LAC2 Lactose permease PICST_57768 0 0 NULL LAC3 Putative lactose permease PICST_35662 0 0 NULL MFS5 Hexose transporter PICST_36948 1 1 1 QUP2 High-affinity hexose (glucose) transporter (HXT1) PICST_91253 17 1099 5.957E-21 RGT2 Glucose transporter/sensor PICST_85501 31 66 0.073 SNF3 High-affinity glucose transporter PICST_57820 52 36 0.391 STL1 Sugar transporter PICST_66414 3861 3383 0.810 SUT1 Hexose transporter PICST_27983 311 120 0.018 SUT2 Sugar uptake (tentative) PICST_27984 854 341 0.040 SUT3 Sugar uptake (tentative) PICST_64741 2571 1105 0.098 SUT4 Sugar transporter protein PICST_33008 3 34 6.981E-6 XUT1 Putative high-affinity sugar transporter PICST_40173 0 0 NULL XUT2 Sugar transporter PICST_39560 1 1 1 XUT3 Putative sugar transporter PICST_16856 8 10 0.816 XUT4 Putative high-affinity xylose transporter (HGT3) PICST_33078 2 1 1 XUT5 Putative xylose transporter PICST_33971 2 4 0.688 XUT6 Sugar transporter, similar to STL12 PICST_39517 13 11 0.845 XUT7 High-affinity xylose transporter, similar to STL13 PICST_56933 40 60 0.341 YBR1 General substrate transporter PICST_65135 6 8 0.792 YBR2 Hexose transporter of the major facilitator

superfamily

17 a DEGs of significance (p < 0.05) are shaded.

18 Table S6 Transcription of genes involved in peroxisomes of S. stipitis

Locus_tag RPKM RPKM p value Gene Function

(glucose) (xylose) PICST_51364 2 4 0.688 ADR1.1 Protein similar to protein that controls the expression of ADH2, peroxisomal protein genes, and

genes required for ethanol, glycerol, and fatty acid utilization PICST_55523 1 0 1 AMO1 Peroxisomal copper amine oxidase PICST_40055 9 19 0.150 ATG1 Serine/Threonine-protein kinase (Autophagy-related protein 1) PICST_29200 4 3 1 ATG12 Autophagy-related protein 12 (Autophagy-related ubiquitin-like modifier) PICST_83006 3 6 0.510 ATG13 Autophagy-related protein 13 PICST_61658 15 26 0.215 ATG17 Autophagy-related protein 17 PICST_52962 10 11 1 ATG18 Autophagy-related protein 18 PICST_62474 5 11 0.214 ATG21 Autophagy-related protein 21 PICST_191 5 6 0.755 ATG26 Sterol 3-β-glucosyltransferase (Autophagy-related protein 26) (UDP-glycosyltransferase 51) PICST_36624 5 8 0.584 ATG3 Protein involved in autophagocytosis during starvation PICST_42095 6 8 0.792 ATG7 Autophagy-related protein 7 (Autophagy-related E1-like activating enzyme) PICST_34219 40 66 0.236 ATG8 Autophagy-related protein 8 precursor (Autophagy-related ubiquitin-like modifier) PICST_40324 50 156 0.005 CAT1 Peroxisomal catalase (PXP-9) PICST_84618 62 61 0.968 GCN1 Translational activator of GCN4 PICST_68683 41 38 0.898 GCN20 ATP-binding cassette (ABC) family, regulator of translational elongation PICST_61090 72 62 0.737 GCN3 Translation initiation factor eIF-2B α subunit PICST_43870 5 17 0.018 IDP2 Isocitrate dehydrogenase (NADP+) peroxisomal oxalosuccinate decarboxylase (IDH, CtIDP2) PICST_52187 18 25 0.479 IPM1 Pex24p integral peroxisomal membrane peroxin PICST_83848 10 18 0.223 PEX1 AAA ATPase, peroxisomal biogenesis

19 Locus_tag RPKM RPKM p value Gene Function

(glucose) (xylose) PICST_85722 15 23 0.353 PEX14 Peroxisomal membrane protein PER10 (Peroxin-14) PICST_85253 128 194 0.293 PEX19 40 kDa farnesylated protein associated with peroxisome PICST_34278 3 9 0.149 PEX2 Peroxisome assembly protein (Peroxin-2) PICST_84357 39 61 0.292 PEX3 Peroxisomal biogenesis factor 3 (Peroxin-3) PICST_31786 9 21 0.068 PEX6 Peroxisomal assembly protein PICST_49430 103 127 0.605 PIK1 Phosphatidylinositol 4-kinase (PI4-kinase, PtdIns-4-kinase) PICST_28993 62 63 1 PMP47 Peroxisomal membrane protein PMP47 PICST_55180 0 2 0.501 POT13 Peroxisomal Acetyl-CoA C-acyltransferase PICST_78783 33 50 0.329 PRB2 Vacuolar protease B PICST_82035 2 8 0.111 PXA2 Peroxisomal long-chain fatty acid import protein 1 (Peroxisomal ABC transporter 2) PICST_40664 78 141 0.138 PXP18 Oleate-induced peroxisomal protein POX18 (Lipid-transfer protein) PICST_38255 35 54 0.308 SOU1 Peroxisomal 2,4-dienoyl-CoA reductase, sorbitol utilization protein PICST_86346 20 198 1.413E-7 SOU2 Peroxisomal 2,4-dienoyl-CoA reductase, sorbitol utilization protein PICST_43336 2 4 1 SPS23 Glucose 1-dehydrogenase, peroxisomal 2,4-dienoyl-CoA reductase PICST_61314 1 0 0.013 SPS24 Peroxisomal 2,4-dienoyl-CoA reductase PICST_67034 9 19 1 THIA Acetyl-CoA acetyltransferase IA (Peroxisomal acetoacetyl-CoA thiolase, Thiolase IA) PICST_40781 4 3 NULL URO1 Peroxisomal urate oxidase PICST_74520 3 6 0.444 VAC8 Vacuole membrane protein required for vacuole inheritance PICST_58712 15 26 1 VAM6 Vacuolar carboxypeptidase Y PICST_75133 10 11 0.611 VPS13 Vacuolar sorting protein PICST_34149 5 11 0.424 VPS17 Vacuolar sorting protein PICST_63641 5 6 0.242 VPS21 Rab5-like GTPase PICST_41057 5 8 0.552 VPS24 Endosomal Vps protein complex subunit

20 Locus_tag RPKM RPKM p value Gene Function

(glucose) (xylose) PICST_36619 6 8 0.463 VPS27 Vacuolar protein sorting-associated protein hydrophilic protein PICST_80500 40 66 0.740 VPS29 Protein involved in endosome to Golgi protein transport PICST_42745 50 156 1 VPS34 1-Phosphatidylinositol 3-kinase PICST_82683 62 61 1 VPS45 Vacuolar protein sorting associated protein PICST_80446 41 38 1 VPS5 Protein involved in Golgi retention and vacuolar sorting PICST_28179 72 62 0.751 VPS53 Protein required for protein sorting at the late Golgi PICST_46785 5 17 1 VPS60 Protein involved in vacuolar protein sorting PICST_37519 18 25 0.872 VPS62 Vacuolar protein sorting protein 62 PICST_33956 10 18 0.500 VPS70 Membrane protein involved in vacuolar protein sorting PICST_80203 15 23 0.485 VPS72 Vacuolar targeting protein PICST_82218 128 194 0.508 VPS8 Vacuolar sorting protein PICST_71522 3 9 0.824 YPT1 GTP-binding protein of the Rab family PICST_51244 39 61 0.691 YPT52 GTP-binding protein of the Rab/Ypt family PICST_52636 9 21 0.186 YPT53 GTP-binding protein of the Rab family

21 Table S7 Transcription of genes of iron transporters in S. stipitis and in S. cerevisiae

Iron transporters S. cerevisiae S. stipitisa RPKM RPKM

(glucose) (xylose) Siderophore-dependent iron transporters ARN1 ARN1 18 16 TAF1 ARN2 1 1 (Fe3+ complex) SIT1 ENB1 Reductases FRE1 FRE1.1 27 20 FRE2 FRE1.2 0 0 (Fe3+, Cu2+ free or complex) FRE3 FRE1.3 135 87 FRE4 FRE1.4 48 9 FRE5 FRE1.5 0 0 FRE6 FRE2.1 4 3 FRE7 FRE3.1 10 14 FRE4.1 0 0 FRE4.2 0 0 FRE7 185 138 Ferroxidases FET3 FET3.1 76 52 FET5 FET3 51 65 (free Fe2+ complex) FET5 37 23 Iron permeases FTR1 FTR1 34 16 FTH1 FTR2 616 403 (ferroxidase-generated Fe3+) Permeases SMF1 SMF1 54 41 SMF2 SMF2 26 28 (free Fe2+) SMF3 SMF3 9 10 FET4 a DEGs of significance (p < 0.05) are shaded.