MOESM1 of Increasing jojoba-like wax ester production in Saccharomyces cerevisiae by enhancing very long-chain, monounsaturated fatty acid synthesis
Creators: name: Wenning, Leonie
name: Ejsing, Christer
name: David, Florian
name: Sprenger, Richard
name: Nielsen, Jens
name: Siewers, Verena
Publisher: name: figshare
Year: 2019
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Plant Biology

description: Additional file 1: Table S1. Codon optimized sequences of genes used in this study. Table S2. Sequences of oligonucleotides used in this study. Table S3. Distribution of wax ester species (mol%) in strains LW23 and LW24. The values represent the mean ± SD of three biological replicates of strains LW23 and LW24, respectively. Figure S1. DNA pathway assembly constructs. Genes coding for a heterologous fatty acyl reductase (FAR), a wax synthase (WS), a fatty acid elongase (FAE) or a fatty acid desaturase (FAD) were synthesized with a codon optimization for S. cerevisiae. The ELO2 gene and the OLE1 gene were amplified based on g-DNA from S. cerevisiae CEN.PK 113-5D. The promoter pTPI1 and the terminator pYX212t are homologous to the respective promoter and terminator on the pYX212 plasmid. All plasmids were constructed using the modular pathway engineering strategy [22]. Figure S2. Integration constructs. The gene coding for CaKCS was synthesized with a codon optimization for S. cerevisiae. The IFA38, PHS1, TSC13, ACB1, FAA1, ELO1 and OLE1 genes were amplified based on g-DNA from S. cerevisiae CEN.PK 113-5D. The ACC1** linear fragment, carrying a kanMX marker under the Ashbya gossypii TEF1 promoter/terminator and flanked by loxP sites, was integrated at position X-2. The TSC13/PHS1/IFA38/ACB1 linear fragment, carrying a Kluyveromyces lactis URA3 marker flanked by direct repeats, was integrated at position X-3. The FAA1/ELO1 linear fragment was integrated at position X-4. The Cas9 linear fragment was integrated at position XI-5 and the CaKCS/OLE1 linear fragment at position XII-5 in the genome. Integrative plasmids were constructed based on the EasyClone(-Marker Free) vector toolkit [35–37]. Figure S3. Growth behavior of strains LW21, LW22, LW23 and LW24 in minimal medium containing 20 g/L glucose. Figure S4. Thin layer chromatography. 1, TLC standard = 100 µg cholesterol, 100 µg oleic acid, 100 µg triolein, 100 µg methyl oleate, 100 µg cholesteryl oleate; 2, wax ester standard = 50 µg lauryl laurate (C24:0); 3, strain LW21 clone1; 4, strain LW21 clone 2; 5, strain LW21 clone 3; 6, strain LW23 clone 1; 7, strain LW23 clone 2; 8, strain LW23 clone 3. The TLC was performed as described in Materials and Methods. Figure S5. Thin layer chromatography. 1, TLC standard = 100 µg cholesterol, 100 µg oleic acid, 100 µg triolein, 100 µg methyl oleate, 100 µg cholesteryl oleate; 2, wax ester standard = 50 µg lauryl laurate (C24:0); 3, strain LW22 clone 1; 4, strain LW22 clone 2; 5, strain LW22 clone 3; 6, strain LW24 clone 1; 7, strain LW24 clone 2; 8, strain LW24 clone 3. The TLC was performed as described in Materials and Methods. Figure S6. GC-FID chromatograms of fatty alcohols (FOHs) isolated from strains LW21 (A), LW22 (B), LW23 (C) and LW24 (D). The peak labeled with (I) corresponds to the internal standard used (C17:0-FOH). A mixture of standards (c = 200 µg/mL) containing the saturated FOHs (II), C16:0-FOH; (III), C18:0-FOH; (IV), C20:0-FOH; (V), C22:0-FOH and (VI), C24:0-FOH is shown in (E). A mixture of standards (c = 200 µg/mL) containing the monounsaturated FOHs (II), C16:1-FOH; (III), C18:1-FOH; (IV), C20:1-FOH; (V), C22:1-FOH and (VI), C24:1-FOH is shown in (F). The chromatogram in (G) shows a combination of the standards (E), (c = 50 µg/mL); (F), (c = 50 µg/mL) and C17:0-FOH (c = 250 µg/mL). The strains were grown for 48 h in minimal medium containing 20 g/L glucose. Figure S7. GC/MS chromatograms of wax esters (WEs) isolated from strains LW21 (A), LW22 (B), LW23 (C) and LW24 (D). The peak labeled with (I) corresponds to the internal standard used (C24:0-WE). A mixture of standards (c = 10 µg/mL) containing the saturated WEs (I), C24:0-WE; (II), C30:0-WE; (III), C32:0-WE; (IV), C34:0-WE; (V), C36:0-WE, (VI), C38:0-WE; (VII), C40:0-WE and (VIII), C42:0-WE is shown in (E). A mixture of standards (c = 10 µg/mL) containing the diunsaturated WEs (III), C32:2-WE; (IV), C34:2-WE; (V), C36:2-WE; (VI), C38:2-WE; (VII), C40:2-WE; (VIII), C42:2-WE and (IX), C44:2-WE is shown in (F). The strains were grown for 48 h in minimal medium containing 20 g/L glucose. Figure S8. Mass spectra of selected peaks of total ion chromatograms of lipids extracted from strain LW24. Mass spectrum of the C38:2-WE (MW = 561 g/mol), eluting after 27.95 min with the specific m/z peak of 561 (A). Mass spectrum of the C40:2-WE (MW = 589 g/mol), eluting after 29.23 min with the specific m/z peak of 589 (B). Mass spectrum of the C42:2-WE (MW = 617 g/mol), eluting after 30.73 min with the specific m/z peak of 617 (C). Mass spectrum of the C44:2-WE (MW = 645 g/mol), eluting after 32.53 min with the specific m/z peak of 645 (D).