Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi

Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi Haiqin Chen, Guangfei Hao, Lei Wang, Hongchao Wang, Zhennan Gu, Liming Liu, Hao Zhang, Wei Chen, and Yong Q. Chen Fold change Genes 5 - - 2 2-3 3-4 4-5 9 78 452 55 3 Log Fold -4 : 4 A B E K L M a b A B E K L M c d e f g h Figure S Overall transcriptional regulation during lipogenesis in M. alpina. Gene transcription changes were compared among samples A, B, E, K, L and M using

sample E (3 min prior to nitrogen exhaustion) as the reference point. The smallest non zero FPKM was.53. Therefore,.5 FPKM was considered as the detection limit and used to replace for log transformation. The number of genes with >5 fold change is indicated at the top. Hierarchical clustering of differentially expressed genes (3328) and samples (A, B, E, K, L, and M) is shown on the left. Eight gene clusters (a, b, c, d, e, f, g, h) were identified and the average expression pattern for each cluster is shown on the right.

Group Gene PK (2.7..4) PCK (4...49) 4 PDH (.2.4.) 5 OGDH (.2.4.2) SUCLG (6.2..4) 7 ACLY (2.3.3.8) 8 ACS (6.2..) 9 ALDH (.2..3) ADH (...2) GPAT (2.3..5) DGAT (2.3..2) ACSL (6.2..3) 4 2 3 G6PD (...49) PGD (...44) HK (2.7.., ALDO (4..2.3) GAPDH (.2..2) ENO (4.2..) ACO (4.2..3) IDH (...42) 6 MDH (...37) 2 CRAT (2.3..7) 3 LIP (3...3) up-regulation down-regulation unchanged not identified M. alpina M. circinelloides R. oryzae C. reinhardtii A. nidulans similarity % 95% 75% 36% Figure S2 Similarity in core gene expression. Transcriptional regulation of the 32 core genes involved in carbon flux and NADPH metabolism illustrated in Figure 2 were compared among M. circinelloides, R.oryzae, C. reinhardtii and A. nidulans and M. alpina.

Expression level (log 2 -ΔΔCt ) Expression level (log 2 -ΔΔCt ) 2.5..5. -.5 3.5..5 -. -.5 -..5. PK (2.7..4) 2 PCK (4...32, 4...49) 7. ACLY (2.3.3.8) Expression level (log 2 -ΔΔCt ) - 2 ACO (4.2..3) IDH (...42) - 8. ACS (6.2..).5. -.5 4 -..5. -.5 9 2..5..5. HK/GCK (2.7.., 2.7..2) PDH (.2.4.) - ALDO (4..2.3) -2 5.5..5. -.5 2 ALDH (.2..3) ADH (...2) OGDH (.2.4.2) SUCLG (6.2..4) 2 -.5..5. -.5 GAPDH (.2..2) 2 3 4. 2 ACSL (6.2..3) CRAT (2.3..7).5 LIP (3...3) 2..5. -.5 -.5. G6PD (...49) PGD (...44) -. -. -.5 A B E K L M A B E K L M A B E K L M A B E K L M A B E K L M Expression level (log 2 -ΔΔCt ).5. -.5 -. ENO (4.2..) -.5 6.5 MDH (...37)..5. -.5 -. -.5 2 GPAT (2.3..5) DGAT (2.3..2) Figure S3 Expression similarities of the core genes in M. alpina, M. circinelloides and R. oryzae. The twenty-three genes involved in carbon flux and NADPH metabolism illustrated in Figure 2 of M. alpina, M. circinelloides and R. oryzae were analyzed by qrt-pcr during lipogenesis. Different symbols indicate different genes with the same annotation. The error bars represent standard deviations. The full names of the enzymes are indicated in the legend of Figure 2.

.4.2 AA (nmol/g DW /h)..8.6.4.2. WT ΔG6PD ΔPGD ΔALDH4A ΔPYCR ΔNIT-6 ΔargC ΔHMGCR Figure S4 The effect of different NADPH-generating genes on AA production. The effect of different NADPH generating genes on AA production were simulated by single gene deletion using MOMA. WT: wide type, G6PD: glucose-6-phosphate dehydrogenase (EC...49), PGD: phosphogluconate dehydrogenase (EC...44), ALDH4A: -pyrroline-5-carboxylate dehydrogenase (EC.5..2), PYCR: pyrroline-5- carboxylate (EC.5..2), NIT-6: nitrite (EC.7..4), argc: N- Acetyl-gamma-glutamyl-phosphate (EC.2..38), HMGCR: hydroxymethylglutaryl-coa (EC...34).

G6PD G6PD2 G6PD3 PGD ME ME2 Expression level (change fold).7.6.5.4.3.2.. MA-G6PD-S MA-G6PD-S2 MA-G6PD-S3 MA-G6PD-S MA-G6PD-S2 MA-G6PD-S3 MA-G6PD-S MA-G6PD-S2 MA-G6PD-S3 MA-PGD-S MA-PGD-S2 MA-PGD-S3 MA-ME-S MA-ME-S2 MA-ME-S3 MA-ME2-S MA-ME2-S2 MA-ME2-S3 Figure S5 Confirmation of RNA knockdown. Three mutants from each RNAi experiments were analyzed by qrt-pcr with the wild-type M. alpina as control. Samples were taken from cultures growing in 5 ml flasks with ml of Kendrick medium stirred at 2 rpm for 96 h at 28 C. Three independent experiments were performed and the bars represent the standard deviations. p <.5 compared to the wild-type control.

Table S. Summary of transcriptome data Library Sample A B E K L M Raw RNA sequencing reads (million) 35.73 37.3 56.66 55.6 52.49 33.2 Mapped RNA sequencing reads (million) 27.33 25.24 44.4 45.62 44.47 24.23 Predicted gene models covered 974 967 394 724 785 9945 A: 2 h, B: -2 h, E: -3 min, K: + h, L: +2 h and M: +48 h

Table S2. Predicted NADPH anabolic reactions Rxn EC Enzyme Formula μ=.3 R.2..5 aldehyde dehydrogenase [NAD(P)[c] +] + nadp[c] + acal[c] -> 2 h[c] + nadph[c] + ac[c].4 R2...42 Isocitrate dehydrogenase (NADP[c] +) nadp[c] + icit[c] -> akg[c] + nadph[c] + co2[c] R3...42 Isocitrate dehydrogenase (NADP[c] +) nadp[c] + icit[c] -> akg[c] + nadph[c] + co2[c] R4...49 glucose-6-phosphate dehydrogenase nadp[c] + dg6p[c] <=> h[c] + nadph[c] + d6pgl[c].4 R5...44 Phosphogluconate dehydrogenase nadp[c] + d6pgc[c] -> co2[c] + nadph[c] + rl5p[c].4 (decarboxylating) R6...4 malate dehydrogenase (oxaloacetatedecarboxylating) nadp[m] + mal[m] -> pyr[m] + nadph[m] + co2[m].555 (NADP[m] +) R7...4 malate dehydrogenase (oxaloacetatedecarboxylating) nadp[c] + mal[c] -> pyr[c] + nadph[c] + co2[c] (NADP[m] +) R8...- unknown hcoa[m] + nadp[m] -> nadph[m] + h[m] + 3ocoa[m] R9 no ec malcoa[m] + nadph[m] + 2 h[m] <=> 3ocoa[m] + nadp[m] + h2o[m] R no ec malcoa[c] + nadph[c] + 2 h[c] <=> 3ocoa[c] + nadp[c] + R...- unknown hcoa[m] + nadp[m] -> nadph[m] + h[m] + 3ocoa[m] R2.2..6 succinate-semialdehyde dehydrogenase h2o[m] + succsal[m] + nadp[m] -> 2 h[m] + nadph[m] + succ[m] R3...2 L-arabinose h[c] + nadph[c] + larab[c] <=> nadp[c] + laol[c] R4...2 D-Ribose h[c] + nadph[c] + rib[c] <=> nadp[c] + ribol[c] R5.5..2 -pyrroline-5-carboxylate dehydrogenase sp5c[c] + nadp[c] + 2 -> glu[c] + nadph[c] + h[c] R6.5..2 -pyrroline-5-carboxylate dehydrogenase sp5c[m] + nadp[m] + 2 h2o[m] -> glu[m] + nadph[m] + h[m] R7.5..2 -pyrroline-5-carboxylate dehydrogenase glugsal[c] + nadp[c] + -> glu[c] + nadph[c] + 2 h[c].56 R8.2..4 glutamate-5-semialdehyde dehydrogenase 2 h[c] + nadph[c] + glup[c] <=> nadp[c] + pi[c] + glugsal[c] R9.5..2 -pyrroline-5-carboxylate dehydrogenase sp5c[c] + nadp[c] + 2 -> glu[c] + nadph[c] + h[c] R2.5..2 Pyrroline-5-carboxylate pro[c] + nadp[c] <=> 2 h[c] + nadph[c] + sp5c[c].56 R2.3..3 Prephenate dehydrogenase (NADP[c] +) nadp[c] + phen[c] -> co2[c] + nadph[c] + 4hppyr[c] R22.4..2 aspartate dehydrogenase asp[c] + nadp[c] -> 2 h[c] + nadph[c] + iasp[c].3 R23.8..2 ferredoxin-nadp+ nadp[c] + h[c] + refdox[c] -> nadph[c] + oxfdox[c] R24.5..3 Dihydrofolate nadp[c] + dhf[c] <=> h[c] + nadph[c] + fot[c] R25 4.2.3.2;...53 6-pyruvoyltetrahydropterin synthase/sepiapterin ahtd[c] + 2 nadph[c] + 2 h[c] -> bh4[c] + 2 nadp[c] + pppi[c] R26.5..34 6,7-dihydropteridine bh4[c] + nadp[c] -> bh2[c] + nadph[c] + h[c]

R27.5..5 methylenetetrahydrofolate dehydrogenase nadp[c] + metthf[c] <=> nadph[c] + methf[c].9 (NADP[c] +) R28.3..- Oxidos ccdol[m] + nadp[m] -> techol[m] + nadph[m] + h[m] R29.3..- Oxidos cdtbe[m] + nad[m] -> dtbe[m] + nadh[m] + h[m] R3...2 alcohol dehydrogenase (NADP[c] +) 6hyac[c] + nadp[c] -> adsde[c] + nadph[c] + h[c] R3.2..5 aldehyde dehydrogenase [NAD(P)[m] +] aldmde[m] + nadp[m] + h2o[m] -> capmde[m] + nadph[m] + h[m] R32.4.3. salicylate hydroxylase + co2[c] + nadp[c] + ccl[c] <=> o2[c] + 2 h[c] + nadph[c] + sali[c] R33.2..5 aldehyde dehydrogenase [NAD(P)[c] +] + nadp[c] + acal[c] -> 2 h[c] + nadph[c] + ac[c].4 R34...7 C-4 sterol decarboxylase (cg26);sterol-4alphacarboxylate nadp[c] + dcda[c] -> co2[c] + nadph[c] + cdol[c] 3-dehydrogenase (decarboxylating) R35.4.2.6 lathosterol oxidase epst[c] + nadp[c] -> ergod[c] + nadph[c] + h[c] R36...2;...72 NADP-dependent alcohol nadp[c] + gl[c] <=> h[c] + nadph[c] + glyal[c] dehydrogenase/glycerol dehydrogenase R37.5.. Saccharopine dehydrogenase (NADP[c] +, L- glutamate forming) h2o[m] + nadp[m] + sacp[m] <=> glu[m] + h[m] + nadph[m] + amasa[m].5

Table S3. Predicted NADPH catabolic reactions Rxn EC Enzyme Formular μ=.3 R...2 Alcohol dehydrogenase (NADP[c] +) h[c] + nadph[c] + acal[c] -> nadp[c] + eth[c] R2...27 GDP-L-fucose synthase gdman[c] + nadph[c] + h[c] -> glfuc[c] + nadp[c] R3...79 glyoxylate (NADP[c] +) glx[c] + nadph[c] + h[c] -> glya[c] + nadp[c] R4...2 D-Xylose (xyra) (xylitol h[c] + nadph[c] + xyl[c] -> nadp[c] + xol[c] dehydrogenase) R5.4..3 glutamate synthase (NADH) akg[c] + gln[c] + h[c] + nadph[c] -> 2 glu[c] + nadp[c] R6.4..3 glutamate dehydrogenase [NAD(P)[m] +] akg[m] + nh3[m] + nadph[m] + h[m] -> glu[m] + nadp[m] + h2o[m] R7.4..3 glutamate dehydrogenase [NAD(P)+] akg[c] + nh3[c] + nadph[c] + h[c] -> glu[c] + nadp[c] + R8...86 (R)-2,3-Dihydroxy-3-methylbutanoate:NADP+ hmobut[m] + nadph[m] + h[m] -> nadp[m] + dmbut[m] oxido R9.2..3 L-aminoadipate-semialdehyde dehydrogenase 2 h[m] + nadph[m] + ama[m] -> h2o[m] + nadp[m] + amasa[m] R.5..2 Pyrroline-5-carboxylate 2 h[c] + nadph[c] + phc[c] -> hpro[c] + nadp[c] R.4.3.9 Kynurenine 3-monooxygenase o2[c] + h[c] + nadph[c] + kyn[c] -> + nadp[c] + hkyn[c] R2...25 shikimate dehydrogenase h[c] + nadph[c] + dhsk[c] -> nadp[c] + sme[c] R3...86 (R)-2,3-Dihydroxy-3-methylpentanoate:NADP+ rhmopt[m] + nadph[m] + h[m] -> dmvat[m] + nadp[m].6 oxido (isomerizing) R4...86 (R)-2,3-Dihydroxy-3-methylpentanoate:NADP+ rhmopt[c] + nadph[c] + h[c] -> dmvat[c] + nadp[c] oxido (isomerizing) R5.8..9 thioredoxin (NADPH) h[c] + nadph[c] + othio[c] -> nadp[c] + rthio[c] R6.8..7 glutathione-disulfide h[c] + nadph[c] + gssg[c] -> nadp[c] + 2 gsh[c] R7.7..4 nitrite [NADPH] 5 h[c] + 3 nadph[c] + hno2[c] -> + 3 nadp[c] + nh4oh[c] R8.8..2 Sulfite (NADPH) 3 h[c] + 3 nadph[c] + slfi[c] -> 3 + 3 nadp[c] + h2s[c] R9...65 pyridoxine 4-dehydrogenase pdxal[c] + nadph[c] + h[c] -> vb6[c] + nadp[c] R2...69 2-dehydropantoate 2- h[m] + nadph[m] + akp[m] -> nadp[m] + pant[m].3 R2... B-ketoacyl-ACP synthase (c,), fatty acyl 6 h[c] + 4 nadph[c] + 3 malcoa[c] -> 2 + 2 co2[c] + 4 CoA synthase R22... B-ketoacyl-ACP synthase (c,), fatty acyl CoA synthase nadp[c] + 2 coa[c] + chcoa[c] 6 h[m] + 4 nadph[m] + 3 malcoa[m] -> 2 h2o[m] + 2 co2[m] + 4 nadp[m] + 2 coa[m] + chcoa[m]

R23.5..3 Dihydrofolate h[c] + nadph[c] + dhf[c] -> thf[c] + nadp[c] R24.5..3 Dihydrofolate 2 h[c] + 2 nadph[c] + fot[c] -> 2 nadp[c] + thf[c] R25.4.3.- 2-octaprenyl-6-methoxyphenol hydroxylase o2[m] + nadph[m] + hm[m] -> h2o[m] + nadp[m] + h[m] + hmb[m] R26.4.3.- ubiquinone biosynthesis monooxygenase hmmb[m] + o2[m] + nadph[m] + h[m] -> hmhmb[m] + Coq7 nadp[m] + h2o[m] R27 2.5..2 Squalene synthase 2 fpp[c] + nadph[c] + 3 h[c] -> 2 ppi[c] + sql[c] + nadp[c] R28.4.3.32 squalene monooxygenase sql[c] + o2[c] + nadph[c] + h[c] -> s23e[c] + nadp[c] + R29.4.3.9 zeaxanthin epoxidase atxin[c] + nadph[c] + h[c] + o2[c] -> vlxin[c] + nadp[c] + R3.4.3.9 zeaxanthin epoxidase zextin[c] + nadph[c] + h[c] + o2[c] -> axtin[c] + nadp[c] + R3...33 dtdp-4-dehydrorhamnose dtdp6m[c] + h[c] + nadph[c] -> nadp[c] + dtdpdm[c] R32...57 3-hydroxybutyryl-CoA dehydrogenase c4hcoa[c] + nadp[c] <=> aaccoa[c] + nadph[c] + h[c] R33.4.-.- Sterigmatocystin biosynthesis monooxygenase o2[m] + h[m] + nadph[m] + avn[m] -> h2o[m] + nadp[m] + havn[m] R34.4.-.- Sterigmatocystin biosynthesis monooxygenase o2[c] + h[c] + nadph[c] + avn[c] -> + nadp[c] + havn[c] R35.4.3.7 phenol 2-monooxygenase 3csol[m] + o2[m] + nadph[m] + h[m] -> 23dlne[m] + nadp[m] + h2o[m] R36.4.3.7 phenol 2-monooxygenase pnol[m] + o2[m] + nadph[m] + h[m] -> ccl[m] + nadp[m] + h2o[m] R37.4.3.7 phenol 2-monooxygenase rsnol[m] + o2[m] + nadph[m] + h[m] -> btol[m] + nadp[m] + h2o[m] R38.4.3.7 phenol 2-monooxygenase 3csol[c] + o2[c] + nadph[c] + h[c] -> 23dlne[c] + nadp[c] + R39.4.3.7 phenol 2-monooxygenase pnol[c] + o2[c] + nadph[c] + h[c] -> ccl[c] + nadp[c] + R4.4.3.7 phenol 2-monooxygenase rsnol[c] + o2[c] + nadph[c] + h[c] -> btol[c] + nadp[c] + R4.4.3.- Oxidos 4eol[m] + nadph[m] + h[m] + o2[m] -> eal[m] + nadp[m] + h2o[m] R42.4.3.- Oxidos bendl[m] + nadph[m] + h[m] + o2[m] -> btol[m] + nadp[m] + h2o[m] R43.4.3.- Oxidos sali[m] + nadph[m] + o2[m] + h[m] -> 25dhba[m] + nadp[m] + h2o[m] R44.4.3.8 dimethylaniline monooxygenase tafen[c] + o2[c] + nadph[c] + h[c] -> tafnox[c] + nadp[c] +

R45.4.3.8 dimethylaniline monooxygenase tafen[p] + o2[p] + nadph[p] + h[p] -> tafnox[p] + nadp[p] + h2o[p] R46..-.- Aflatoxins biosynthesis;,3,6,8-tetra hydroxy h[c] + nadph[c] + vera[c] -> nadp[c] + dmst[c] naphthalene R47..-.- Aflatoxins biosynthesis;,3,6,8-tetra hydroxy h[c] + nadph[c] + verb[c] -> + nadp[c] + dhdmst[c] naphthalene R48... beta-ketoacyl-[acyl-carrier protein](acp) c4hacp[m] + nadp[m] <=> aacacp[m] + nadph[m] + h[m] R49... beta-ketoacyl-[acyl-carrier protein](acp) c6hacp[m] + nadp[m] <=> c6oacp[m] + nadph[m] + h[m] R5... beta-ketoacyl-[acyl-carrier protein](acp) c8hacp[m] + nadp[m] <=> c8oacp[m] + nadph[m] + h[m] R5... beta-ketoacyl-[acyl-carrier protein](acp) chacp[m] + nadp[m] <=> coacp[m] + nadph[m] + h[m] R52... beta-ketoacyl-[acyl-carrier protein](acp) c2hacp[m] + nadp[m] <=> c2oacp[m] + nadph[m] + h[m] R53... beta-ketoacyl-[acyl-carrier protein](acp) c4hacp[m] + nadp[m] <=> c4oacp[m] + nadph[m] + h[m] R54... beta-ketoacyl-[acyl-carrier protein](acp) c6hacp[m] + nadp[m] <=> c6oacp[m] + nadph[m] + h[m] R55... 3-oxoacyl-[acyl-carrier-protein] aacacp[c] + nadph[c] + h[c] <=> c4hacp[c] + nadp[c].62 R56 2.3..86 fatty-acyl-coa synthase c4dacp[c] + nadph[c] + h[c] <=> c4acp[c] + nadp[c].62 R57... 3-oxoacyl-[acyl-carrier-protein] c6oacp[c] + nadph[c] + h[c] <=> c6hacp[c] + nadp[c].62 R58 2.3..86 fatty-acyl-coa synthase c6dacp[c] + nadph[c] + h[c] <=> c6acp[c] + nadp[c].62 R59... 3-oxoacyl-[acyl-carrier-protein] c8oacp[c] + nadph[c] + h[c] <=> c8hacp[c] + nadp[c].62 R6 2.3..86 fatty-acyl-coa synthase c8dacp[c] + nadph[c] + h[c] <=> c8acp[c] + nadp[c].62 R6... 3-oxoacyl-[acyl-carrier-protein] coacp[c] + nadph[c] + h[c] <=> chacp[c] + nadp[c].62 R62 2.3..86 fatty-acyl-coa synthase cdacp[c] + nadph[c] + h[c] <=> cacp[c] + nadp[c].62 R63... 3-oxoacyl-[acyl-carrier-protein] c2oacp[c] + nadph[c] + h[c] <=> c2hacp[c] + nadp[c].62 R64 2.3..86 fatty-acyl-coa synthase c2dacp[c] + nadph[c] + h[c] <=> c2acp[c] + nadp[c].62 R65... 3-oxoacyl-[acyl-carrier-protein] c4oacp[c] + nadph[c] + h[c] <=> c4hacp[c] + nadp[c].62 R66 2.3..86 fatty-acyl-coa synthase c4dacp[c] + nadph[c] + h[c] <=> c4acp[c] + nadp[c].62 R67... 3-oxoacyl-[acyl-carrier-protein] c6oacp[c] + nadph[c] + h[c] <=> c6hacp[c] + nadp[c].62 R68 2.3..86 fatty-acyl-coa synthase c6dacp[c] + nadph[c] + h[c] <=> c6acp[c] + nadp[c].62

R69...33 very-long-chain 3-oxoacyl-CoA c8ocoa[c] + nadph[c] + h[c] <=> c8hcoa[c] + nadp[c].56 R7.3..93 very-long-chain enoyl-coa c8dcoa[c] + nadph[c] + h[c] <=> c8coa[c] + nadp[c].56 R7...33 very-long-chain 3-oxoacyl-CoA c2ocoa[c] + nadph[c] + h[c] <=> c2hcoa[c] + nadp[c].2 R72.3..93 very-long-chain enoyl-coa c2dcoa[c] + nadph[c] + h[c] <=> c2coa[c] + nadp[c].2 R73...33 very-long-chain 3-oxoacyl-CoA c22ocoa[c] + nadph[c] + h[c] <=> c22hcoa[c] + nadp[c]. R74.3..93 very-long-chain enoyl-coa c22dcoa[c] + nadph[c] + h[c] <=> c22coa[c] + nadp[c]. R75...33 very-long-chain 3-oxoacyl-CoA c24ocoa[c] + nadph[c] + h[c] <=> c24hcoa[c] + nadp[c] R76.3..93 very-long-chain enoyl-coa c24dcoa[c] + nadph[c] + h[c] <=> c24coa[c] + nadp[c] R77.4.3.7 sterol 4-demethylase lnst[c] + 3 o2[c] + 3 nadph[c] + 2 h[c] -> dctol[c] + for[c] + 3 nadp[c] + 4 R78.3..72 lanosterol delta24- lnst[c] + nadph[c] + h[c] -> dhstro[c] + nadp[c] R79.3..7 delta4-sterol ;C-4 sterol h[c] + nadph[c] + dctol[c] -> nadp[c] + dcdol[c] (cg24) R8.4.3.72 methylsterol monooxygenase;c-4 sterol methyl 3 o2[c] + 2 h[c] + 3 nadph[c] + dcdol[c] -> 4 + 3 oxidase (cg25) nadp[c] + dcda[c] R8...27 3-keto-steroid h[c] + nadph[c] + cdol[c] -> nadp[c] + mzymst[c] R82.3..72 delta24-sterol zymst[c] + nadph[c] + h[c] -> ac8bol[c] + nadp[c] R83.4.2.6 lathosterol oxidase ac7bol[c] + nadph[c] + h[c] + o2[c] -> pvd3[c] + nadp[c] + 2 R84.3..72 delta24-sterol acdbol[c] + nadph[c] + h[c] -> ac7bol[c] + nadp R85.3..2 7-dehydrocholesterol pvd3[c] + nadph[c] + h[c] -> chtrol[c] + nadp[c] R86.3..72 delta24-sterol 7strol[c] + nadph[c] + h[c] -> pvd3[c] + nadp[c] R87.4.3.7 sterol 4-demethylase obl[c] + 3 o2[c] + 3 nadph[c] + 2 h[c] -> amt3bol[c] + for[c] + 3 nadp[c] + 4 R88.3..7 Delta4-sterol amt3bol[c] + nadph[c] + h[c] -> mefol[c] + nadp[c] R89.3..2 7-dehydrocholesterol dhrsol[c] + nadph[c] + h[c] -> isol[c] + nadp[c] R9.3..7 delta24(24())-sterol h[c] + nadph[c] + egteol[c] -> nadp[c] + egstr[c] R9.4.2.6 C-5 sterol desaturase (ERG3) nadph[c] + epst[c] -> 3 h[c] + nadp[c] + ergod[c] R92.4.2.6 lathosterol oxidase acdbol[c] + nadph[c] -> 7dstrol[c] + nadp[c] + 3 h[c] R93.3..2 7-dehydrocholesterol 7dstrol[c] + nadph[c] + h[c] -> nadp[c] + dmstrol[c] R94... Acylglycerone-phosphate 4 h[c] + nadph[c] + at3p2[c] -> nadp[c] + agl3p[c] R95...2 3-Dehydrosphinganine h[c] + nadph[c] + dhsph[c] -> nadp[c] + sph[c] R96.4.-.- C4-hydroxylase sph[c] + nadph[c] + h[c] + o2[c] -> nadp[c] + psph[c] +

R97.4.-.- C4-hydroxylase o2[c] + h[c] + nadph[c] + dcer2[c] -> + nadp[c] + pcer2[c] R98.3..27 hexadecanal:nadp+ delta2-oxido c6e[c] + nadph[c] + h[c] -> c6a[c] + nadp[c] R99.4.-.- sphingolipid delta-4 desaturase o2[c] + h[c] + nadph[c] + dcer2[c] -> + nadp[c] + pcer2[c] R.4.4. dimethylallyl diphosphate c24(6)[c] + o2 + nadph[c] + 2 h[c] -> 45eet[c] + nadp[c] + R.4.4. Cytochrome P45 c24(6)[c] + o2 + nadph[c] + 2 h[c] -> 2eet[c] + nadp[c] + R2.4.4. Cytochrome P45 c24(6)[c] + o2 + nadph[c] + 2 h[c] -> 89eet[c] + nadp[c] + R3.4.4. Cytochrome P45 c24(6)[c] + o2 + nadph[c] + 2 h[c] -> 56eet[c] + nadp[c] + R4.4.4. Cytochrome P45 c24(6)[c] + o2 + nadph[c] + 2 h[c] -> 6hete[c] + nadp[c] + R5.4.9. delta 9 desaturase c8acp[m] + o2[m] + nadph[m] + 3 h[m] -> c8acp[m] + 2 h2o[m] + nadp[m] R6.6.2.4 NADPH--cytochrome P45 nadph[c] + 2 feri[m] -> h[m] + nadp[c] + 2 fero[m] R7.4.- C4-hydroxylase sph[c] + nadph[c] + h[c] + o2[c] -> nadp[c] + psph[c] + R8.4.9. delta 9 desaturase c6coa[c] + o2[c] + nadph[c] + h[c] -> c6coa[c] + 2 + nadp[c] R9.4.9. delta 9 desaturase c8coa[c] + o2[c] + nadph[c] + h[c] -> c8coa[c] + 2.52 + nadp[c] R.4.9.- delta 2 desaturase c8coa[c] + o2[c] + nadph[c] + h[c] -> c82coa[c] + 2.42 + nadp[c] R.4.9.- delta 5 desaturase c82coa[c] + o2[c] + nadph[c] + h[c] -> c83(3)coa[c] + 2 + nadp[c] R2.4.9.3 delta 6 desaturase c82coa[c] + o2[c] + nadph[c] + h[c] -> c83(6)coa[c] +.39 nadp[c] + 2 R3.4.9.3 delta 6 desaturase c83(3)coa[c] + o2[c] + nadph[c] + h[c] -> c84(3)coa[c] + nadp[c] + 2 R4.4.9. delta 9 desaturase c2coa[c] + o2[c] + nadph[c] + h[c] -> c2coa[c] + 2 + nadp[c] R5...33 very-long-chain 3-oxoacyl-CoA c22ocoa[c] + nadph[c] + h[c] <=> c22hcoa[c] + nadp[c]

R6.3..93 very-long-chain enoyl-coa c22dcoa[c] + nadph[c] + h[c] <=> c22coa[c] + nadp[c] R7...33 very-long-chain 3-oxoacyl-CoA c23(6)ocoa[c] + nadph[c] + h[c] <=> c23(6)hcoa[c] +.35 nadp[c] R8.3..93 very-long-chain enoyl-coa c23(6)dcoa[c] + nadph[c] + h[c] <=> c23(6)coa[c] +.35 nadp[c] R9...33 very-long-chain 3-oxoacyl-CoA c24(3)ocoa[c] + nadph[c] + h[c] <=> c24(3)hcoa[c] + nadp[c] R2.3..93 very-long-chain enoyl-coa c24(3)hcoa[c] <=> c24(3)dcoa[c] + R2.4.9.- delta 5 desaturase c23(6)coa[c] + o2[c] + nadph[c] + h[c] -> c24(6)coa[c] +.35 nadp[c] + 2 R22.4.9.- delta 5 desaturase c24(3)coa[c] + 2 o2[c] + nadph[c] + 3 h[c] -> c25(3)coa[c] + nadp[c] + 4 R23.4.9.- delta 5 desaturase c24(6)coa[c] + o2[c] + nadph[c] + h[c] -> c25(3)coa[c] + 2 + nadp[c] R24...33 very-long-chain 3-oxoacyl-CoA c225(3)ocoa[c] + nadph[c] + h[c] <=> c225(3)hcoa[c] + nadp[c] R25.3..93 very-long-chain enoyl-coa c225(3)hcoa[c] <=> c225(3)dcoa[c] + R26...33 very-long-chain 3-oxoacyl-CoA c245(3)ocoa[c] + nadph[c] + h[c] <=> c245(3)hcoa[c] + nadp[c] R27.3..93 very-long-chain enoyl-coa c245(3)hcoa[c] <=> c245(3)dcoa[c] + R28.4.9.3 delta 6 desaturase c245(3)coa[c] + o2[c] + nadph[c] + h[c] -> c246(3)coa[c] + nadp[c] + 2 R29.2..38 N-Acetyl-gamma-glutamyl-phosphate 2 h[m] + nadph[m] + naglup[m] <=> nadp[m] + naglus[m].552 + pi[m] R3.2.. aspartate-semialdehyde dehydrogenase 2 h[c] + nadph[c] + basp[c] <=> nadp[c] + pi[c] + aspsa[c] R3...3 homoserine dehydrogenase h[m] + nadph[m] + aspsa[m] <=> nadp[m] + hser[m] R32...3 homoserine dehydrogenase h[c] + nadph[c] + aspsa[c] <=> nadp[c] + hser[c] R33.2..8 malonate-semialdehyde dehydrogenase oppa[m] + nadp[m] + coa[m] <=> h[m] + nadph[m] + malcoa[m] R35.5..2 methylenetetrahydrofolate h[c] + nadph[c] + metthf[c] <=> nadp[c] + mthf[c] [NAD(P)H] R36...34 hydroxymethylglutaryl-coa (NADPH) 2 h[c] + 2 nadph[c] + hmgcoa[c] <=> 2 nadp[c] + coa[c] + mvl[c].62