The majority of genes governing fatty acid biosynthesis were identified as being overexpressed in nitrogen limited cells as shown in the global meta­bolic pathway level and fatty acid biosynthesis module. The fold-change and abundances of identified transcripts for the components of fatty acid biosynthesis at the gene level are presented in Figure 5A. The first step in fatty acid biosynthesis is the transduction of acetyl-CoA into malonyl — CoA by addition of carbon dioxide. This reaction is the first committing step in the pathway and catalyzed by Acetyl-CoA Carboxylase (ACCase). While the gene encoding ACCase was repressed under the — N condition, the biotin-containing subunit of ACCase, biotin carboxylase (BC), was significantly up-regulated in response to nitrogen starvation. The BC cata­lyzes the ATP-dependent carboxylation of the biotin subunit and is part of the heteromeric ACCase that is present in the plastid—the site of de novo fatty acid biosynthesis [27]. To proceed with fatty acid biosynthe­sis, malonyl-CoA is transferred to an acyl-carrier protein (ACP), by the action of malonyl-CoA ACP transacylase (MAT). This step is followed by a round of condensation, reduction, dehydration, and again reduction reactions catalyzed by beta-ketoacyl-ACP synthase (KAS), beta-ketoac — yl-ACP reductase (KAR), beta-hydroxyacyl-ACP dehydrase (HAD), and enoyl-ACP reductase (EAR), respectively. The expression of genes cod­ing for MAT, KAS, HAD, and EAR were up-regulated, whereas the KAR encoding gene was repressed in — N cells. The synthesis ceases after six or seven cycles when the number of carbon atoms reaches sixteen (C16:0- [ACP]) or eighteen (C18:0-[ACP]). ACP residues are then cleaved off by thioesterases oleoyl-ACP hydrolase (OAH) and Acyl-ACP thioesterase A (FatA) generating the end products of fatty acid synthesis (i. e. palmitic (C16:0) and stearic (C18:0) acids). Genes coding for these thioesterases, i. e. FatA and OAH, were overexpressed in -N cells. The up-regulation of these thioesterase encoding genes, as previously reported in E. coli and the microalga P. tricornutum, is associated with reducing the feedback inhibi­tion that partially controls the production rate of fatty acid biosynthesis

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FIGURE 5: Differential expression of genes involved in (A) the fatty acid biosynthesis; (B) triacylglycerol biosynthesis; (C)P-oxidation; and (D) starch biosynthesis. Pathway were reconstructed based on the de novo assembly and quantitative annotation of the N. oleoabundans transcriptome. (A) Enzymes include: ACC, acetyl-CoA carboxylase (EC:

6.4.1.2) ; MAT, malonyl-CoA ACP transacylase (EC: 2.3.1.39); KAS, beta-ketoacyl-ACP synthase (KAS I, EC: 2.3.1.41; KASII, EC: 2.3.1.179; KAS III, EC: 2.3.1.180); KAR, beta-ketoacyl-ACP reductase (EC: 1.1.1.100); HAD, beta-hydroxyacyl-ACP dehydrase (EC: 4.2.1.-); EAR, enoyl-ACP reductase (EC: 1.3.1.9); AAD, acyl-ACP desaturase (EC:

1.14.19.2) ; OAH, oleoyl-ACP hydrolase (EC: 3.1.2.14); FatA, Acyl-ACP thioesterase A (EC: 3.1.2.-); A12D, A12(ro6)-desaturase (EC: 1.4.19.6); A15D, A15(ro3)-desaturase (EC: 1.4.19.-); (B) Enzymes include: GK, glycerol kinase (EC: 2.7.1.30); GPAT, glycerol-3- phosphate O-acyltransferase (EC: 2.3.1.15); AGPAT, 1-acyl-sn-glycerol-3-phosphate O-acyltransferase (EC:2.3.1.51); PP, phosphatidate phosphatase (EC: 3.1.3.4); DGAT, diacylglycerol O-acyltransferase (EC: 2.3.1.20); and PDAT, phopholipid:diacy glycerol acyltransferase (EC 2.3.1.158); (C) Enzymes include: ACS, acyl-CoA synthetase (EC: 6.2.1.3); ACOX1, acyl-CoA oxidase (EC: 1.3.3.6); ECH, enoly-CoA hydratase (EC: 4.2.1.17); HADH, 3-hydroxyacyl-CoA dehydrogenase (EC: 1.1.1.35); ACAT, acetyl-CoA C-acetyltransferase (EC: 2.3.1.16, 2.3.1.9); (D) Enzymes include: PGM, phosphoglucomutase (EC: 5.4.2.2); AGPase, ADP-glucose pyrophosphorylase (EC:
2.7.7.27); SS, starch synthase (EC: 2.4.1.21); BE, a-1,4-glucan branching enzyme (EC: 2.4.1.18); and HXK, hexokinase (2.7.1.1). Starch catabolism enzymes include: a-AMY, a-amylase (EC: 3.2.1.1); O1,6G, oligo-1,6-glucosidase (EC: 3.2.1.10); P-AMY, P-amylase (EC: 3.2.1.2); and SPase, starch phosphorylase (EC: 2.4.1.1). Ethanol fermentation via pyruvate enzymes include: PDC, pyruvate decarboxylase (EC: 4.1.1.1); and ADH, alcohol dehydrogenase (EC: 1.1.1.1). Enzymes aceE, pyruvate dehydrogenase E1 component (EC 1.2.4.1); aceF, pyruvate dehydrogenase E2 component (EC: 2.3.1.12); and pdhD, dihydrolipoamide dehydrogenase (EC 1.8.1.4), transforms pyruvate into acetyl-CoA. Key enzymes are shown with an asterisk (*) next to the enzyme abbreviations, and dashed arrows denote reaction(s) for which the enzymes are not shown. All presented fold changes are statistically significant, q value < 0.05.

[7,8], and results in the overproduction of fatty acids [9]. It has also been suggested that an increase in FatA gene expression and the associated acyl-ACP hydrolysis may aid in increased fatty acid transport from the chloroplast to the endoplasmic reticulum site where TAG assembly occurs [10,28]. Finally, for supplying reducing equivalents via NADPH to power fatty acid biosynthesis, genes encoding for the pentose phosphate pathway were strongly up-regulated in the — N condition (Table 2).

The altered expression of genes associated with the generation of dou­ble bonds in fatty acids reflects the observed increase in the proportion of unsaturated of fatty acids (Figure 1D), and the enrichment of C18:1 during nitrogen limitations. The acyl-ACP desaturase (AAD), which intro­duces a one double bond to C16:0/C18:0, and delta-15 desaturase, which converts C18:2 to C18:3, were significantly up-regulated in the — N case, whereas the delta-12 desaturase catalyzing the formation of C18:2 from C18:1was repressed during nitrogen limitation.

Under nitrogen limitations, 10 of the 13 genes associated with fatty acid degradation (a and P-oxidation pathways for saturated and unsatu­rated acids) were significantly repressed. Figure 5C demonstrates the typi­cal P-oxidation pathway for saturated fatty acids, while Table 3 displays expression levels for additional peroxisomal genes associated with fatty acid oxidation, but not shown in Figure 5C. Before undergoing oxidative degradation, fatty acids are activated through esterification to Coenzyme A. The activation reaction, is catalyzed by acyl-CoA synthetase (ACSL), which was up-regulated in — N cells. The acyl-CoA enters the P-oxidation pathway and undergoes four enzymatic reactions in multiple rounds. The first three steps of the pathway; oxidation, hydration and again oxidation of acyl-CoA are catalyzed by acyl-CoA oxidase (ACOX1), enoly-CoA hydratase (ECH), and hydroxyacyl-CoA dehydrogenase (HADH), respec­tively. In the last step of the pathway, acetyl-CoA acetyltransferase (ACAT) catalyzes the cleavage of one acetyl-CoA, yielding a fatty acyl-CoA that is 2 carbons shorter than the original acyl-CoA. The cycle continues until all the carbons are released as acetyl-CoA. The expression level of ECH and HADH were unchanged and genes encoding for enzymes ACOX1 and ACAT catalyzing the first and last reactions in the cycle were identified as significantly repressed in — N cells.

Table 2. N. oleoabundans genes involved in the pentose phosphate pathway

Pentose phosphate pathway Log2FC

Phosphogluconate dehydrogenase (decarboxylating) (PGD, EC: 1.1.1.44) -1.13

Glucose-6-phosphate dehydrogenase (G6PD, EC: 1.1.1.49) -1.41

Transketolase (tktA, EC: 2.2.1.1) 2.55

Transaldolase (talA, EC: 2.2.1.2) -0.66

6-phosphofructokinase (PFK, EC: 2.7.1.11) -0.45

Gluconokinase (gntK, EC: 2.7.1.12) 0.10

Ribokinase (rbsK, EC: 2.7.1.15) 0.11

Ribose-phosphate diphosphokinase (PRPS, EC: 2.7.6.1) -0.10

Gluconolactonase (GNL, EC: 3.1.1.17) -0.67

6-phosphogluconolactonase (PGLS, EC: 3.1.1.31) 0.07

Fructose-bisphosphatase (FBP, EC: 3.1.3.11) -0.24

Fructose-bisphosphate aldolase (fbaB, EC: 4.1.2.13) 0.17

Ribulose-phosphate 3-epimerase (RPE, EC: 5.1.3.1) -0.11

Ribose-5-phosphate isomerase (rpiA, EC: 5.3.1.6) -0.34

Glucose-6-phosphate isomerase (GPI, EC: 5.3.1.9) -1.21

Phosphoglucomutase (pgm, EC: 5.4.2.2) -0.83

Negative Log2FC values represent up-regulation under nitrogen limitation. All presented fold changes are statistically significant, q value < 0.05.

TABLE 3: N. oleoabundans genes involved in catabolic pathways related to peroxisomal fatty acid oxidation, lysosomal lipases, and the regulation of autophagy

Enzyme encoding gene Log2FC

Peroxisome

a-oxidation

2-hydroxyacyl-coa lyase 1 (HACL1, EC: 4.1.-.-) 0.35

Unsaturated fatty acid p-oxidation

Peroxisomal 2,4-dienoyl-coa reductase (DECR2, EC: 1.3.1.34) 0.21

A(3,5)-A(2,4)-dienoyl-coa isomerase (ECH1, EC: 5.3.3.-) -0.27

ATP-binding cassette, subfamily D (ALD), member 1 (ABCD1) 0.25

Long-chain acyl-coa synthetase (ACSL, EC: 6.2.1.3) 0.25

Other oxidation

Peroxisomal 3,2-trans-enoyl-coa isomerase (PECI, EC: 5.3.3.8) 0.59

Carnitine O-acetyltransferase (CRAT, EC: 2.3.1.7) 0.30

NAD+diphosphatase (NUDT12, EC: 3.6.1.22) 0.47

Glycerolipid metabolism

Triacylglycerol lipase (EC: 3.1.1.3) 0.33

Acylglycerol lipase (MGLL, EC: 3.1.1.23) -0.13

Glycerophospholipid metabolism

Phospholipase A1 (plda, EC: 3.1.1.32) -1.26

Phospholipase A2 (PLA2G, EC: 3.1.1.4) -0.31

Phospholipase C (plcc, EC: 3.1.4.3) -0.10

Lysosome Lipases

Lysosomal acid lipase (LIPA, EC: 3.1.1.13) -0.48

Lysophospholipase III (LYPLA3, EC: 3.1.1.5) 0.20

Regulation of autophagy

Unc51-like kinase (ATG1, EC: 2.7.11.1) -0.53

5′-AMP-activated protein kinase, catalytic alpha subunit (snrk1, PRKAA) -0.05

Vacuolar protein 8 (VAC8) 0.13

Beclin 1 (BECN1) -0.59

TABLE 3: cont.

Enzyme encoding gene Log2FC

Phosphatidylinositol 3-kinase (VPS34, EC: 2.7.1.137) -1.26

Phosphoinositide-3-kinase, regulatory subunit 4, p150 (VPS15, EC: 2.7.11.1) 0.11

Autophagy-related protein 3 (ATG3) 0.11

Autophagy-related protein 4 (ATG4) -0.16

Autophagy-related protein 5 (ATG5) -0.27

Autophagy-related protein 7 (ATG7) 0.17

Autophagy-related protein 8 (ATG8) -0.50

Autophagy-related protein 12 (ATG12) -0.58

Negative log2 fold change (Log2FC) values represent up-regulation under nitrogen limitation. All presented fold changes are statistically significant, q value < 0.05.