Abstract
Microbial oil triacylglycerol (TAG) from the renewable feedstock attract much attention. The oleaginous yeast Yarrowia lipolytica has become the most studied for lipid biosynthesis. Fatty acid desaturases catalyze the introduction of a double bond into fatty-acid hydrocarbon chains to produce unsaturated fatty acids. Desaturases are known to enhance lipid accumulation. In this study, we have achieved a significant increase in lipid production and increase the unsaturated fatty acids content in Y. lipolytica. By comparing the expression of the native genes of △-9 stearoyl-CoA desaturase (SCD) and △12 desaturase (△12D), and an exogenous △15 desaturase (△15D) from flax in the strain with deleted peroxisomal biogenesis factor 10 (PEX10) and overexpressed diacylglyceride acyl-transferase (DGA1), we found that the strain with overexpressed △15 desaturase accumulated 30.7% lipid. Simultaneously, we explored the effect of two copies of desaturase genes (12D-SCD, 15D-SCD, 12D-15D) on lipid production, and found co-expression of △12D and △15D accumulated 42.6% lipid. The lipid content was further increased by 56.3% through the deletion of the multifunctional enzyme (MFE1) and the overexpression of acetyl-CoA carboxylase (ACC1). Finally, the lipid productivity of 50 g/L and maximal lipid content of 77.8% DCW are obtained using a 5-L stirred-tank bioreactor during the stationary phase in the engineered YL-10. Our result demonstrated that the △12 and △15 desaturases play an important role in lipid production in Y. lipolytica and provides an effective strategy for biodiesel development.
Highlights
Biofuels are a promising alternative to reduce the dependence on fossil fuels and alleviate environmental pressure (Stephanopoulos, 2007)
Increasing lipid biosynthesis can be achieved through enhancing the supply of pathway precursors, which is primarily initiated by the activity of the malic enzyme (ME), ATP citrate lyase (ACL), and acetyl-CoA carboxylase (ACC1) (Tai and Stephanopoulos, 2013; Zhang et al, 2013; Blazeck et al, 2014; Ledesma-Amaro and Nicaud, 2015)
We created the peroxisomal biogenesis factor 10 (PEX10) deletion strain (PEX10 disruption cassette was constructed as described in the Materials and Methods), which accumulated lipid at 14.5% of DCW, about 1-fold increase compared to 7.2% for the control strain POlf (Figure 2A)
Summary
Biofuels are a promising alternative to reduce the dependence on fossil fuels and alleviate environmental pressure (Stephanopoulos, 2007). The deletion of either six acyl-CoA oxidase enzymes (POX16), the multifunctional enzyme (MFE1), and the thiolase POT1 (YALI0E18568g) involved in peroxisomal β-oxidation or peroxisomal biogenesis factor 10 (PEX10) resulted in blocking the fatty acid degradation pathway, increased lipid accumulation (Dulermo and Nicaud, 2011; Tai and Stephanopoulos, 2013; Ledesma-Amaro and Nicaud, 2015). Increasing lipid biosynthesis can be achieved through enhancing the supply of pathway precursors, which is primarily initiated by the activity of the malic enzyme (ME), ATP citrate lyase (ACL), and acetyl-CoA carboxylase (ACC1) (Tai and Stephanopoulos, 2013; Zhang et al, 2013; Blazeck et al, 2014; Ledesma-Amaro and Nicaud, 2015). Elevated expression of the key enzymes DGA1 and DGA2 (acyl-CoA: diacylglycerol acyl-transferases I and II) are known to promote TAG biosynthesis and transport TAG into the lipid droplet (Gajdoš et al, 2015; Qiao et al, 2015; Friedlander et al, 2016)
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