Engineering of a high lipid producing Yarrowia lipolytica strain

Abstract

Background: Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways.

Results: We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h.

Conclusions: The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.

Keywords: Lipid accumulation; Metabolic engineering; Oleaginous yeast; Yarrowia lipolytica.

Fig. 1

Major steps of lipid biosynthesis in Y. lipolytica. Overexpression genetic targets screened in this study for their effect on lipid production are shown in green boxes. Deletion genetic targets are shown in red boxes

Fig. 2

Overexpression of DGA1 genes in Y. lipolytica strain NS18. Nine DGA1 genes (Table 2) under the control of the Y. lipolytica GPD1 promoter were randomly integrated into the NS18 genome and 8 transformants for each gene were analyzed by fluorescence-based lipid assay after 72 h of growth in nitrogen-limited media. The average with standard deviation from triplicate experiments is shown for the parent strain. Fluorescence was measured at excitation 486 nm and emission 510 nm and normalized by cell optical density (OD) at 600 nm

Fig. 3

Overexpression of DGA2 genes in Y. lipolytica. Six DGA2 genes (Table 2) under the control of the Y. lipolytica TEF1 promoter were overexpressed in NS125 (a) and NS281 (b) strains that overexpress DGA1 from Y. lipolytica and R. toruloides, respectively. Fifteen transformants for each gene were analyzed by fluorescence-based lipid assay after 72 h of growth in nitrogen-limited media. The average with standard deviation from triplicate experiments is shown for the parent strain. Fluorescence was measured at excitation 486 nm and emission 510 nm and normalized by cell optical density (OD) at 600 nm

Fig. 4

Comparison of lipid accumulation in Y. lipolytica strains with different target combinations by different methods. a Strains analyzed by fluorescence assay after 96 h of fermentation in a 48-well plate. Two or three transformants were analyzed for each construct and average with standard deviation is shown. b Strains analyzed by fluorescence assay and GC after 96 h of fermentation in 50-mL flasks. The measurement was done in triplicates and average with standard deviation is shown. c Strains analyzed by GC after 140 h of fermentation in 1 L bioreactors. With exception for NS450 the measurement was done in duplicates and average with standard deviation is shown

Fig. 5

Evaluation of high lipid Y. lipolytica strain NS432 performance in 1 L bioreactor as compared to the parental strain NS18 in batch (a) and fed-batch (b) fermentation. Dry cell weight, glucose consumption, citrate and lipids (reported as fatty acid methyl esters) were monitored throughout the fermentation. All experiments except for NS18—batch were performed twice and the average with standard deviation from duplicate experiments are shown