The 'LipoYeasts' project: using the oleaginous yeast Yarrowia lipolytica in combination with specific bacterial genes for the bioconversion of lipids, fats and oils into high-value products

Abstract

The oleochemical industry is currently still dominated by conventional chemistry, with biotechnology only starting to play a more prominent role, primarily with respect to the biosurfactants or lipases, e.g. as detergents, or for biofuel production. A major bottleneck for all further biotechnological applications is the problem of the initial mobilization of cheap and vastly available lipid and oil substrates, which are then to be transformed into high-value biotechnological, nutritional or pharmacological products. Under the EU-sponsored LipoYeasts project we are developing the oleaginous yeast Yarrowia lipolytica into a versatile and high-throughput microbial factory that, by use of specific enzymatic pathways from hydrocarbonoclastic bacteria, efficiently mobilizes lipids by directing its versatile lipid metabolism towards the production of industrially valuable lipid-derived compounds like wax esters (WE), isoprenoid-derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs) and free hydroxylated fatty acids (HFAs). Different lipid stocks (petroleum, alkane, vegetable oil, fatty acid) and combinations thereof are being assessed as substrates in combination with different mutant and recombinant strains of Y. lipolytica, in order to modulate the composition and yields of the produced added-value products.

Figure 1

Scanning electron and fluorescence microscope visualization of lipid accumulation and lipid body formation depending on genotype of Y. lipolytica cells grown on glucose (YPD), triglyceride (YNB‐Triolein) and fatty acid (YNB‐Oleic acid). Strains used are the wild type (WT), a quadruple lipid body‐deficient mutant deleted for all acyl transferases (Δdga1, Δlro1, Δare1and Δare2) (JMY1877) (A. Beopoulos, R. Haddouche, P. Kabran, T. Chardot and J.‐M. Nicaud, in preparation) and an «obese» lipid‐overproducing mutant strain deleted for GUT2 and the POX genes (JMY1367) (Δpox1–6 andΔgut2) (Beopoulos et al., 2008). Upper panel (no staining) and lower panel (lipid are stained with lipidTox^TMgreen neutral strain).

Figure 2

Fatty acid‐fatty alcohol (top) and isoprenoid WE (bottom).

Figure 3

Short‐ and medium‐chain‐length PHAs. SCL‐PHA's: polyhydroxybutyrate (C4), polyhydroxyvalerate (C5). MCL‐PHA's: poly(3‐hydroxyhexanoate) (C6), poly(3‐hydroxyoctanoate), poly(3‐hydroxydecanoate) (C10), poly(3‐hydroxydodecanoate) (C12), poly(3‐hydroxytetradecanoate) (C14), and poly(3‐hydroxyhexadecanoate) (C16).

Figure 4

R‐3‐HFA.

Figure 5

Examples of carotenoid structures.

Figure 6

Proposed structure of stacked polyenic carotenoid ester chains.

Figure 7

Metabolic engineering of Yarrowia lipolytica for production of added‐value products. Lipids such as triacylglycerols (TAGs), fatty acids and n‐alkanes are used as carbon sources. They get hydrolysed (TAGs) extracellularly and get transported into the cell, where fatty acids get either stored as TAGs in lipids bodies or get further oxidized in ω‐ or β ‐oxidation cycles. Storage of fatty acids as TAGs will be blocked. β‐Oxidation cycle provides precursors for hydroxylated fatty acids (HFAs) and polyhydroxyalkanoates (PHAs). Modifications of Pox1–6 genes catalysing the first step of β‐oxidation will be explored for modulating monomer composition of the produced HFAs and PHAs. Finally, the ω‐oxidation will be also blocked to avoid the production of undesired dicarboxylic fatty acids. The resulted pool of fatty acids and fatty acid alcohols will be used for the production of WEs, carotenoids or carotenoid esters by expressing the corresponding bacterial biosynthetic genes.