Table 1.
Strains used in this study.
Fig 1.
Overview of campesterol biosynthesis pathway and the corresponding genetic modification in Y. lipolytica.
(a) Overview of campesterol biosynthesis pathway in Y. lipolytica. Campesterol was produced via expression of DHCR7 and disruption of ERG5 based on the ergosterol synthesis pathway in Y. lipolytica. Acetyl-CoA, the precursor of campesterol, is provided either by glycolysis from hydrophilic carbon sources (i.e. glucose and glycerol), or by β-oxidization from hydrophobic materials (e.g. oil). Meanwhile, acetyl-CoA from either hydrophilic or hydrophobic substrates can also generate cellular fatty acids and subsequently triacylglycerols (TAG), consisting of the de novo and the ex novo lipid accumulation process in Y. lipolytica respectively. (b) The method for expression of DHCR7 and disruption of ERG5 at the same time. The middle section of gene ERG5 was replaced by the DHCR7 expression cassette (URA3-EXP1p-DHCR7-XPR2t) in chromosome A. (c) GC-TOF/MS profile of the wild-type Y. lipolytica. ATCC 201249 and campesterol producing strain SyBE_Yl1070030. Wild-type strain (blue) showed an ergosterol peak at 21 min, while Y. lipolytica SyBE_Yl1070028 (red) showed a campesterol peak at 21.7 min.
Fig 2.
Campesterol production in strains with DHCR7 from different species.
Y. lipolytica strains with DHCR7 from X. laevis, O. saliva and R. norvegicus were named as SyBE_Yl1070028, SyBE_Yl1070029 and SyBE_Yl1070030 respectively. The amount of campesterol produced by these three strains was measured after 120 h shake flask culture in YPD medium.
Fig 3.
Sequences and architecture differences among DHCR7 from X. laevis, O. saliva and R. norvegicus.
(a) Overview the structure model of DHCR7 from X. laevis. NADPH (blue) was docked into the NADPH pocket and ergosta-5,7-dienol (yellow) modelled into the substrate binding pocket in the DHCR7 structure. The pocket for substrate binding was enlarged and present below, indicating that Y278 and D409 clamp the hydroxyl of the substrate through a hydrogen-bonding network. (b) Sequences alignment of DHCR7 from X. laevis, O. saliva and R. norvegicus. The putative cholesterol hydroxyl group binding sites and cofactor NADPH binding sites were marked by red squares and blue stars, respectively. Mutations which might attribute to the lowest activity of DHCR7 from R. norvegicus were boxed with red lines. (c) Campesterol production in Y. lipolytica strains with wild-type and mutated X. laevis DHCR7 (named as SyBE_Yl1070028 and SyBE_Yl1070031 respectively). The campesterol production for these strains was measured after 120 h shake flask culture in YPD medium.
Fig 4.
Shake flask cultivation with different carbon sources.
(a) Cells growth for Y. lipolytica strain SyBE_Yl1070028 with glucose, glycerol and sunflower seed oil as the carbon source. (b) Substrate (glucose, glycerol and sunflower oil) consumed by strain SyBE_Yl1070028. (c) The final campersterol titers produced by strain SyBE_Yl1070028 with glucose, glycerol and sunflower seed oil as the carbon source. (d) Cells growths specific rate (μ, h-1), biomass conversion yield (YX/S) and campesterol production yield (YP/S) for strain SyBE_Yl1070028 with different carbon source. μ is determined as ln(X/X0)/ (t-t0), where X is for total biomass (g/L) and t is for time (h). Meanwhile, YX/S represents the yield of total biomass produced per unit of substrate consumed and YP/S represents the yield of product formed per unit of substrate consumed.
Fig 5.
Bioreactor fermentation with different carbon sources.
Campesterol producing strain SyBE_Yl1070028 was fed-batch cultured in glucose (a) or in sunflower seed oil (b) with carbon source restriction strategy. YX/S (yield of total biomass produced per unit of substrate consumed) and YP/S (yield of product formed per unit of substrate consumed) for fed-batch bioreactor cultures were compared with those from the shake-flask experiments (c).
Fig 6.
Lipid accumulation in Y. lipolytica strain SyBE_Yl1070028 cultured with different carbon sources.
(a) The morphology and the lipid body (indicated by arrows) of the engineered Y. lipolytica strain in shake flask cultivation with glucose, glycerol and sunflower seed oil as the carbon source. (b) The lipid accumulation on the dry cell weight (DCW) basis for the shake flask trials.