2.1. Strains and culture conditions
Escherichia coli NEB5α high efficiency competent cells were obtained from NEB for plasmid construction, preparation, propagation and storage. The Y. lipolytica wild type strain W29 was purchased from ATCC (ATCC20460). The auxotrophic Po1g (Leu−) was obtained from Yeastern Biotech Company (Taipei, Taiwan). All strains and plasmids are listed in supplementary Table S2.
LB broth or agar plate with 100 µg/mL ampicillin was used to cultivateE. coli strains. Yeast rich medium (YPD) was prepared with 20 g/L Bacto peptone (Difco), 10 g/L yeast extract (Difco), and 20 g/L glucose (Sigma-Aldrich), and supplemented with 15 g/L Bacto agar (Difco) for solid plates. YNB seed medium was made with 1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate) (Difco), 5 g/L ammonium sulfate (Sigma-Aldrich), 0.69 g/L CSM-Leu (Sunrise Science Products, Inc.) and 20 g/L glucose. Selective YNB plates were made with YNB media supplemented with 15 g/L Bacto agar (Difco). In fermentation process, YNB fermentation medium with glucose as substrate and carbon/nitrogen molar ratio of 80:1 was made with 1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate) (Difco), 1.1 g/L ammonium sulfate (Sigma-Aldrich), 0.69 g/L CSM-Leu (Sunrise Science Products, Inc.) and 40 g/L glucose. YNB fermentation medium with sodium acetate as substrate and carbon/nitrogen molar ratio 80:1 was made with 1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate) (Difco), 0.825 g/L ammonium sulfate (Sigma-Aldrich), 0.69 g/L CSM-Leu (Sunrise Science Products, Inc.), 41 g/L sodium acetate. Glacial acetic acids were purchased from Sigma-Aldrich.
Phosphoric buffer solution (PBS) with pH 6.0 was made with 0.2 M Na2HPO4 and 0.2 M Na2HPO4, which was used to replace water to make YNB- glucose-PBS fermentation medium. Bromocresol purple was a pH-sensitive indicator which could change its color with the pH from 5.2-7.0 (El-Ashgar et al., 2012) and 40 mg/L bromocresol purple was added into fermentation medium to indicate pH variation. The pH of medium was regulated to 6.0 by 6. 0 M HCl in the fermentation process. The components in glucose-YNB media with C/N ratio 60:1, 40:1, 20:1, 10:1 were as same as them in C/N ratio 80:1 except the content of ammonium sulfate changed to 1.47 g/L, 2.2 g/L, 4.4 g/L, 8.8 g/L respectively, to explore the effect of C/N ratio on squalenen accumulation. And 1 mg/L cerulenin solution prepared with dimethylsulfoxide (DMSO) was added into fermentation medium to inhibit precursor (fatty acids biosynthesis) competing pathway.