Mapping Condition Dependent Regulation of Lipid Metabolism in Saccharomyces Cerevisiae

Mapping Condition Dependent Regulation of Lipid Metabolism in Saccharomyces cerevisiae

Michael C. Jewett*,§,1, Christopher T. Workman**,1, Intawat Nookaew*,§§,†, Francisco A. Pizarro*,‡, Eduardo Agosin*,‡, Lars I. Hellgren**, Jens Nielsen*, §§, 2

* Center for Microbial Biotechnology, DTU Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, DK-2800 Kgs. Lyngby, Denmark. § Department of Chemical and Biological Engineering and Chemistry of Life Processes Institute Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA **Center for Biological Sequence Analysis, DTU Systems Biology, Technical University of Denmark, Building 208, DK-2800 Kgs. Lyngby, Denmark. §§Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-412 96 Göteborg, Sweden † Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand ‡ Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Macul, Santiago, Chile.

1 These authors contributed equally 2 To whom correspondence should be addressed. Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-412 96 Göteborg, Sweden E-mail: [email protected]

DOI: 10.1534/g3.113.006601

COt CAt NOt NAt

2% 2% 1% 1% 19% 27% 28% 21% 34% 53%

7% 4% 2% 5% 6% 67%

10% 68% 1% 42%

COT CAT NOT NAT

3% 3% 1% 4% 20% 30% 31% 40% 23% 4%

4% 44% 65% 53% 4% 1% 13% 12% 1% 45%

Sterols Sphingolipids Phospholipids Free fatty acids Storage lipids (TAG/SE)

Figure S1 Pie chart representation of measured lipid classes for each experimental condition based on µmol/gDCW (DCW = dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). For example, “COt” stands for C-limited, aerobic, and 15°C. These data highlight major changes across different conditions. In one example, nitrogen-limited aerobic conditions (NOt & NOT) have the largest percentage of storage lipids in the total lipid pool (42-45%). TAG: triacylglycerol, SE: steryl ester.

2 SI M. C. Jewett et al.

A. B. Triacylglycerol (TAG) Steryl esters (SE) 5 8 4 6 mol/ 3 mol/ g DCW 4 2 g DCW 1 2 0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT

Figure S2 Triacylglycerol (A) and steryl ester (B) content for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). The content of TAG and SE is increased under nitrogen-limited aerobic conditions (NOt/NOT).

M. C. Jewett et al. 3 SI A. Phosphatidylcholine (PC) B. Phosphatidylcholine di-substituted medium acyl chain (PCS) 10 4 8 3 mol/ 6 mol/ g DCW g DCW 2 4 2 1 0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT

Figure S3 Phosphatidylcholine (A) and di-substituted medium acyl-chain phosphatidylcholine (B) content for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N- limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). Di-substituted medium acyl-chain phosphatidylcholine is only present under anaerobic conditions.

4 SI M. C. Jewett et al.

A. Phosphatidylinositol (PINS) B. Phosphatidylinositol di-substituted medium acyl chain (PINSS)

8 2.5

6 2 mol/ mol/ 1.5 4 g DCW g DCW 1 2 0.5 0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT

Figure S4 Phosphatidylinositol (A) and di-substituted medium acyl-chain phosphatidylinositol (B) content for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N- limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). Di-substituted medium acyl-chain phosphatidylinositol is only present under anaerobic conditions.

M. C. Jewett et al. 5 SI

A. B. Phosphatidylserine (PS) Phosphatidylethanolamine (PE) 1 2.5 0.8 2 mol/ 0.6 mol/ 1.5 g DCW g DCW 0.4 1 0.2 0.5 0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT

Figure S5 Phosphatidylserine (A) and phosphatidylethanolamine (B) content for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). Phosphatidylethanolamine increases at lower temperatures.

6 SI M. C. Jewett et al.

A. B. Free fatty acids (FFA) Ergosterol (ERGOST) 2 8

1.5 6 mol mol/ g DCW 1 g DCW 4

0.5 2

0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT

Figure S6 Free fatty acids (A) and ergosterol (B) content for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). The concentration of free fatty acid is increased under anaerobic conditions, low temperature. Ergosterol levels are significantly higher under aerobic conditions, as expected since ergosterol biosynthesis is not active without oxygen. Note that anaerobic ergosterol levels represent amounts absorbed from the medium.

M. C. Jewett et al. 7 SI

A. B. Phospholipids Storage lipids 20 12 15 10 8 mol/ 10 mol/ g DCW g DCW 6 5 4 2 0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT

Figure S7 Total phospholipid (A) and storage lipid (B) content for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). Total phospholipid content (phosphatidylinositol, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine) is most dramatically regulated by temperature and oxygen availability. Storage lipids (triacylglycerol and steryl esters) demonstrate a major change in metabolism under N-limited, aerobic conditions.

8 SI M. C. Jewett et al.

Total fatty acids in all lipids 40

30 mol/ g DCW 20

0 COt COT CAt CAT NOt NOT NAt NAT

Figure S8 Total fatty acid content in all lipid species for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). Total fatty acid content is increased at low temperature and under nitrogen-limitation.

M. C. Jewett et al. 9 SI

A. B. C. c10:0 c12:0 c14:0 5 3 3 4 2.5 2.5 2 2 mol/ 3 mol/ mol/ 1.5 g DCW 2 g DCW g DCW 1.5 1 1 1 0.5 0.5 0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT 0 COt COT CAt CAT NOt NOT NAt NAT D. E. F. c16:0 c18:0 c14:1 10 2 1.2 8 1 1.5 6 0.8 mol/ mol/ mol/ 1 0.6 g DCW 4 g DCW g DCW 0.4 0.5 2 0.2 0 0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT

G. H. c16:1 c18:1 20 12 10 15 8 mol/ mol/ 10 6 g DCW g DCW 4 5 2 0 0 COt COT CAt CAT NOt NOT NAt NAT COt COT CAt CAT NOt NOT NAt NAT

Figure S9 Total acyl chain composition from all measured lipid species (phosphatidylinositol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, triacylglycerol, steryl esters, and free fatty acids) for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). The notation for acyl-chain length is given as c10:0, where the first number (in this case “10”) is the number of carbon atoms and the second number (in this case “0”) is the number of double bonds. Key observations include: medium chain acyl groups (C10:0, C12:0, and C14:0) are greater under anaerobic conditions, C16:0 is greater under anaerobic conditions, C18:0 is greater under nitrogen limitation, C14:1 and C16:1 are greater at lower temperatures where both a decreased chain length and the cis double bond will reduce the interaction between the acyl chains, and C18:1 is generally greater under nitrogen limitation.

10 SI M. C. Jewett et al.

A. B. C. mRNA Lipids Metabolites

C vs N O vs A C vs N O vs A C vs N O vs A

525 145 234 7 21 20 3 9 4 53 15 6 187 84 4 9 6 2

627 2 3

T vs t 3781 T vs t 19 T vs t 17

Figure S10 The overlap amongst significant genes (A), lipids (B), and metabolites (C) at a threshold of P ≤ 0.001 following Bonferroni correction across experimental conditions. C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”. See Table S4 for the list of genes from the Multi-way ANOVA.

M. C. Jewett et al. 11 SI Supplementary'Figure'11'

The TCA cycle and

TRP1 TRP2 trp

TRP3 TRP4 PHA2 TRP1 TRP2 amino acid biosynthesis trp PHES phe TRP5 TRPS ARO7 TRP3 ARO9 TRP4 PHA2 pphn ARO8 PHES phe ARO4 g3p TYRS tyr TRP5 ARO7 ARO3 PPHNS TRPS TYR1 ARO9 pphn ARO8 ARO2 ARO1 e4p ARO4 g3p TYRS tyr ARO3 PPHNS SER3 SER1 chor TYR1 CHORS aald 3pg ARO2 ARO1 SER33 SER2 e4p SER3 SER1 SHM2 chor CHORS pep aald SERS SHM1 GLY1 3pg GLYS2 SER33 SER2 GLYS1 SHM2 ser PRO1 pep SERS GLY1 PRO3 SHM1 PEPS2 ala gly GLYS2 THR4 PRO2 PCK1 GLYS1 thr ser pro PRO1 PRO3 PEPS2 SERDG THR1 gly ala PROS ile ILV5 ILV3 THR4 THRS ALT2 PRO2 PCK1 CHA1 YLR089C thr ILV1 pro CALAS ILV6 SERDG THR1 PROS ile ILV5 ILV3 GLN1 ILV2 THRS YLR089C ALT2 ASN1 ILES CHA1 glu ILV1 CALAS ILV6 GDH2 BAT2 GLNS ASN2 GLUD GLYS3 GLN1 ILV2 ASN1 ILES pyr AKIVS glu ASNS AGX1 val GDH2 BAT2 GLUS2 GLNS ASN2 asn GLUS VALS GLUD GLYS3 HOM2 gln pyr AKIVS HOM3 GLT1 GDH3 BAT1 AGX1 akiv ASNS GDH1 GLUS2 val HOM6 CYS4 CYS3 asn akg GLUS VALS LEUS2 HSERS leu HOM2 STR3 MAE1 gln STR2 ASPS HOM3 GLT1 GDH3 BAT1 ASPS2 akiv MAE LEU2 ASP1 OAAS2 GDH1 ippmal CYS3 HOM6 asp AAT2 LEU1 CYS4 ASP3-1 PYC1 akg HSERS LEUS2 leu ASP3-2 AAT1 PYC2 STR3 ASPS MAE1 cys CYSS STR2 ASP3-4 ASP3-3 LEUS1 ASPS2 oaa LEU9 MAE LEU2 IDH1 ASP1 OAAS2 ippmal asp AAT2 LEU1 ASP3-1 PYC1 hser accoa ASP3-2 AAT1 PYC2 LEU4 LYS9 cys IDP1 AKGS LYS12 CYSS ASP3-3 LEUS1 ASP3-4 LEU9 ARG4 oaa IDH1 IDP3 ARG3 MDH1 LYS21 accoa MDH3 hser IDH2 IDP2 LYS1 LEU4 LYS20 LYS9 ac ARG1 IDP1 AKGS LYS12 ORT1 ARGS ARG4 OAAS1 mal LYSS MDH2 IDP3 coa ARG3 MDH1 LYS21 LYS2 LYS4 hcys HCYSS CPA2 MDH3 IDH2 IDP2 LYS1 MET16 LYS20 CPA1 CIT3 ac ARG1 ORT1 ARGS CITS GLYXS2 mal CIT2 MDH2 OAAS1 LYSS ECM17 CIT1 icit coa LYS2 MET17 MET3 HCYSS LYS4 SUL2 MET10 lys hcys CPA2 arg MET16 ARG2 DAL7 CPA1 CIT3 SUL1 MET14 ECM40 glx MET2 MLS1 CIT2 CITS GLYXS2 ECM17 ARG5,6 CIT1 MET6 METS MET17 MET3 icit SUL2 MET10 lys MALS cit ARG8 arg ARG2 DAL7 CAR1 SUL1 MET14 ECM40 ARGD glx MET2 MLS1 orn ORNS GLYXS1 FUM1 ARG5,6 ISCITS MET6 METS met ARG8 MALS cit ACO1 ICL1 SPE4 SPE3 ACO2 CAR1 ARGD orn ORNS GLYXS1 FUM1 SPE2 ISCITS SPE1 KGD2 LSC1 met ACO1 ICL1 SPE4 ACO2 SAMS SPE3 SUCS KGD1 LSC2

sam SPE2 SAM1 SPES SPE1 KGD2 LSC1 fum SAM2 YEL047C OSM1 SAMS suc SUCS KGD1 LSC2 spe FUMR sam SAM1 SPES

SAM2 fum YEL047C OSM1 suc FUMS spe FUMR

SDH4 FUMS SDH2 SDH1 YJL045W SDH3 SDH4 SDH2 SDH1 YJL045W CvsN Tvst SDH3

TRP1 TRP2 trp

TRP3 TRP4 PHA2

PHES phe TRP5 TRPS ARO7

ARO9 pphn ARO8

ARO4 g3p TYRS tyr ARO3 PPHNS TYR1

ARO2 ARO1 e4p SER3 SER1 chor CHORS aald 3pg

SER33 SER2

SHM2 pep SERS SHM1 GLY1 GLYS2 GLYS1 Node color ser PRO1 PRO3 PEPS2 gly ala THR4 PRO2 PCK1 log ratio X/Y thr pro THR1 2 SERDG PROS ile ILV5 ILV3 THRS ALT2 CHA1 YLR089C ILV1 10 CALAS ILV6 Node and edge type key: GLN1 ILV2 ASN1 ILES glu

GDH2 BAT2 GLNS ASN2 GLUD GLYS3 AKIVS 5 pyr ASNS AGX1 GLUS2 val Gene asn GLUS VALS HOM2 gln HOM3 GLT1 GDH3 BAT1 akiv GDH1 CYS4 CYS3 HOM6 akg HSERS LEUS2 leu 0 STR3 MAE1 STR2 ASPS ASPS2 MAE LEU2 ASP1 OAAS2 ippmal Metabolite/lipid asp AAT2 LEU1 ASP3-1 PYC1 ASP3-2 AAT1 PYC2 cys CYSS ASP3-3 LEUS1 ASP3-4 LEU9 oaa IDH1

hser accoa LEU4 -5 LYS9 IDP1 AKGS LYS12 ARG4 Reaction IDP3 ARG3 MDH1 LYS21 MDH3 IDH2 IDP2 LYS1 LYS20 ac ARG1 ORT1 ARGS OAAS1 mal LYSS -10 MDH2 coa LYS2 LYS4 hcys HCYSS CPA2 MET16 CPA1 CIT3 Distribution CIT2 CITS GLYXS2 ECM17 CIT1 MET17 MET3 icit SUL2 MET10 lys arg ARG2 DAL7 SUL1 MET14 ECM40 glx Node border MET2 MLS1 ARG5,6 MET6 METS ARG8 MALS cit CAR1 ARGD Gene-reaction log GLYXS1 P (X/Y) orn ORNS FUM1 ISCITS met 10 ACO1 ICL1 SPE4 SPE3 ACO2

SPE2 SPE1 KGD2 LSC1 metabolite/lipid-reaction SAMS SUCS KGD1 LSC2 sam -17 SAM1 SPES

SAM2 fum YEL047C OSM1 suc spe FUMR lipid-distribution

FUMS

SDH4 SDH2 SDH1 YJL045W SDH3 -2 OvsA 0

' ' '

' Figure'S11:'The!condition!dependent!response!of!the!TCA!cycle!and!amino!acid!biosynthesis! Figure S11 The condition dependent response of the TCA cycle and amino acid biosynthesis comparing C-Limited versus N- limited, comparing!30°C versus 15°C, and aerobic CRLimited! versus!versus anaerobic conditions. The areas highlighted show different up NRlimited,! 30°C! versus! 15°C,! and! aerobic! versus!- anaerobic!and down- regulation patterns conditions.!The!areas!highlighted!show!different!upillustrating how Cytoscape visualization enables the targeted and rapid identification of changes across the R!and!downR!regulation!patterns!illustrating! cell. Chow!C-limited, “C”; Nytoscape!visualization!enables!the!targeted!-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”and!rapid!identification!of!changes!across!the!. Measurement ratios were visualized with a logcell.! !2 CcolorRlimited,!-bar and the color of each node border represents the log “C”;! NRlimited,! “N”;! aerobic,! “O”;! anaerobic,!10(p-value) (see node and edge color key). Gray “A”;! 30°C,! “T”;! and! 15°C,! “t”.! coloring indicates the lack of a measurement for that node. Measurement!ratios!were!visualized!with!a!log !colorRbar!and!the!color!of!each!node!border! 2 represents!the!log10(pRvalue)!(see!node!and!edge!color!key).!!Gray!coloring!indicates!the!lack!of! a!measurement!for!that!node.! ! !

! Jewett!et#al.!! 17!SI!

12 SI M. C. Jewett et al.

Phospholipid biosynthesis

INP51INP52 INP53 INP54 INP51INP52 INP53 INP54

MSS4 PIK1 FAB1 MSS4 PIK1 p l FAB1 p l PIPSS tag141tag161 tag181 PIPSS STT4 tag141 tag161 tag181 STT4 ag160 VPS34 tag180 PLC1 VPS34 TAGDIS tag160 tag180 ! TAGDIS PLC1 LSB6 tag100 tag120 tag140 CKI1 LSB6 PCT1 tag100 tag120 tag140 CKI1 CPT1 PCT1 PLIPC pips PINK CPT1 PLIPC pips PINK TAGSN TAGSN tag PTDCHOS1 tag PTDCHOS1

pc181 pc141 pc161 pc161 pc181 LRO1 pc141 LRO1 pc160 pc180 cho pc160 pc180 cho pc100 pc120 pc140 pc100 pc120 pc140 pcs141 pcs161 pcs181 pcs141 pcs161 dag PCDIS pcs181 DGA1 dag PCDIS pcs160 pcs180 DGA1 DAGAT pcs160 PCSDIS DAGAT pcs180 PCSDIS pcs100 pcs120 pcs140 GDE1 pins141 pins161 pins181 pcs100 pcs120 pcs140 GDE1 pins141 pins161 pins181 coa pc pins pins160 pins180 coa pc PINSDIS pins160 pins180 DAGEST PINSDIS pins100 pins120 pins140 DAGEST pins100 pins120 pins140

CHO2 acylcoa PAH1 pins pinss14p1inss161pinss181 CHO2 PTDCHOS2 OPI3 acylcoa PAH1 pins pinss14p1inss161pinss181 DAGS PTDCHOS2 OPI3 GUT1 PINSSDIS DAGS pinss160pinss180 GUT1 LPAS1 DPP1 PINSSDIS pinss160pinss180 gro MUQ1 LPAS1 DPP1 GPT2 GLYCK pinss100 pinss140 MUQ1 PTDETNS1 pinss120 gro pinss100 sam GPT2 GLYCK PTDETNS1 pin ss120pinss140 AYR1 sam SCT1 LPP1 AYR1 SCT1 LPP1 g l EPT1 lpa EPT1 LPAS2 lpa g l LPAS2 PAS PAS adhap pe161 pe141 adhap pe161 SLC1 pe181 pe141 CRD1 EKI1 etnp pe pe181 SLC1 pe PEDIS pe160 pe180 CRD1 EKI1 etnp PGS1 PEDIS pe160 pe180 PGS1 CLRS ETNPS pe120 pe140 INM1 pe100 INO1 CLRS pe120 pe140 YDR287W ETNPS pe100 INM1 INO1 YDR287W pa etn pa PSD1 etn cl PSD1 PTDETNS2 cl G3PDH INSS g6p PTDETNS2 PSD2 PISS ins G3PDH INSS g6p ADHAPS PSD2 PISS ins ADHAPS GPD2 GPD1 GPD2 GPD1 cdpdag PIS1 CDPDAGS cdpdag PIS1 DAK1 DAK2 CDPDAGS GUT2 DAK1 DAK2 GUT2

glyon DHAPK dhap CDS1 ps glyon DHAPK dhap CDS1 ps PTDSERS PTDSERS ser ser

CHO1 PSDIS CHO1 PSDIS

ps100 ps120 ps140 ps100 ps120 ps140 ps160 ps180 ps160 ps180 ps141 ps161 ps181 CvsN ps141 ps161 ps181 Tvst

INP51INP52 INP53 INP54

MSS4 PIK1 FAB1 p l PIPSS tag141 tag161 tag181 STT4 VPS34 tag160 tag180 TAGDIS PLC1 Node color LSB6 tag100 tag120 tag140 CKI1 ! PCT1 CPT1 PLIPC pips PINK log ratio X/Y TAGSN 2 tag PTDCHOS1 10 pc181 pc141 pc161 LRO1 pc160 pc180 cho

pc100 pc120 pc140 pcs141 pcs161 pcs181 5 dag PCDIS Node and edge type key: DGA1 pcs160 DAGAT pcs180 PCSDIS pcs100 pcs120 pcs140 GDE1 pins141 pins161 pins181 pc coa pins160 180 PINSDIS pins DAGEST 0 pins100 pins120 pins140 Gene

CHO2 acylcoa PAH1 pins pinss14p1inss161pinss181 PTDCHOS2 OPI3 DAGS GUT1 PINSSDIS pinss160pinss180 LPAS1 DPP1 MUQ1 gro pinss100 GPT2 GLYCK PTDETNS1 pinss120pinss140 -5 sam Metabolite/lipid AYR1 SCT1 LPP1 EPT1 lpa g l LPAS2 PAS -10 adhap pe141 pe161 SLC1 pe181 CRD1 EKI1 etnp pe Reaction PEDIS pe160 pe180 PGS1 CLRS ETNPS pe100 pe120 pe140 INM1 INO1 YDR287W pa Node border etn PSD1 cl Distribution PTDETNS2 G3PDH INSS g6p PSD2 PISS ins ADHAPS log P (X/Y) GPD2 GPD1 10 PIS1 CDPDAGS cdpdag DAK1 DAK2 GUT2 Gene-reaction glyon DHAPK dhap CDS1 ps -17 PTDSERS ser

CHO1 metabolite/lipid-reaction PSDIS

ps100 ps120 ps140 ps160 ps180 lipid-distribution ps141 ps161 ps181 OvsA -2 0

Figure S12 The condition dependent response of phospholipid biosynthesis comparing C-Limited versus N-limited, 30°C versus 15°C, and aerobic versus anaerobic conditions. The areas highlighted show different up- and down- regulation patterns illustrating how Cytoscape visualization enables the targeted and rapid identification of changes across the cell. C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”. Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key). Gray coloring indicates the lack of a measurement for that node.

M. C. Jewett et al. 13 SI

HXT1 HXT4 HXT2 HXT5 ERG10 GLK1 ERG9 . . ERG20 ERG13 ERG12 HMG2 A HXK1 HXT3 HXT7 HXT10 B IDI1 MVD1 ERG8 HMG1

HIS2 HXT6 HXT17 HXT14 HXT13 ZWF1 GND2 SOL3 HIS1 his mev5p MEV5PS mev MEVS hmgcoa HMGS accoa HXT16 HXT11 HXT15 SQLS HXK2 HXT9 RKI1 PRS2 HIS6 HIS3 HXT8 SOL4 GND1 HIS4

PRS4 PRS5 PRS1 HISS coa gluc G6PS g6p OPP rb5p R5PS sql r5p HIS7 PRS3 HIS5 ERG26 ERG28 NCP1

TPS1 TPS3 TPS2 gln X5PS akg SQLE TSL1 UDPglc ERG1 ERG24 ERG4 ERG7 TRES x 5 p TAL1 RPE1

NTH1 G3PS7PS1 ERG11 ERG6 ERG2 F6PS PGI1 TKL2 epxsql sam NTH2 TKL1 TRED trehalose E4PF6PS G3PF6PS ERG27 ERG3 ERG5 ERG25 ATH1 ERGS f6p

s7p ARE2 PFK2 ARE1 PFK FBP FBP1 acylcoa STRES PFK1 e4p ergost

f16bp c100

c141 c120 FBA1 FBA ffa FFADIS SEHYLS se c161 c140

c181 c160 dhap G3PS1 g3p c180 SEDIS TPI1 YEH2 TGL1 YEH1

TDH1 se100 se120se140se160 se180 TDH3 3PGS TDH2 se141 se161 se181 PGK1

3pg

Central Metabolism: C-limited vs. N-limited Sterol Metabolism: aerobic vs. anaerobic

3pg

ENO1 ENO2 C. PEPS1 GPM1 D. GPM2 GPM3

pep tag LRO1 pc181 pc141 pc161 cho pc160 pc180 LPD1 pc100 dag pc120 pc140 PDA1 DGA1 pcs141 pcs161 pcs181 YDR531W ECM31 YIL083C DAGAT PCDIS PDX1 PYK2 PDB1LAT1 pcs160 pcs180 PYRS1 GDE1 pins141 pins161 pins181 YGR277C PAN6 PAN5 PCSDIS SIS2 pcs120 pcs140 CDC19 coa pcs100 pins160 pins180 ACCOAS1 PINSDIS coa VHS3 YKL088W DAGEST pc YDR196C pins100 pins120 pins140 FMS1

pins pinss161pinss181 DLD1 DLD2 ACS2 acylcoa PAH1 pinss141 DLD3 DAGS spe CHO2 GUT1 PINSSDIS pinss180 pyr pinss160 COAS DPP1 PTDCHOS2 OPI3 lac LACTS ACH1 MUQ1 accoa gro pinss100 GLYCK PTDETNS1 pinss120pinss140 ACCOAS2 akiv LPP1 ACS1 g l EPT1 sam

pe141 pe161 pe181 CRD1 EKI1 etnp pe PEDIS pe160 pe180 PGS1 ALD3 CLRS ac ETNPS pe100 pe120 pe140 PDCR INO1 PDC1 INM1 YDR287W ALD2 pa etn PSD1 cl PTDETNS2 PDC6 PDC5 INSS g6p PSD2 PISS ins

OACS cdpdag PIS1 aald CDPDAGS ps ALD4 ALD5 ALD6 PTDSERS ADH1 ADH2 ser

ADH3 ADH4 ALDH ADH5 SFA1

etoh

Central Metabolism: C-limited vs. N-limited Phospholipid metabolism: aerobic vs. anaerobic

Node color Node border log ratio X/Y log P (X/Y) Node and edge type key: 2 10 10 Gene Gene-reaction -17 5 Metabolite/lipid metabolite/lipid-reaction 0 Reaction lipid-distribution -5 -2 Distribution -10 0

14 SI M. C. Jewett et al.

E. F. C16ald etnp

ECI1 SPS19 DCI1 DPL1 PSPHPL1 PSPHPL2 SUR4 FEN1

IFA38 TCOA2 TSC13 tencoa dhs1p phs1p POT1 CRC1

TCOA1 FOX2 vlcfa VLFAS YSR3 POX1 FAO LCB4 CTA1 SBPP1 SBPP2 SLCBK2 SLCBK1 LCB5 LCB3 TSC3 LCB1 SUR2 acylcoa coa accoa TSC10 OLE1 OLE1R LCB2 palmcoa

SERPT kdh5 KDH5R sph PSPHS psph HFA1 ser

MALCOS BPL1 LAC1 FAT1 FAA3 FAA4

BOXD coa CERS LAG1 ACC1 YDC1 FAA1 PXA2 CERAFS malcoa LIP1 CERAS2 CERAS ffa FAS YPC1 FAA2 PXA1 ACP1 FAS2 lacylcoa OAR1PPT2 FFADIS vlcfa MCT1 ELO1 FAS1 CEM1

c100 c120 c140 ACB1 ETR1 HTD2 c160 cerD CER2S cerP c180 M2CS mip2c

c141 c161 c181 SCS7 CSG2 IPT1 AUR1

CSH1 SUR1 Fatty Acid Metabolism: 30C vs. 15C Sphingolipid Metabolism: 30C vs 15C G. H. cho pa

gro

LIPS3 SPO14

AGX1 GLUS2 val ffa LIPS2 pc GLUS pyr VALS gln HOM3 GLT1 GDH3 BAT1 akiv GDH1 PLB1 GDE1 NTE1 HSERS LEUS2 leu ASPS MAE1 akg OAAS2 MAE LEU2 ippmal asp AAT2 PYC1 LEU1 TGL3 PLB2 PLB3 SPO22 AAT1 PYC2 TGL2 LEUS1 oaa LEU9 IDH1 TGL4 accoa LIPS1 LEU4 LYS9 IDP1 AKGS LYS12 ARG4 TGL5 IDP3 dag ARG3 MDH1 LYS21 MDH3 IDH2 IDP2 LYS1 tag ARG1 LYS20 ORT1 ARGS mal MDH2 OAAS1 LYSS coa LYS2 LYS4 CPA2 CPA1 CIT3

CIT2 CITS GLYXS2 CIT1 icit lys arg ARG2 DAL7 ECM40 glx MLS1 ARG5,6

ARG8 MALS cit CAR1 ISCITS ARGD GLYXS1 Lipase degradation: aerobic versus anaerobic orn ORNS ACO1 FUM1 ACO2

TCA cycle: 30C vs. 15C

Node color Node border Node and edge type key: log ratio X/Y log P (X/Y) 2 10 10 Gene Gene-reaction -17 Metabolite/lipid metabolite/lipid-reaction 5 0 Reaction lipid-distribution -2 Distribution -5 -10 0

Figure S13 The condition dependent response of small cellular networks as visualized using Cytoscape. (A) Central metabolism: glycolysis, CvsN. (B) Sterol metabolism: OvsA. (C) Central metabolism: pyruvate metabolism, CvsN. (D) Phospholipid metabolism: OvsA. (E) Fatty acid metabolism: Tvst. (F) Sphingolipid metabolism: Tvst. (G) Central metabolism: TCA cycle, Tvst. G. Lipase metabolism: OvsA. C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”. Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key). Gray coloring indicates the lack of a measurement for that node.

M. C. Jewett et al. 15 SI Non-polar lipids/phospholipids 6

5 Dimensionless 4 (mol/gDCW) 3 (mol/gDCW) 2

0 COt COT CAt CAT NOt NOT NAt NAT

Figure S14 The ratio of non-polar lipids (free fatty acids, sterols, and steryl esters) to phospholipids (phosphatidylinositol, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine) for each experimental condition based on µmol/gDCW (dry cell weight). Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). There is a shift away from phospholipid synthesis under nitrogen-limited aerobic conditions (i.e., higher ratio above).

16 SI M. C. Jewett et al.

Glucose

100.00 100.00 1.59 100.00 1.85 100.00 0.32 0.44 CO 16.83 51.10 19.56 41.69 1.57 14.61 BIOMASS G6P P5P 16.97 15.64 1.69 11.56 13.82 18.19 32.07 4.81 1.45 38.76 15.57 3.75 1.59 83.83 12.20 86.74 4.68 3.62

1.40 1.62 F6P 0.13 E4P 0.13 64.61 64.78 16.97 3.84 93.31 13.82 5.01 94.12 4.81 0.40 3.75 0.40 S7P G3P

140.95 136.74 0.00 176.98 v47 0.00 179.30 13.91 GLYC 2.81 12.15 3.28 v45 0.26 0.26 PEP ETH 138.14 133.47 0.00 176.72 0.00 172.83 5.62 179.04 15.64 174.96 6.53 18.19 0.52 174.28 0.53 33.70 CO 15.64 15.64 CO 176.55 24.12 18.19 18.19 OAAcyt 1.01 PYRcyt ACA 1.45 ACE 1.45 AcCoAcyt 1.03 1.59 1.59 88.81 91.15 -28.08 1.43 -17.59 1.46 10.10 -0.49 11.73 -0.50 0.94 CO 0.96 CO 101.51 90.90 COt PYRmit 0.49 0.50 22.81 COT 11.48 101.51 0.00 90.90 CAt 0.00 0.49 AcCoAmit 0.50 CAT

OAAmit

161.68 MALmit 192.40 ICIT 0.45 0.99 101.51 90.90 0.49 0.50 227.12 300.00 0.91 CO 1.99 FUM AKG 96.25 84.79 0.00 0.00 5.27 6.11 CO 0.49 0.50

Figure S15 13C-flux analysis demonstrates carbon flux through the pentose phosphate pathway is significantly decreased under anaerobic conditions (CAt & CAT relative to COt & COT). For example, flux from glucose-6-phosphate (G6P) to pentose-5- phosphate is approximately 3.5-fold higher in the aerobic case. All abbreviations are directly taken from Gombert et al., 2001. Carbon fluxes, measured here, are normalized to glucose uptake. Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”).

Jewett et al. 17 SI

PIP2

MIG1 UPC2

OAF1 GCN4 DAL81 HAP1

DAL80 C C:O O NRG1 T

GLN3 SNF1 ROX1 HSF1

- Transcription Factor

- Experimental condition

Figure S16 Network of translation factors enriched for different growth factors. Edge thickness represents the number of known transcription factor regulatory targets observed in the experimental condition (with thicker lines indicated more genes) (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”).

18 SI M. C. Jewett et al.

PINSS120 C-limited versus N-limited: IML3 NFU1 PC160 negative PCC correlations YLR267W YAT1 PS140 ATP20 COR1 POX1 FRE7 OMA1

ATP5 NCE103 PC100 CTA1 SIP18 HAP3 TAG140 KGD1 KGD2 SDH2 MCR1 YCP4 NDE1 RIP1 PLB2 SE161 GRE1 SPG1 PINSS180 HAP4 SUL1 UGO1 ATP18 CAF17 YOR356w IZH1 ATP17 COX5a CAT8 EAF3 YDR161W YDR493W MRPL49 YIL158W YJR080C MRPS9 C180 IZH4 TAG181 C181 SCM4 YBR269C ALD4 CBP4 YPL113C ADR1 CIT1 TIM11 COX12 STM1 RIM1 SMD1 YOR021C HAP5 MRPL11 GIS2 TAG161 DSF1 TAG100 PDH1 ATP3 YNL100W NDE2 ACN9 YKR016W SDH1 GUT2 ACH1 QRI5 MHR1 MRPL31 TIF35 YGL117W MNP1 SE181 GSY1 TSA2 PC140 NCB2 PINS180 HAP2 LHP1 RBG2 SUT1 YBL059W MRP13 MAM33 MHT1 KNH1 ATP15 JID1 SRL3 C161 NDI1 PINSS140 RML2 MRPL17 MRPL16 PET117 COX23 MDM38 YLR243W YGR287C ATP4 COX7 HAP1 TIP1 SE180 OM14 MTO1 RSM25 COX11 LIP2 RSM24 RRF1 MRPS28 MEF1 EMI2 PINSS160 ATP7 ATP16 BAP2 TAG180 TAG120 PHB1 PPA2 HXK1 HXK2 PINSS100 MDH1 AGX1 POS5 YML050W THI73 MAL32 PE141 MRPS18 PS180 YJR100C TAG141 YER067W PCS180 ATP14 YFH1 YGR146C PS181 CPR3 LAP4 PE161 SUC2 OXR1 SDH3 PCS160

TAG160 YPR011C PS160 PRM5 RIB1 HSP12 TPK2 ZTA1 PC181 PCS140 ERG7 YNR034W-A YGR110w

C160 AFT1 STR3 MLS1 FTR1 PS120

PCS100 DAL3 GAP1

C140 B. YJL171C LRO1

YMR226C ALG8 STT3 NIT1 NUP60 RCE1

PUT2 TAG100 LEM3 MGS1 YHI9

RXT2 YPL014W YPL245W YIL165C AVT1 TAG141 YNL311C YNR065C HNM1 VPH1 GAS5

C-limited versus N-limited: DAL2 TAG140 LAS21 TPN1 PAN5 GAS3

positive PCC correlations YPL199C PC100 ATG22

TUP1 VMA2 GAL83 UGA3 YCR024C-B PLB1 ECM32 C100

C181 YLR301W PMP1 SYN8 MUQ1 TAG160 PDC5

YDR090C YPL056C YDR089W CCR4 DSE2

MNT4 TFP1 TAG120 PS120 YFL052W MUP3 DSF2 KIP2

SE120 BAP3 C180 VHR1 TEA1 NIT3 MYO5

SE140 BSC6 RIM101

EDC2 MGA1 YGL140C BRO1 YNR047W PINS180

PS140 HMS2 SML1 SKN1 KOG1 MNR2 EKI1 ICT1 GAT1 PPQ1

DUR1,2 YGK3 AMD2 WHI4 VTC3 YHR112C PS161 STL1

YHR113W IML2 BEM3 YLR257W PS180 YDR540C IML1

NPR1 RVS161 APE2 TOR1 KIC1 NAB6 UGA4 YOR283W

AZF1 YDL237W

Node color Node border log ratio X/Y log P (X/Y) 2 10 10 Node and edge type key: -17 5 Gene Negative correlation Metabolite Positive correlation 0 Lipid Transcription factor Transcription Factor interaction -5 -2 -10 0

Figure S17 Correlation analysis demonstrates significant (P ≤ 0.001 following Bonferroni correction) relationships between genes and lipids as characterized by length when comparing carbon-limited versus nitrogen-limited conditions. (A) Negative Pearson Correlation Coefficients (PCC). (B) Positive Pearson Correlation Coefficients (PCC). For example, C18:0 is negatively correlated to PDH1. Enriched transcription factors are shown (yellow edges). Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key).

M. C. Jewett et al. 19 SI A. NCE102 C141 YAL049C PINS100 RIM4 PE100 UFE1 Aerobic versus Anaerobic: PUT4 YBR138C PINS141 PCS181 SLM1 PE120

PC141 PNS1 YOR154W PE140 negative PCC correlations PINS140 EST2 PINSS181 PCS141 AQY2 YME2 PE160 ZEO1 YLL053C ANT1 YTP1 POX1 SPG1 PINSS161 PET10 TAG140 PCS100 CTA1 GRE1 UBC5 PCS160 YLR152C YER053C-A MDV1 AFT1

YOL048C SUE1 PCS140 CCC2 PCS161 YGR287C LSP1 PINS160 NDE2 MDG1 TPK2 PEX21 FET3 SOD2 PINSS100 YGR110w C140 TAG100 YBR269C SUT1 YLR168c C161 PINS181 YGL160w YFL061W HPA2 HAP4 GSY1 DSF1 ECM13 SDH3 PCS180 INH1 JID1 STR3 GAP1 PRP11 PS120 YER067W TAG141 EMI2 C160 HAP1 GVP36 PINSS160 NDI1 MLS1 DAL3 YDR248C YGR266wYOR215C ATP14 YGL039W YGR146C PC181 POP4 NDE1 HAP2 SLC1 FAA1 AAC1 PINSS140 HSP12 ATP16 C180 PLB2 TAG161 COX4 HXK2 HXK1 NCE103 YBR047W ISU1 ROX3 PCS120 PC140 ATP4 PDH1 IZH1 OM14 HAP3 TAG160 OMA1 ATP20 ERG7 ZTA1 LSB6 TIP1 HAP5 PINS180 KNH1 COX5a PINSS180 COX12 AGX1 TAG181 MIC17 YPL107W MAL32 SCM4 PPH3 COX6 PC100 SDH2 YNL100W AGP1 KGD2 ALD4 SRL3 COX8 PC161 SE181 PC160 C181 MSB2 TAG180 ATP7 RIP1 YKR016WYPL113C IZH4 IDI1

PS140 COR1 ATP17 YOR356w GUT2 SE161 DAN3 PS161 AQR1 PET9 YPL272C PC120 TIM11 POS5 SE180 PE161 PDR15 OLE1 IML3 PINSS120

B. GAL80

HMS2

PINSS120

PS140 DAN4 PS120 YFL052W PC100 PC120 PDR11 Aerobic versus Anaerobic: SE140

BAP3 PINSS141 SRO77 HES1 positive PCC correlations ERG24 SE120 PCS180 EXG2 YCL021W-AYML083c ERG28

YMR226C PC140 TAG141 PC141 SET4 ARE1 AUS1 RER2 PINSS180 KAR2 TAD2 SE161 GPX2 YGR131w SSK22 PINS141 PINSS140 ROX1 ERG1 CSR1 YLR413W ARC40 YAL049C PINSS160 PINSS181 TAG161 IZH2 TIR3 ADH5 HAP1 YBL095W PINSS100 MPD1 PCS120 PMT3 YGL039W MAE1 PINSS161 DAN1 C161 PPH3 YJR116W HEM13 ROX3 PCS181 MOT3 PCS140 SMA1 HUG1 TIR2 COQ6 PS161 STL1 UPC2 AAC3 PC181 PINS160 LEU2 YHL044W ANB1 C180 VHR1 PCS160 TUP1 SLC1 PTP1 PMT5

YDR540C PINS180 C140 INM1 YOR012W TIR4 TIR1 YHR112C PCS100

TAG140 PINS140 YPL014W RIB4

PCS141 TAG100 RHR2

PCS161 BAT1 TAG120 YDR090C ADH3 CKS1

KTR5 PINS181

ECM32 SCW11YFR026C YHP1 YOR214C C181 C120

MUQ1 UNG1

PINS100

Figure S18 Correlation analysis demonstrates significant (P ≤ 0.001 following Bonferroni correction) relationships between genes and lipids as characterized by length when comparing aerobic “O” versus anaerobic “A” conditions. (A) Negative Pearson Correlation Coefficients (PCC). (B) Positive Pearson Correlation Coefficients (PCC). Enriched transcription factors are shown (yellow edges). Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key).

20 SI M. C. Jewett et al.

KNH1 YLR243W TOM22 MRPL31 YOR021C

A. RML2 MNP1 COX11 MRPS28 MRPS9

PINS180 STM1 YGL117W ATP12 TIF35

OAF1 YIL158W SMD1 YDR161W MRPL11 PC120 PET9 MTO1 SEC5 30C versus 15C: RPB3 GIS2 RIM1 LIP2 ECM30 PS140 PINS141 negative PCC correlations AGP1 MSB2 PS180 EST2

PC161 YJL133C-A RGR1 PC141 KCS1

YFL061W PINS181 SE180 OLE1 PCS161 ZEO1 HAP4 POS5 HAP1 PCS141 PC180 TOM6 HAP3 SHE9 PE161 YOR066W SDH2

PINS100 HAP5 ORC6 YGR287C HAP2 PINSS161 BNA5 ADR1 C161 C181 PINS161

HSP31 YBR269C SUT1 PE100 PCS140 YTP1 GUT2 TAG160

YCR007C PE140 APC4 MAL32 TAG100 C140 YER053C-A

TAG180 NDE2 PE160 CAT8 PCS100

SPG1 PCS160 TAG140 YGR110w SIP18

C160

POX1

UIP5 SCS22 PINS141 B. TAD2

ERG28 DFG10 ERG24 PC141 C180 YHR112C IML1 PMD1 ADH5

PINS140 VHR1 YLR257WYOR283WYDR540C TIR1 UPC2

30C versus 15C: EKI1 YGK3 ICT1 PCS120

positive PCC correlations PINS180 WHI4 BRO1 BEM3 BAT1 PCS141

PPQ1 TOR1 KIC1 AMD2

YOR214C PCS161 DUR1,2 PS180

PINS100 UNG1

PC120 DAN4 C120

PC161 DFG5

STT3 LRO1

PC180 RTA1 TAG100

ALG8 YJL171C

C100 GAL83

YPL014W MGS1 HNM1 GAS3

LAS21 YHI9 PAN5 TAG140

RCE1 YPL245W AVT1

NIT1 TPN1 GAS5

Figure S19 Correlation analysis demonstrates significant (P ≤ 0.001 following Bonferroni correction) relationships between genes and lipids as characterized by length when comparing high temperature (30°C) versus low temperature (15°C) conditions. (A) Negative Pearson Correlation Coefficients (PCC). (B) Positive Pearson Correlation Coefficients (PCC). Enriched transcription factors are shown (yellow edges). Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key).

M. C. Jewett et al. 21 SI PRR2 PINSS A. NCE103

TIM11 RIP1 YNL100W NDE1

COX12 COX5a AGX1 JID1 LNST HHT2 YML050W FFA AKG DUR1,2 CSF1 VPS54 ATP7 YPL105C

HSP26 SER PE AMD2 ATP4 CPR5 MNN1 MALxt HIP1 PYR YCL012C DMZYMST GOR1 AGP3 YOL138C BRO1 YKL187C MAS6 MAM33RSM25 STE2 ICY1 GLG2 GCY1 YCP4 VMA10 ASP VIP1 QDR3 MHR1 MRPL17 PEP HNT2 PHM6YGR035C YPR036W-A

PYRxt HRB1 MMP1 ADY3 SCW4 GLU OM14 YLR460YCR102C C MFA2 VMA8 YMR124WACH1 ATP5 MSS18 GUT2 ETHxt HAP4 YOR342C HSP12 PHD1 YHR122WSPS19 ATP17 YPL113C KGD1 TPO1 MSN4 GLN1 BUD9 CIT CITMxt MHT1 NAGLU PHM8 PHB1YOR356WGRX2 CAF17 OMA1 EEB1 YBL029WFAR1 FET4 HMS1 ATP15 CBP4 NDI1 HAP3 YGL117W LHP1 RIM2 RCL1 MAL33 MCR1 ATP14 ATP18 KGD2 IMD3 SUCCxt MSN2 GLX LPD1 CIT1 UGO1 SDH3 DPM1 ILS1 KTR3 COX23 ECM3 COX7 ATP16 NCA2 HAP5 YLR243W CRC1 SDH1 SDH2 GRE2 AKA

ERGOST STF1 GPT2 GLxt YMR31 SDH4 ITC TIF35 YIL158W BIO2YGL262W PFK27 ALD4 HAP1 MLS1 MBR1YHR198C CSR2 YKR075C ARO4 HMT1 STM1

DAL3 MIG2 MEP2 YFL054YMR206C WYGL146YNR034W-C MRPL2A 8YPL230W PDS1 STE6 TYR CITC GIS2 RMT2 ACxt YNK1 YIL024C COX20 SIP2 HXK1 SNF3YDR018C ECM38 SIA1YMR155W MDM3YOR0218 C

PRP45 RIB1 MTH1 ISF1 YOR285W MDL2 MEP1 VAL YSP3 GAP1 PRM5 TIP1 DSF1 YIL057C GLG1 ARG82YFR017C ADR1

RPI1 YNL234W SCM4 ERG7 EMI2 NCA3 AST2 HXT7 TMA10 CAT2 STR3 PROxt TAG

SIP18 MRK1 POX1 CST9 GAL3

IZH4 TSA2

ERDEOL SRL3 IZH1 SE

C-limited versus N-limited: negative PCC correlations

DAL80

DAL81 LRO1 URE2 FFA GLN3 SPG4 B. YLR301W YOR032W-A HXT1 YER140W PUT2 AVT4 CAN1 DAP2 YJL045WECM4 SPO74 BTT1 ILE MALxt TOR1 EDC2 CHA1 JIP4 YBR139W KOG1 SSA3 PMP1 DAL7 GAT1 RMD11 ECM38YNR047W BRO1 YDR520C YGR287C YJR015W SCH9 VPS54 MUB1 NPR1 DAL4 YGR125w

OAF1 ASP3-1 RVS167 DAL5 BRE4 AZF1 ASE1 YSP3 CSM4 AKA TRS23 RAD30YMR253C DHH1 VAC8 PIP2 GLxt MAL YNL040W HST1 STD1 DDC1 TFC4 YMR226C PRB1 YFL052W MBF1 QDR3 YOR051C SPS4 ARO9 CIT

MNR2 CAT8 ADY2 ARO1 CITMxt

ADP1 SML1 NPR2 IML1YOR283W RIM101 ACxt PEP UBR1 TEA1 PTR3YHR113W YLL007C LRG1 YNR066FCCY21YGL157W FDH1YNR064CHEF3 SCO1 GLX ARG81 PHO8&a1mpDUR;quo3t;DUR1,2YOL138&quoC tB;SC6 KIP2 PUT1 ECM37 HCM1YCR100YCR099C CTHI80 FOX2 IDP3 DAK2 RIM15 APE2 YMR086W AMD2YGL140C VTC3 STL1 ITC YNR065YNR068C CBSC5 YGL159W

YOR052C WHI4 UGA4 YGK3 HPA3 ERDEOL AVT6

BAP3 FAA3 FEST

PROxt YAT2YGR067CPXA1 YGR043C ECI1 YLR257w DMZYMST SUCCxt GZF3 ARO10 RBG1 CRF1 ICS2 ADH2 IDP2 GTT1 ISR1 NAGLU VAL ALD3 MGA1 LNST HMS2 GSG1 YMR147WACS1 DBR1 YHR126C RSA1 CITC KHA1 GNP1 HUL5 YLR307C-A YMR018WSFC1 SPS19 JEN1 ZTA1 ETHxt ICT1 AKG YHL042WSUN4 FAR1 CTA1 BNS1 ICL2 LAC1 STE4 YDR090C TYR PE YGL117W ICL1 YPL199C TAG GIC2 ECM32YFR055W

GLN1 MUQ1

C-limited versus N-limited: positive PCC correlations

Figure S20 Correlation analysis demonstrates significant (P ≤ 0.001 following Bonferroni correction) gene-lipid and gene- metabolite relationships when comparing carbon-limited versus nitrogen-limited conditions. (A) Negative Pearson Correlation Coefficients (PCC). (B) Positive Pearson Correlation Coefficients (PCC). Enriched transcription factors are shown (yellow edges). Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key).

22 SI M. C. Jewett et al.

IZH4 SRL3 IZH1 SE

MALxt SCM4 YPL272CDAN3 YGR035C DSF1 TIP1 A. TAG STE2 EMI2 ERG7 PEP BUD9 TPO1 SCS7 HXK1 ICY1

ROX1 FET4 FAR1 YBL029W ACxt POX1 Aerobic versus anaerobic: SCW4 HAP1 PDR15YLR460C ADE17 UPC2 HMS1 OCT1 GUT2 AGP3 MSS18 MBR1 EEB1YCR102C negative PCC correlations NAGLU MFA2 ATP17 GRX2 YPL113COMA1 HAP3 GLN1 MOT3 ALD4

PFK27 ADI1YOR356WSPS19 AKA HAP5 TYR ECM3 WTM2 SDH2 YEL047CYML083cHEM13YOR012W CKS1 SDH3

HAP4 ERGOST TIR1 TIR3 TGL5 COS2 TIR4 HSP12 OM14

NDI1 ATP16 YGL039WYHL044W ANB1 MRS3 AUS1 COX12 RIP1

NCE103 KGD2 NCA2 ATP14 SSK22 AAC3 YLR413W ARE1 DAN1 COX5a TIM11

HMX1 LEU2 TIR2 ATP4 ATP7

YGL160w GOR1 GCY1 COX4 LSB6 ETHxt

YLR168c PINSS COX8 COX6 LNST PUT4 JID1 YGR266w NDE1

GRE2 GLxt AGX1 YNL100W INH1 MIC17 MDG1

GAP1 MLS1 GLG2 FTH1 CTR3 DMZYMST ECM38 AQY2 PINS HNT2 YDR248C VAL PRR2 YNL234W AHP1 ECM13

DAL3 LSP1 YAL049C PROxt UBC5 PET10 YLR108C

STR3 ERDEOL

YDR367W

YJL107C YLL053C AAC1 YOL048CFUM1

FET3 YLR356W YLR152C YCL012C YGL262W FFA SUE1 PCS MXR1 ITC

BIO2

YME2 LAC YER053C-A

YJL193W SPG4 YTP1

CCC2 CITM SLM1 ASP HPA2 GLN UFE1 LACxt

GLY CITMxt

SPG1 ZEO1

SER

B. YAR029W HAP1

SPH DAN4

GLN3 TUP1 OIVAL YNL040WYMR226C GAL80

DAL80 HEM13 FUM UPC2 RMI1 ECM38 DAL4 CIT YGL039W ANB1 YML083c PYRxt DAL81

DAN1 SLC1 TIR2 URE2 DUR3 SUCCxt

UBC5 PET10 LSP1 TIR3 TIR1 TIR4 AUS1 ITC ECM37 FAR1 PFK2 GLN1 ERGOST MRS3 SSK22 HUG1 PINS MIC17 YDR248C FAA1 CRF1 YFR055W YDR090C YMR018WYAT2 PXA1 ARE1 YOR012W IZH2 CKS1 PPH3 DPL1 AHP1 ICL1 SIP4 ADH2 ACS1 YGR043C GIC2 MUQ1 ECM32 TYR PMT3 ERG1 ICL2 PINSS ETHxt YGR067C ICS2 YMR147W ECI1 BNA2 LAC1 STE4 ERDEOL YLR307C-A EXG2 YGR131w UTH1 DMZYMST BAP3 SPS19 GTT1 IDP2 CTA1 YHL042W SUN4 PROxt THI71 CSR1 KAR2 JEN1 ZTA1 SFC1 BNS1 NAGLU PRO YOR214C

FAA3 SMA1 CITM GLX STL1 FEST BAT1 ALD3 SCO1 HMS2 SER AAC3 CITMxt ARO10 MAL YFL052W LNST YEL047C THR CAT8 DAK2 IDP3 ECM34 UNG1 GLxt ADY2 YJL127C-B GLN ARO9 FOX2 PIP2 KTR5 PEP YGR287C OAF1 FDH1YNR064C GLY SNF11 RHR2

CSM4 PCS RIB4

MALxt YJL045W ECM4 YDL218W INM1

Aerobic versus anaerobic: YOR032W-A

SPG4 positive PCC correlations PIG1

Figure S21 Correlation analysis demonstrates significant (P ≤ 0.001 following Bonferroni correction) gene-lipid and gene- metabolite relationships when comparing aerobic “O” versus anaerobic “A” conditions. (A) Negative Pearson Correlation Coefficients (PCC). (B) Positive Pearson Correlation Coefficients (PCC). Enriched transcription factors are shown (yellow edges). Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key).

M. C. Jewett et al. 23 SI A. HAP3 HAP5 SDH2 AKA MBR1

POX1 MTH1 SNF3 AST2 CSR2 ACxt HAP1 GUT2 ETHxt CIT SIP18 ISF1 YIL057CADR1

RCL1 MRK1 YIL024CYFL054CNCA3 YGR035C NAGLU YGL117W RIM2 TMA10YOR285WYFR017C

DUR1,2 SSN2 IMD3 KTR3 MNN1 PEP SUCCxt GRR1 BRO1 PDR3 ISW2 MDM1 YJL193W RPS23B RKI1 RPL26B CBF5 GLN DUS3 YLR243W STT4 AMD2 TUS1 MALxt TSC11 NUP159

PE GLN3 PWP1 BIO2 ARO4 YGL262W YLR247C PDR1 YSP1 YOR296W CSF1 BNA5 ITC GLY SPG4 AEP1 SEC66 NOP13 THS1 NOP1 YOL138C QDR3 CPR5 STM1 HSP31 HSP26 HMT1 SIK1 YIL158W SER ZEO1 NIP7 TIF35 CYS FUI1 CITC CITMxt UPC2 TIR1 YOR021C DPH1 YNL157W SPG1 APC4 GLX ERGOST GIS2 YDL121C YDR367W ADY3 ASP CITM YTP1 ARX1 RMT2 PHM8 ERDEOL GLxt YJL107C LAC YER053C-A GLU

PCS

30C versus 15C: negative PCC correlations

B. GLN3 DAL81

DAL80

BTT1 ILE

YAR029W UPC2 TIR1 PINS

SPH DAN4

YGR287C GLxt HAP1 ERR3 SPG4 ORC4 YGL117W PE ARP10 PIG1 SPO20 QDR3 TOR1 SUCCxt LRO1 FFA TRP4 MAL GLN SSA4 BRO1 YOR032W-A

YGL157W WHI4YOR283WYOR052C YNL181W DAL4 FRM2 SSA3 SER SPO74 CIT MALxt AMD2 NPR2 DUR3 YCR100C URE2 ACxt CSM4 TCO89 PTR3 SPO11 GLY ADY2 YOL138CPHO81 ARG81 PTC3 YCR099C

PEP YGK3 DUR1,2 IML1 YNL176CPUT1 ARO10 PRO YOR214C ITC DHH1 AKA UBR1 ECM37 BAT1 THR YGR043C NAGLU YLR257w FUM PIP2 CITM UNG1 ECI1 KHA1 RTA1 RMI1 OIVAL OAF1 CITC

YIR018C-AICT1 ICL2 TYR PYRxt

30C versus 15C: positive PCC correlations

Figure S22 Correlation analysis demonstrates significant (P ≤ 0.001 following Bonferroni correction) gene-lipid and gene- metabolite relationships when comparing high temperature (30°C) versus low temperature (15°C) conditions. (A) Negative Pearson Correlation Coefficients (PCC). (B) Positive Pearson Correlation Coefficients (PCC). Enriched transcription factors are shown (yellow edges). Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key).

24 SI M. C. Jewett et al.

ROX1 MOT3 UPC2

ARE1 ERG1 PIS1 AAC3 LEU2 DAN1 ERG2 TIR1 TIR2 TIR4 LSC1 DPL1 CMD1

CDC53 HEM13 ANB1 AUS1 YML083C TIR3 NCE102 ISU1

AHP1 PPH3 VPS60

PET10 YDR248C AAC1 ergost YLL053C FAA1 ARC15

PEX21 MIC17 PUS5 YGL039WYNR014W YLR413W EXG2 AQR1 MRS3 COS2 YHL044W SPO22 YBR138C UBC5

HUG1 MPD1 COQ6 YFR018C CIS3 ADE3 DIF1 OXA1 PLP1 YLR108C AQY2 LYP1 COS10 YEL047C SLC1 SSK22 MSC7 AUR1 PPG1 LSP1

WTM2 CKS1 NRT1 TGL5 YJR116W YOR012W PYK2 YNL046W

PMT5 ECM3 YHP1

Node color Node border log ratio O/A log P (O/A) 2 10 10 Node and edge type key: -17 5 Gene Negative correlation Metabolite Positive correlation 0 Transcription Factor Transcription factor interaction -5 -2 -10 0

Figure S23 Correlation analysis demonstrates significant gene-ergosterol relationships (P ≤ 0.01 following Bonferroni correction). As indicated in Table 1, the total number of interactions is 76 (all are shown). Measurement ratios for aerobic versus anaerobic conditions were visualized with a log2 color-bar and the color of each node border represents the log10(p- value) (see node and edge color key). Transcription factors identified in the enrichment analysis are shown (see node and edge key).

M. C. Jewett et al. 25 SI

PLB2 TGL5 LRO1 TGL3 TGL1 acylcoa POT1 OAF1 C-limited versus N-limited ffa TGL4 tag se1!6!1 YEH1 DGA1 ! PIP2 coa CAT2 YEH2 TGL2 FAA2 FAT1 tag100 tag140

p l

TAGSN LRO1 accoa tag160 tag100 tencoa

CAT2 B. tag161 tag120 FAO POT1 dag OAF1 TAGDIS tag coa tag180 tag140 CARNSHUTTLE

PIP2 tag181 tag141 DAGAT C-limited versus N-limited acylcoa LIPS1 FAA2 BOXD

c160 c100 FAT1

cho c161 c120

FFADIS ffa LIPS2 gro c180 c140

pc c181 c141 PLB2 SEHYLS ergost Node and Node color Node border log ratio X/Y log P (X/Y) edge type key: 2 10 se Gene 10 Metabolite -17

SEDIS Lipid 5 Transcription Factor Distribution se100se120 0 se140 se141 Positive correlation se160 se161 -5 se180 se181 Negative correlation -2 Transcription factor -10 0 interaction Metabolite/Lipid - Reaction

Lipid distribution

1st neighbor

2nd neighbor

Cenrtral Node

Figure S24 Integrative method for correlation of omics data reveals global regulatory signatures. Correlation networks for steryl ester 16:1 (se161), triacylglycerol 10:0 (tag100), and triacylglycerol 14:0 (tag140) show 1st (green highlight) and 2nd (blue highlight) significantly linked genes under aerobic versus anaerobic conditions. In (A), genes in small white boxes were not identified as significantly correlated to se161, tag100, and tag140, but are represented as “connector nodes” between metabolites. TFs implicated by the enrichment analysis are shown. The co-regulated gene neighborhood network from (A) was expanded to include genes and metabolites necessary to carry out the metabolic transformations indicated (B). This provides a more integrated perspective of cellular regulation. Measurement ratios were visualized with a log2 color-bar and the color of each node border represents the log10(p-value) (see node and edge color key). Gray coloring indicates the lack of a measurement for that node.

26 SI M. C. Jewett et al.

Literature Cited:

Gombert AK, Moreira dos Santos M, Christensen B, Nielsen J (2001) Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression. Journal of Bacteriology 183: 1441- 1451.

Nookaew I, Jewett MC, Meechai A, Thammarongtham C, Laoteng K, Cheevadhanarak S, Nielsen J, Bhumiratana S (2008) The genome-scale metabolic model iIN800 of Saccharomyces cerevisiae and its validation: a scaffold to query lipid metabolism. BMC systems biology 2: 71.

Jewett et al. 27 SI Tables S1-S2 Available for download at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.113.006601/-/DC1

Table S1 Metabolic reconstruction of iIN800

Table S2 Normalized mRNA, metabolite, and lipid data

Jewett et al. 28 SI Table S3 Physiological yield data and steady-state nutrient concentrations in chemostat cultures. See below, and download the Excel file at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.113.006601/-/DC1.

Nutrient Oxygen T Biomass D Residual Glucose Yxs Yxn Limitation Condition [ºC] (g/L) (hr-1) (g/L) (Cmol/Cmol) (Cmol/Nmol) Carbon Anaerobic 30 1.18 ± 0.06 0.047 ± 0.002 b.d. 0.14 ± 0.003 n.d. Carbon Anaerobic 15 1.14 ± 0.13 0.051 ± 0.003 b.d. 0.14 ± 0.003 n.d. Carbon Aerobic 30 5.93 ± 0.53 0.052 ± 0.005 b.d. 0.59 ± 0.031 n.d. Carbon Aerobic 15 6.84 ± 0.49 0.050 ± 0.003 b.d. 0.68 ± 0.032 n.d. Nitrogen Anaerobic 30 3.05 ± 0.20 0.050 ± 0.001 18.3 ± 0.8 0.07 ± 0.001 11.6 ± 0.4 Nitrogen Anaerobic 15 1.80 ± 0.20 0.049 ± 0.001 16.0 ± 0.8 0.07 ± 0.011 8.3 ± 1.5 Nitrogen Aerobic 30 3.84 ± 0.17 0.049 ± 0.000 17.9 ± 0.5 0.11 ± 0.003 15.8 ± 0.3 Nitrogen Aerobic 15 2.72 ± 0.39 0.050 ± 0.002 17.0 ± 0.6 0.16 ± 0.014 10.4 ± 1.1

Nutrient Oxygen T Yace/s Ysuc/s Yeth/s Ygly/s Ypyr/s Limitation Condition [ºC] (Cmol/Cmol) (Cmol/Cmol) (Cmol/Cmol) (Cmol/Cmol) (Cmol/Cmol) Carbon Anaerobic 30 0.000 ± 0.000 0.000 ± 0.000 0.53 ± 0.00 0.055 ± 0.002 0.00031 ± 0.00017 Carbon Anaerobic 15 0.000 ± 0.000 0.000 ± 0.000 0.52 ± 0.00 0.069 ± 0.0052 0.00022 ± 5.5E-05 Carbon Aerobic 30 0.000 ± 0.000 0.000 ± 0.000 0.00 ± 0.00 0.000 ± 0 0.00024 ± 1.3E-05 Carbon Aerobic 15 0.000 ± 0.000 0.000 ± 0.000 0.00 ± 0.00 0.000 ± 0 0.00000 ± 0 Nitrogen Anaerobic 30 0.003 ± 0.000 0.003 ± 0.001 0.62 ± 0.00 0.002 ± 4E-05 0.00080 ± 0.00036 Nitrogen Anaerobic 15 0.006 ± 0.002 0.000 ± 0.000 0.56 ± 0.02 0.004 ± 0.0008 0.00071 ± 0.00037 Nitrogen Aerobic 30 0.004 ± 0.000 0.009 ± 0.000 0.49 ± 0.00 0.001 ± 3E-06 0.00919 ± 0.00051 Nitrogen Aerobic 15 0.015 ± 0.001 0.002 ± 0.000 0.43 ± 0.04 0.000 ± 0.0001 0.00329 ± 7.4E-05

Nutrient Oxygen T Yco2/s Carbon Balance Limitation Condition [ºC] (Cmol/Cmol) Carbon Anaerobic 30 0.20 ± 0.006 0.92 ± 0.007 Carbon Anaerobic 15 0.19 ± 0.031 0.92 ± 0.032 Carbon Aerobic 30 0.32 ± 0.048 0.90 ± 0.053 Carbon Aerobic 15 0.33 ± 0.004 1.01 ± 0.031 Nitrogen Anaerobic 30 0.28 ± 0.017 0.97 ± 0.017 Nitrogen Anaerobic 15 0.31 ± 0.033 0.95 ± 0.042 Nitrogen Aerobic 30 0.35 ± 0.019 0.96 ± 0.02 Nitrogen Aerobic 15 0.35 ± 0.038 0.99 ± 0.025 b.d. - below detection, n.d. - not determined, ace - acetate, suc – succinate, eth – ethanol, gly – glycerol, pyr – pyruvate, co2 – carbon dioxide

Jewett et al. 29 SI Table S4 Multi-way ANOVA results for single factors: carbon-limited vs nitrogen-limited, “CN”; aerobic vs anaerobic, “OA”; and 30C vs 15C “Tt”; with associated log2-fold-change (LFC) and p-values. This supplemental file also contains data on interaction factors (between pairs of single factors): CN:OA; CN:Tt; OA:Tt; and the interaction between all factors, CN:OA:Tt. Table S4 is available for download as an Excel file at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.113.006601/- /DC1.

Jewett et al. 30 SI Table S5 Percent variance captured by each Principle Component (PC) dimension

PC PC1 PC2 PC3 mRNA 36.7 23.6 15.2 Metabolites 62.9 19.3 8.2 Lipids 56.8 21.8 9.8

Jewett et al. 31 SI Tables S6 and S7 are available for download as Excel files at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.113.006601/-/DC1.

Table S6 Metabolic model for cytoscape visualization used in this study. This model centers the genome scale metabolic network iIN800 on our metabolite and lipid measurements. As described in the manuscript, integrated analyses we performed used the complete iIN800 model described by Nookaew et al. (2008). All reaction, metabolite, and lipid abbreviations are also described.

Table S7 In silico fluxes under the constraints of maximized biomass production, a steady state metabolic network, and fixed protein composition. Fluxes were normalized to the glucokinase reaction G6PS, which was set to 100. Each experiment is given a three letter code (C-limited, “C”; N-limited, “N”; aerobic, “O”; anaerobic, “A”; 30°C, “T”; and 15°C, “t”). For reaction abbreviations, see Table S6.

Jewett et al. 32 SI Table S8 Significant lipids, metabolites, and genes when comparing nitrogen-limited aerobic conditions (NOx = NOT & NOt) versus all other conditions (i.e., COT, COt, CAT, CAt, NAT, & NAt) are shown. Significance was determined by P ≤ 0.01 following Bonferroni correction.

Type Label log10pvalue log2(NOx/others) LIP SE161 -13.0 3.8 LIP TAG -11.4 2.5 LIP TAG181 -11.1 3.0 LIP SE181 -9.8 3.0 LIP TAG161 -9.0 3.1 LIP SE -8.8 2.7 LIP TAG141 -5.8 1.9 LIP TAG180 -5.4 2.7 LIP TAG160 -4.8 1.9 LIP SE180 -4.1 2.1 LIP PSPH -3.6 -1.6 MET PYRxt -3.5 3.6 MET PYR -3.1 2.7 MET AKG -2.5 2.5 GENE SRL3 -8.6 -0.4 GENE IZH4 -8.1 -1.9 GENE TIP1 -7.4 -0.3 GENE TSA2 -5.7 -0.7 GENE HSP150 -5.7 -0.1 GENE DAN3 -5.2 -1.3 GENE IZH1 -4.6 -0.2 GENE YPL272C -4.4 -1.0 GENE SCM4 -4.2 -0.2 GENE PLB2 -3.8 -0.5 GENE DAP1 -3.4 -0.2 GENE GPX2 -3.2 0.2 GENE IDS2 -3.2 0.1 GENE YGR146C -3.0 -0.6 GENE YBL095W -2.9 0.2 GENE ERG6 -2.9 -0.1 GENE ERG7 -2.6 -0.1 GENE YDR352W -2.6 0.2 GENE YPL199C -2.4 0.1 GENE PTP3 -2.3 0.2 GENE CRH1 -2.2 -0.1 GENE YMR226C -2.1 0.1 GENE TEP1 -2.1 0.4 GENE HES1 -2.1 -1.4 GENE MID2 -2.0 -0.2 GENE YEL070W -2.0 -0.4

Jewett et al. 33 SI Table S9 Direct connections between genes and lipids or metabolites in the iIN800 metabolic network and the correlation network.

Gene Lip/Met PCC

ARE1 Ergosterol Neg

AUS1 Ergosterol Neg

LSB6 Phosphatidylinositol di-substituted medium acyl- Neg chain (PINSS)

LSB6 Phosphatidylinositol di-substituted medium acyl- Neg chain 14:0 (PINSS140)

GAP1 Valine Neg

Lip: lipid Met: metabolite PCC: Pearson correlation coefficient Neg: negative correlation

Jewett et al. 34 SI Table S10 Based on measured lipids and metabolites that were identified in the correlation analysis, we observed that sterol levels were most highly correlated with 1st and 2nd gene neighbors (P ≤ 0.01, Benjamini Hochberg p-value adjustment). Whereas ~63% of sterols measured were highly correlated to 1st and 2nd gene neighbors, only ~17% of amino acids were. These data suggest that sterol biosynthesis is more regulated at the transcriptional level than amino acid biosynthesis. Within the phospholipid category, we note that 3 of 9 (or 33%) major phosphatidylinositol species were highly correlated to 1st and 2nd gene neighbors (PINS, PINS100, PINS120, PINS140, PINS141, PINS160, PINS161, PINS180, PINS181; significant species in italics and underlined).

Significant Total species P ≤ 0.01

sterol 8 5

organic acid 20 5

currency metabolite 2 2

phospholipid 53 4 neutral lipid 16 3 amino acids 18 3 sphingolipid 2 0 fatty acid 8 0 alcohols 2 0

Total metabolite and lipid species (first column): sterol: ERGOST, EPST, LNST, DMZYMST, ZYMST, ERG722OST, ERTEOL, FEST organic acid: PEP, MAL, SUCC, PYRxt, PYR, ACxt, GABA, MALxt, AKG, ORN, FUM, LACxt, ICIT, NAGLUm, GLX, OIVAL, CIT, ITCm, LAC, IPPMAL currency metabolite: NADPH, NADP phospholipid: PC, PC100, PC120, PC140, PC141, PC160, PC161, PC180, PC181, PCS, PCS100, PCS120, PCS140, PCS141, PCS160, PCS161, PCS180, PCS181, PE, PE100, PE120, PE140, PE141, PE160, PE161, PE180, PE181, PINS, PINS100, PINS120, PINS140, PINS141, PINS160, PINS161, PINS180, PINS181, PINSS, PINSS100, PINSS120, PINSS140, PINSS141, PINSS160, PINSS161, PINSS180, PINSS181, PS, PS100, PS120, PS140, PS160, PS161, PS180, PS181 neutral lipid: TAG, TAG140, TAG100, SE161, TAG160, TAG120, TAG180, SE, SE181, TAG181,TAG141, SE180, TAG161, SE141, SE140, SE120, SE160, SE100 amino acids: LYS, TYR, PROxt, ALA, HIS, ASP, CYS, VAL, PHE, THR, ILE, GLU, ASN, PRO, GLY, SER, LEU, GLN sphingolipid: PSPH, SPH fatty acid: FFA, C10, C12, C18, C14, C161, C141, C181, C16 alcohols: ETHxt, GLxt

Significant metabolites and lipids (second column): Amino acids: alanine, proline (extracellular), lysine Currency metabolites: NADPH, NADP Neutral lipids: SE161, TAG100, TAG140 Organic acids: phosphoenolpyruvate, succinate, malate, pyruvate (extracellular) Phospholipids: PINS181, PINS160, PINSS181, PINS Sterols: ergosterol, lanosterol, episterol, 4,4-dimethylzymosterol, zymosterol

For metabolite and lipid abbreviations, see Table S6.

Jewett et al. 35 SI Table S11 KEGG pathways whose gene neighbors for sets of metabolites have a bias to be significantly correlated or anti- correlated. “KEGG:” KEGG pathway; “p.BH:” P ≤ 0.01, Benjamini Hochberg p-value adjustment; “Genes in pathway:” the total number of genes in the defined KEGG pathway; “Genes in pathway and in CN:” the total number of genes in the defined KEGG pathway that are also in the correlation network (CN); “METS/LIPS in pathway:” the total number of metabolites and lipids in the defined KEGG pathway; “METS/LIPS in pathway and in CN:” the total number of metabolites and lipids in the defined KEGG pathway that are also in the correlation network.

Genes in METS/LIPS in Genes in Pathway and METS/ LIPS in Pathway and in KEGG p.BH Pathway in CN Pathway CN Pyrimidine metabolism 0 69 69 84 7 Aminophosphonate metabolism 0 8 8 39 28 Glycerophospholipid metabolism 0 23 23 100 56 Aminoacyl-tRNA biosynthesis 0 37 37 94 17 Glycine, serine and threonine metabolism 1.18E-10 43 42 108 27 One carbon pool by folate 2.89E-10 14 14 42 4 Phenylalanine, tyrosine and tryptophan biosynthesis 3.79E-09 20 19 54 11 Lysine biosynthesis 2.12E-07 15 15 42 7 Purine metabolism 3.47E-06 89 89 106 10 Sphingolipid metabolism 1.37E-05 13 13 19 5 Biosynthesis of steroids 0.000185 21 21 43 9 Alanine and aspartate metabolism 0.000324 34 31 66 10 Biosynthesis of phenylpropanoids 0.000727 35 33 65 8 Drug metabolism - other enzymes 0.000735 8 8 23 2 Valine, leucine and isoleucine biosynthesis 0.000739 18 18 60 10 Porphyrin and chlorophyll metabolism 0.000815 15 15 33 1 Nitrogen metabolism 0.00127 15 12 29 10 Selenoamino acid metabolism 0.00127 19 19 42 6 Histidine metabolism 0.00149 17 17 37 6 Sulfur metabolism 0.00253 12 12 40 5 Glutamate metabolism 0.0046 29 29 60 13 Methionine metabolism 0.00576 17 17 40 4 Glycolysis / Gluconeogenesis 0.00907 47 45 61 5

Jewett et al. 36 SI