Biosynthesis of Fatty Acid Derivatives by Recombinant Yarrowia lipolytica Containing MsexD2 and MsexD3 Desaturase Genes from Manduca sexta.

One of the most interesting groups of fatty acid derivates is the group of conjugated fatty acids from which the most researched include: conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), which are associated with countless health benefits. Sex pheromone mixtures of some insect species, including tobacco horn-worm (Manduca sexta), are typical for the production of uncommon C16 long conjugated fatty acids with two and three conjugated double bonds, as opposed to C18 long CLA and CLNA. In this study, M. sexta desaturases MsexD2 and MsexD3 were expressed in multiple strains of Y. lipolytica with different genotypes. Experiments with the supplementation of fatty acid methyl esters into the medium resulted in the production of novel fatty acids. Using GCxGC-MS, 20 new fatty acids with two or three double bonds were identified. Fatty acids with conjugated or isolated double bonds, or a combination of both, were produced in trace amounts. The results of this study prove that Y. lipolytica is capable of synthesizing C16-conjugated fatty acids. Further genetic optimization of the Y. lipolytica genome and optimization of the fermentation process could lead to increased production of novel fatty acid derivatives with biotechnologically interesting properties.

Conversion of waste materials into very long chain fatty acids by the recombinant yeast Yarrowia lipolytica.

Erucic acid (C22:1Δ13) has several industrial applications including its use as a lubricant, surfactant and biodiesel and composite material constituent. It is produced by plants belonging to the Brassicaceae family, especially by the high erucic acid rapeseed. The ability to convert oleic acid into erucic acid is facilitated by FAE1. In this study, FAD2 (encoding Δ12-desaturase) was deleted in the strain Po1d to increase oleic acid content. Subsequently, FAE1 from Thlaspi arvense was overexpressed in Yarrowia lipolytica with the Δfad2 genotype. This resulted in the YL10 strain producing very long chain fatty acids, especially erucic acid. The YL10 strain was cultivated in media containing crude glycerol and waste cooking oil as carbon substrates. The cells grown using glycerol produced microbial oil devoid of linoleic acid, which was enriched with very long chain fatty acids, mainly erucic acid (9% of the total fatty acids). When cells were grown using waste cooking oil, the highest yield of erucic acid was obtained (887 mg L-1). However, external linoleic and α-linolenic were accumulated in cellular lipids when yeasts were grown in an oil medium. This study describes the possibility of conversion of waste material into erucic acid by a recombinant yeast strain.

Production of Long Chain Fatty Alcohols Found in Bumblebee Pheromones by Yarrowia lipolytica.

Fatty alcohols (FA-OH) are aliphatic unbranched primary alcohols with a chain of four or more carbon atoms. Besides potential industrial applications, fatty alcohols have important biological functions as well. In nature, fatty alcohols are produced as a part of a mixture of pheromones in several insect species, such as moths, termites, bees, wasps, etc. In addition, FA-OHs have a potential for agricultural applications, for example, they may be used as a suitable substitute for commercial insecticides. The insecticides have several drawbacks associated with their preparation, and they exert a negative impact on the environment. Currently, pheromone components are prepared mainly through the catalytic hydrogenation of plant oils and petrochemicals, which is an unsustainable, ecologically unfriendly, and highly expensive process. The biotechnological production of the pheromone components using engineered microbial strains and through the expression of the enzymes participating in the biosynthesis of these components is a promising approach that ensures ecological sustenance as well. The present study was aimed at evaluating the production of FA-OHs in the oleaginous yeast, Yarrowia lipolytica, with different lengths of fatty-acyl chains by expressing the fatty acyl-CoA reductase (FAR) BlapFAR4 from B. lapidarius, producing C16:0-OH, C16:1Δ9-OH, and lower quantities of both C14:0-OH and C18:1Δ9-OH, and BlucFAR1 from B. lucorum, producing FA-OHs with a chain length of 18-26 carbon atoms, in this yeast. Among the different novel Y. lipolytica strains used in the present study, the best results were obtained with JMY7086, which carried several lipid metabolism modifications and expressed the BlucFAR1 gene under the control of a strong constitutive promoter 8UAS-pTEF. JMY7086 produced only saturated fatty alcohols with chain lengths from 18 to 24 carbon atoms. The highest titer and accumulation achieved were 166.6 mg/L and 15.6 mg/g DCW of fatty alcohols, respectively. Unlike JMY7086, the BlapFAR4-expressing strain JMY7090 produced only 16 carbon atom-long FA-OHs with a titer of 14.6 mg/L.

Overexpression of diacylglycerol acetyltransferase from Euonymus europaeus in Yarrowia lipolytica leads to the production of single-cell oil enriched with 3-acetyl-1,2-diacylglycerols.

The 3-acetyl-1,2-diacylglycerols (acTAGs) are the molecules that are structurally similar to triacylglycerols (TAGs). They are naturally produced by plants of the family Celastraceae and animals such as Cervus nippon and Eurosta solidaginis. The presence of acetate in the sn-3 position of the glycerol backbone confers advantages to these compounds, for example, lower viscosity and calorific value compared to classical TAGs. In this work, the gene EeDAcT, which encodes diacylglycerol acetyltransferase in a species of bush (Euonymus europaeus), was overexpressed in strains Po1d (capable of accumulating storage lipids) and JMY1877 (incapable of accumulating storage lipids) of Yarrowia lipolytica, to test the activity of the gene EeDAcT and the production of acTAGs in oleaginous and nonoleaginous genetic backgrounds. It was observed that both the strains containing the gene EeDAcT (YL33 and YL35 for Po1d and JMY1877 strains, respectively) produced acTAGs. The strain YL33 accumulated up to 20% intracellular lipids, 20% of which was acTAGs, and 40% was TAGs. On the other hand, the strain YL35, which showed interrupted TAGs accumulation, produced up to 10% acTAGs as the only storage lipid. Unfortunately, the quantity of acTAGs produced in YL35 was insignificant, as the overall lipid accumulated in the strain was not more than 4% of the biomass. The fatty acid profile of acTAGs produced by the YL33 strain was remarkably similar to TAGs, and both of these structures were rich in oleic (45%) and palmitic (25%) acids.