Engineering caveolin-mediated endocytosis in Saccharomyces cerevisiae

Abstract

As a potential substitute for fatty acids, common low-cost oils could be used to produce acetyl-CoA derivatives, which meet the needs of low-cost industrial production. However, oils are hydrophobic macromolecules and cannot be directly transported into cells. In this study, caveolin was expressed in Saccharomyces cerevisiae to absorb exogenous oils. The expression of caveolin fused with green fluorescent protein showed that caveolin mediated the formation of microvesicles in S. cerevisiae and the addition of 5,6-carboxyfluorescein showed that caveolae had the ability to transport exogenous substances into cells. The intracellular and extracellular triacylglycerol levels were then detected after the addition of soybean oil pre-stained with Nile Red, which proved that caveolae had the ability to absorb the exogenous oils. Lastly, caveolin for oils absorption and lipase from Bacillus pumilus for oil hydrolysis were co-expressed in the naringenin-producing Saccharomyces cerevisiae strain, resulting in naringenin production increasing from 222 mg/g DCW (dry cell weight) (231 mg/L) to 269 mg/g DCW (241 mg/L). These results suggested that the caveolin-mediated transporter independent oil transport system would provide a promising strategy for the transport of hydrophobic substrates.

Keywords: (2S)-Naringenin; Caveolin-1; Oil; Saccharomyces cerevisiae; β-Oxidation.

Fig. 1

Confocal microscopy observation of caveolae formed in S. cerevisiae. Figure A, B, C, D and E: Representative images in control strain Z04, engineered strains Z10, Z11, Z12 and Z13, respectively. A-1, B-1, C-1, D-1 and E−1 are under fluorescence microscopy; A-2, B-2, C-2, D-2 and E−2 are under bright field microscopy; A-3, B-3, C-3, D-3 and E−3 are merged images of fluorescence microscopy and bright field microscopy.

Fig. 2

Endocytosis of 5,6-carboxyfluorescein in S. cerevisiae. A: The endocytosis of 5,6-carboxyfluorescein in control strain C800, A-1, A-2 and A-3 under fluorescence field microscopy, bright field microscopy and a merged image of the two, respectively. B: The endocytosis of 5,6-carboxyfluorescein in engineered caveolin-mediated strain Z01, B-1, B-2 and B-3 under fluorescence microscopy, bright field microscopy and a merged image of the two, respectively.

Fig. 3

Endocytosis of hydrophobic substances in S. cerevisiae. A: The endocytosis of soybean oil stained with Nile Red in control strain C800, A-1 and A-2 under bright field and fluorescence microscopy. B: The endocytosis of soybean oil stained with Nile Red in engineered caveolin-mediated strain Z01, B-1 and B-2 under bright field and fluorescence field microscopy.

Fig. 4

TLC analysis of intracellular and extracellular TAGs and FFAs. Lane 1: TLC analysis of extracellular lipids of engineered strain Z01 in YPDSO medium. Lane 2: TLC analysis of extracellular lipids of control strain C800 in YPDSO medium. Lane 3: TLC analysis of extracellular lipids of engineered strain Z01 in YPD medium. Lane 4: TLC analysis of extracellular lipids of control strain C800 in YPD medium. Lane 5: blank control of YPDSO medium; Lane 6: TAGs standard; Lane 7: free fatty acids (FFAs) standard. Lane 8: TLC analysis of intracellular lipids of engineered strain Z01 in YPDSO medium. Lane 9: TLC analysis of intracellular lipids of control strain C800 in YPDSO medium. Lane 10: TLC analysis of intracellular lipids of engineered strain Z01 in YPD medium. Lane 11: TLC analysis of intracellular lipids of control strain C800 in YPD medium.

Fig. 5

Effects of CAV1 and lipases on naringenin production. A: The endocytosis process of soybean oil by caveolae and (2S)-naringenin biosynthetic pathway in S. cerevisiae. The dotted line represents multistep reactions. TAL, tyrosine ammonia lyase; 4CL, 4-hydroxycinnamoyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase. B: The naringenin production of engineered strains in YPDSO medium. C: The naringenin production of engineered strains in YPD medium. BP: lipase from Bacillus pumilus, TGL1: the gene of sterol ester hydrolase from S. cerevisiae, TGL3: the gene of lipase from S. cerevisiae. Cyan columns represent OD₆₀₀; yellow columns represent the production of naringenin. D: The residues of soybean oil in engineered strains. All data are means ± SD (n ≥ 3). *P < 0.05; **P < 0.01; ***P < 0.001; n. s. P > 0.05. (Student's t-test: two-tailed, two-sample equal variance).