2.2. Bacterial and yeast strains, transformation and culture

All chemicals, reagents, and substrates were purchased from Sigma (Saint Quentin‐Fallavier, France) except if otherwise stated.

The E. coli XL1‐Blue strain from Invitrogen (Paisley, UK) was used to amplify plasmid DNA. E. coli competent cells were made with Mix & Go!^TM Transformation Buffer Set from Zymo Research (Freiburg, Germany) according to the supplier specifications. After transformation, the clones were screened on LB_Kanamycin (10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl, 40 mg/L kanamycin) plates (with 15 g/L agar). Clones were cultured in LB_Kanamycin broth at 37°C during 20 hr under agitation. Plasmids extractions were performed with QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany). All sequences were checked by Sanger sequencing by Eurofins (Ebersberg, Germany) with sequencing primers (Table S3).

The Y. lipolytica chassis was derived from the modified strain JMY2159 (called OleoX) used in previous studies (Beopoulos et al., 2014). The OleoX strain is auxotrophic for leucine and uracil and combines 10 deletions: the two acyl‐CoA:diacylglycerol acyltransferase DGAT1 and DGAT2, the phospholipid:diacylglycerol acyltransferase LRO1, the oleate Δ¹²‐desaturase Yl.FAD2 and the six genes POX1–POX6. The deletion of POX genes disrupts the β‐oxidation which impairs the consumption of lipids. The deletion of the acyltransferases and the Δ¹²‐desaturase should lead to an accumulation of sn2 oleoyl‐phosphatidylcholine. The zeta docking platform was added to OleoX strain to obtain the chassis OleoX zeta (OXZ) strain, as described in Bordes, Fudalej, Dossat, Nicaud, & Marty (2007; Figure S1).

OXZ competent cells were made by Frozen‐EZ Yeast Transformation II Kit^TM from Zymo Research. OXZ strain was transformed with a plasmid digested by NotI‐HF to generate a cassette containing Ura3ex selection marker and the gene of interest surrounded by zeta sequence. After typically 72 hr at 30°C on YNB plates (1.7 g/L YNB_wowo, 5 g/L NH₄Cl, 10 g/L glucose, 50 mM NaKPO₄ buffer at pH 6.8, and 15 g/L agar), transformed OXZ clones were directly picked to start an inoculum for cultures.