Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering

Abstract

Background: Oleaginous yeast Yarrowia lipolytica is an organism of choice for the development of biofuel and oleochemicals. It has become a chassis for metabolic engineering in order to produce targeted lipids. Understanding the function of key-enzymes involved in lipid metabolism is essential to design better routes for enhanced lipid production and for strains producing lipids of interest. Because medium chain fatty acids (MCFA) are valuable compounds for biokerosene production, we previously generated strains capable of producing MCFA up to 12% of total lipid content (Rigouin et al. in ACS Synth Biol 6:1870-1879, 2017). In order to improve accumulation and content of C14 fatty acid (FA), the elongation, degradation and accumulation of these MCFA in Yarrowia lipolytica were studied.

Results: We brought evidence of the role of YALI0F0654 (YlELO1) protein in the elongation of exogenous or de novo synthesized C14 FA into C16 FA and C18 FA. YlELO1 deletion into a αFAS_I₁₂₂₀W expressing strain leads to the sole production of C14 FA. However, because this strain does not provide the FA essential for its growth, it requires being cultivated with essential fatty acids and C14 FA yield is limited. To promote MCFA accumulation in Y. lipolytica without compromising the growth, we overexpressed a plant diglyceride acyltransferase specific for MCFA and reached an accumulation of MCFA up to 45% of total lipid content.

Conclusion: We characterized the role of YlELO1 in Y. lipolytica by proving its involvement in Medium chain fatty acids elongation. We showed that MCFA content can be increased in Yarrowia lipolytica by promoting their accumulation into a stable storage form (triacylglycerides) to limit their elongation and their degradation.

Keywords: Diglyceride acyltransferase; Elongase; Fatty acid synthase; Medium chain fatty acids; Yarrowia lipolytica.

Fig. 1

A Multiple sequence alignment of Saccharomyces cerevisae elongases and Yarrowia lipolytica elongases. Accession numbers: ScELO1, P39540; ScELO2, P25358; ScELO3, P40319, YALI0B20196, Q6CDY7; YALI0F06754, Q6C2L8.The four motifs found in members of the ELO family are boxed (A–D) in red. Box B contains the histidine-rich region common to elongase enzymes. Transmembrane regions predicted using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) are underlined (in green) for the elongase sequences of Yarrowia lipolytica

Fig. 2

Growth of the strains ∆pox ∆fas (a) and ∆pox ∆fas ∆elo1 (b) in rich medium YT2D5 (diamond) complemented with mC14:0 (square), mC16:0 (triangle) or C18:1 (circle)

Fig. 3

a Lipid profiles of the strains ∆pox ∆fas and ∆pox ∆fas ∆elo1 grown in rich medium complemented with mC16:0 at 72 h. b Lipid profiles of the strains ∆pox and ∆pox ∆elo1 grown in rich medium at 72 h. C16 fatty acids are depicted in green, C18 fatty acids in blue. Average and standard deviation are given for two clones cultivated separately

Fig. 4

Kinetic of fatty acids synthesis by the strains ∆pox FAS-I₁₂₂₀W (a), ∆pox FAS-I₁₂₂₀W egDGAT (b) and ∆pox FAS-I₁₂₂₀W ∆DGAT1 ∆DGAT2 egDGAT (c). C18s corresponds to C18 fatty acid species (C18:0, C18:1 and C18:2) and is depicted in blue; C16s corresponds to C16 fatty acid species (C16:0 and C16:1) and is depicted in green. C14s corresponds to C14 fatty acid species (C14:0 and C14:1) and is depicted in orange. C12 FA is depicted in purple. diC12 is in brown and diC14 in pink. OD measure is given in a secondary vertical axis and is represented as black diamond markers on a dashed line. Average and standard deviation are given for two clones cultivated separately

Fig. 5

Lipid metabolism in Y. lipolytica. Schematic representation of the metabolic pathways of lipid synthesis, elongation, accumulation and degradation. Glc (glucose), AcCoa (acetyl-CoA), MaCoA (malonyl-CoA), FFA (free fatty acid), TAG (triacylglycerol), SE (steryl-esters), DAG (diacylglycerol), PL (phospholipids), MCFA (medium chain fatty acids), LCFA (long chain fatty acids), VLCFA (very long chain fatty acids). Gene names are in italic, in blue. ACC1 (acetyl-Coa carboxylase) FAS (fatty acid synthase), ELO (elongase), OLE1 (acyl-CoA desaturase), FAD2 (fatty acid desaturase), LRO (phospholipid DAG acyltransferase), TGL (triacylglycerol lipases), FAA1 (fatty acid-Coa synthetase), DGAT (DAG acyl transferase). Hypothetical routes discussed in this paper are depicted with a dashed arrow