<p> 1Supplementary Table 1 </p><p>2Primers used for this study. The data include sequences and annealing temperatures (Ta) for the primer pairs.</p><p>Primer Primer Name Sequence (5′ → 3′) Ta Set I pAO-Eco-McD6-F CCG GAA TTC ACC ATG GGG GGC GGA GGT CAA 68 °C pAO-Eco-McD6-R CCG GAA TTC TTA TTT ATG GAG ATA CGC ATC CA pAO-Eco-AsELO-F CCG GAA TTC AAA ATG GAG ACC TTC AAT CAC A pAO-Eco-AsELO-R CCG GAA TTC TCA ATC CAC TCT CAG TTT CTT pAO-Eco-PtD5D-F CCG GAA TTC ACC ATG GCT CCG GAT GCG GAT pAO-Eco-PtD5D-R CCG GAA TTC TTA CGC CCG TCC GGT CAA GGG A II Infu-AsELO-BamH-F GAC GTT CGT TTG TGC GGA TCC AGA TCT AAC ATC CAA AGA CGA 68 °C Infu-AsELO-SnaB-R GAT AAA CTA CCG CAT TAT ACG TAG CAC AAA CGA ACG TCT CAC T Infu-PtD5D-SnaB-F GAC GTT CGT TTG TGC TAC GTA AGA TCT AAC ATC CAA A Infu-PtD5D-Mlu-R GAT AAA CTA CCG CAT TAA CGC GTG CAC AAA CGA ACG TC qPCR ACT1 764F TAC CGG ATA GTG AGA CGG AAA 60 °C ACT1 913R TGT ATG GCT CTG ACG GAT TG McD6D 223F TTC CAT CCA GAG CCT GAC TT McD6D 371R CCC TCC CTC TCT ACC TCC TC AsELO 720F CAC GCT AAT TTT CCT GTT CTC A AsELO 871R GTT TCT TGT GTG CAG TGT GCT PtD5D 782F GAT ACT GGT TGT CCG CTG TCT PtD5D 935R ATC ACG TTC ACC GCA ATG TA 3Restriction enzyme sites are underlined. 4</p><p>5</p><p>6</p><p>7</p><p>8</p><p>9</p><p>10Supplementary Table 2 </p><p>11Fatty acid compositions (% w/w) of P. pastoris expressing pAO-D6ELD5</p><p>Fatty acid +ALA</p><p>1 1 pAO815 pAO-D6ELD5</p><p>16:0 14.1±0.3 14.5±0.2 </p><p>16:1 3.0±0.2 2.9±0.2 </p><p>18:0 6.5±0.2 5.6±0.1 </p><p>18:1(n-9) 36.5±0.3 39.9±0.3</p><p>18:1(n-7) 0.3±0.3 2.3±0.2 </p><p>18:2 (LA, n-6) 11.7±0.2 19.2±0.3</p><p>18:3 (GLA, n-6) ND 0.9±0.2 </p><p>20:3 (DGLA, n-6) ND 0.8±0.2</p><p>20:4 (ARA, n-6) ND ND</p><p>22:4 (DTA, n-6) ND ND</p><p>18:3 (ALA, n-3) 27.9± 0.2 10.9±0.3</p><p>18:4 (STA, n-3) ND 0.3±0.2</p><p>20:4 (ETA, n-3) ND 2.5±0.2</p><p>20:5 (EPA, n-3) ND 0.1±0.2</p><p>22:5 (DPA, n-3) ND 0.2±0.3</p><p>18.8± 0.3 18.1±0.2</p><p>Total lipid</p><p>12Each value is the mean ± SD from three independent experiments. </p><p>13These cells were grown for 72 h in a medium fed with 0.5% (v/v) methanol. </p><p>14ND, not detected.</p><p>15+ALA, exogenously supplied fatty acid (0.5 mM ALA).</p><p>16Total lipid is mg lipid g-1 dry cells.</p><p>17</p><p>18</p><p>19</p><p>20</p><p>21</p><p>2 2 22</p><p>23</p><p>24Supplementary Fig. 1. Fatty acid profiles of P. pastoris coexpressing the McD6DES, AsELOVL5, and</p><p>25PtD5DES ORFs in the presence of exogenous ALA substrate. Fatty acid profiles of P. pastoris GS115 cells with</p><p>26integrated empty vectors as a control (pAO815), or pAO815-D6ELD5, and 47015-U and 47085-U standards of</p><p>27upper two panels. The transformants coexpressing McD6DES, AsELOVL5, and PtD5DES ORFs in the presence</p><p>28of ALA substrate accumulated GLA, STA, DGLA, ETA, EPA, and DPA. The new peaks are marked by asterisks</p><p>29in the fatty acid profiles and compared with standards. Other minor peaks are the result of the elongation of</p><p>30endogenous fatty acids.</p><p>3 3</p>