Attenuating the triacylglycerol catabolism enhanced lipid production of Rhodotorula strain U13N3.

Abstract

Enhancing the lipid production of oleaginous yeasts is conducive to cutting the cost of feedstock for biodiesel. To increase the lipid productivity of Rhodotorula sp. U13N3, genes involving lipid degradation were knocked out and fermentation conditions were investigated. Results of transcription analysis demonstrated that genes encoding the ATG15-like lipase (ATG15) and peroxisomal acyl-CoA oxidase (ACOX2) were upregulated significantly at the lipogenesis stage. When ATG15 and ACOX2 were knocked out separately from the genome by the CRISPR/Cas9 method, both ΔATG15 and ΔACOX2 mutants showed better lipid production ability than the parent strain. Flow cytometry and confocal microscopic analyses indicated that simultaneous the knockout of ATG15 and ACOX2 did not impact the cell viability, whereas the lipid production was enhanced markedly as the lipid yield increased by 67.03% in shake flasks. Afterward, the ΔATG15ΔACOX2 transformant (TO2) was cultivated in shake flasks in the fed-batch mode; the highest biomass and lipid yield reached 45.76g/L and 27.14g/L at 216h, respectively. Better performance was achieved when TO2 was cultivated in the 1-L bioreactor. At the end of fermentation (180h), lipid content, yield, yield coefficient, and productivity reached 65.53%, 27.35g/L, 0.277g/g glycerol, and 0.152g/L/h, respectively. These values were at the high level in comparison with Rhodotorula strains cultivated in glycerol media. Besides, fermentation modes did not affect the fatty acid composition of TO2 significantly. In conclusion, blocking the lipid degradation was an applicable strategy to increase the lipid production of Rhodotorula strains without compromising their cell viability. KEY POINTS: • ATG15-like lipase and acyl-CoA oxidase (ACOX2) participated in lipid degradation. • Knockout of ATG15 and ACOX2 increased lipid productivity, and lipid yield coefficient. • Cell viability maintained at high level in the knockout mutants during fermentation.