Abstract
The oleaginous yeast Yarrowia lipolytica has attracted much attention due to its ability to utilize a wide range of substrates to accumulate high lipid content and its flexibility for genetic manipulation. In this study, intracellular lipid metabolism in Y. lipolytica was tailored to produce fatty acid, a renewable oleochemical and precursor for production of advanced biofuels. Two main strategies, including blocking activation and peroxisomal uptake of fatty acids and elimination of biosynthesis of lipids, were employed to reduce fatty acid consumption by the native pathways in Y. lipolytica. Both genetic modifications improved fatty acid production. However, disruption of the genes responsible for assembly of nonpolar lipid molecules including triacylglycerols (TAGs) and steryl esters resulted in the deleterious effects on the cell growth. The gene tesA encoding thioesterase from Escherichia coli was expressed in the strain with disrupted faa genes encoding fatty acyl-CoA synthetases and pxa1 encoding peroxisomal acyl-CoA transporter, and the titer of fatty acids resulted in 2.3 g/L in shake flask culture, representing 11-fold improvement compared with the parent strain. Expressing the native genes encoding acetyl-CoA carboxylase (ACC) and hexokinase also increased fatty acid production, although the improvement was not as significant as that with tesA expression. Saturated fatty acids including palmitic acid (C16:0) and stearic acid (C18:0) increased remarkably in the fatty acid composition of the recombinant bearing tesA compared with the parent strain. The recombinant expressing tesA gene resulted in high lipid content, indicating the great fatty acid producing potential of Y. lipolytica. The results highlight the achievement of fatty acid overproduction without adverse effect on growth of the strain. Results of this study provided insight into the relationship between fatty acid and lipid metabolism in Y. lipolytica, confirming the avenue to reprogram lipid metabolism of this host for overproduction of renewable fatty acids.
Highlights
Increase in greenhouse gas emissions and volatility in fossil fuel prices have prompted development of alternative chemicals and fuels produced by microorganisms from renewable biomass
All the enzymes encoded by these four genes from Y. lipolytica have potential adenosine monophosphate (AMP) binding domains, one of the important characteristics of fatty acyl-CoA synthetases (Shockey et al, 2000), and YlFaa1 and Fat1 genes have been identified and studied previously (Wang et al, 2011; Dulermo et al, 2014)
To investigate the relationship between biosynthesis of lipid molecules including TAGs and steryl esters and fatty acid production, a mutant strain Y. lipolytica YlR2 (∆YlDga1, ∆YlDga2, ∆YlLro1, and ∆YlAre1) bearing multiple disrupted genes involved in lipid assembly was constructed (Table 1)
Summary
Increase in greenhouse gas emissions and volatility in fossil fuel prices have prompted development of alternative chemicals and fuels produced by microorganisms from renewable biomass. Microbial lipids produced from renewable feedstock such as cellulosic sugars can be used as the precursors for sustainable production of fungible fuels including green diesel and biodiesel with high-energy density (Xiong et al, 2012). The model organisms such as Escherichia coli and Saccharomyces cerevisiae have been intensively engineered for production of lipids and lipid-derived chemicals (Steen et al, 2010). Because of its generally recognized as safe (GRAS) status, Y. lipolytica has been metabolically engineered for production of an array of renewable chemicals such as succinic acid (Cui et al, 2017) and triacetic acid lactone (TAL; Markham et al, 2018) and other high-value bioproducts such as β-carotene and limonene (Cao et al, 2016)
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