Bioproduction

Conjugated linolenic acids (CLnAs) are synthetized by bifunctional fatty acid conjugase / desaturase (FadX) enzymes from linoleic acid (incorporated into phosphatidylcholine). The sequences of the enzymes catalyzing the synthesis of 3 of the 7 known CLnAs have been described in the literature and their activities are specific to the position and the stereochemistry of the double bonds:

• punicic acid, C18:3 (9Z, 11E, 13Z) is synthesized by EC: 1.14.19.16 [1,2].

• α-calendic acid, C18:3 (8E, 10E, 12Z), is synthesized by EC: 1.14.19.14 [3,4].

• α-oleosteaic acid, C18:3 (9Z, 11E, 13E) is synthesized by EC: 1.14.19.33 [5].

To achieve a sustainable bioproduction of such fatty acids in order to limit environmental and economical problems, we decided to use as a biological chassis the oleaginous yeast Yarrowia lipolytica. This species has already proven its effectiveness for the production of fatty acids, thanks to its highly developed lipid metabolism [6-8]. As a proof of concept of our project, we decided to focus on one of those CLnAs, the punicic acid, that has interesting properties such as anti-obesity, anti-inflammatory, anti-cancer, anti-diabetes activities [9].

Design

Linoleic acid, C18:2 (9Z,12Z), the substrate of FadX enzymes, is a natural metabolite for our chassis Yarrowia lipolytica. Thus, to convert it into punicic acid, only the presence of a EC: 1.14.19.16 enzyme is necessary (Figure 1). Two EC: 1.14.19.16 enzymes were described in the literature: one from pomegranate / Punica granatum (Pg-FadX, BBa_K2983061) and another one from the chinese cucumber / chinese snake gourd / Trichosanthes kirilowii (Tk-FadX, BBa_K2983062).

To express these two enzymes (Pg-FadX and Tk-FadX) in our chassis, we codon optimized the sequences for Y. lipolytica and placed them under the control of the pTef1 promoter (BBa_K2983052) and of the Lip2 terminator (BBa_K2983055). The resulting FadX transcriptional units (BBa_K2983081 and BBa_K2983082, respectively) were assembled into our YL-pOdd1 plasmid (BBa_K2983030) which is part of our Loop assembly system dedicated to our chassis, the oleaginous yeast Y. lipolytica (for further details on this system, visit the dedicated page on this wiki). Thus, we generated two FadX expression plasmids (BBa_K2983181 and BBa_K2983182, respectively) able to integrate upon transformation, into a Y. lipolytica Po1d stain. All these parts are summarized in Table 1.

Yarrowia lipolytica: Yes, but which strain(s)? Y. lipolytica an ideal chassis for the bio-production of fatty acids in general and we have tried to put the odds on our side by choosing strains favoring even more the storage and production of these fatty acids. It’s for this reason that, to produce punicic acid, we have opted for two strains JMY2159 and JMY3820 (Table 2). In these strains the mechanisms of fatty acids’ degradation through the β-oxidation pathway are disrupted (pox1-6Δ). In addition, in JMY2159 the triacylglycerol synthesis (dga1Δ dga2Δ lro1Δ) is inactivated which favors fatty acids’ accumulation in a free form (R-COOH). Also, the oleic acid to linoleic acid conversion by Δ12 desaturation (fad2Δ) is disrupted, which was shown to favor punicic acid production in yeast Schizosaccharomyces pombe [10]. On the other hand, in strain JMY3820 fatty acids accumulation as triacylglycerols is promoted. In this strain the triacylglycerol mobilisation is inhibited by the disruption of the gene encoding the triglyceride lipase (Δtgl4), the triacylglycerol degradation is inhibited by deleting POX (POX1-6) genes. And two enzymes of the triacylglycerol biosynthetic pathway, the acyl-CoA:diacylglycerolacyltransferase (DGA2) and glycerol-3-phosphate dehydrogenase (GPD1) are overexpressed to push and pull triacylglycerol biosynthesis. As a control, we also use the auxotrophic wild-type strain JMY195. A computational analysis of these Y. lipolytica strains that assisted us in strain selection can be found on the Dry Lab page of this wiki.

All these Y. lipolytica strains were transformed with the NotI digested Pg-FadX and Tk-FadX expression plasmids (BBa_K2983181 and BBa_K2983182) and the genome integrations were confirmed by PCR (using a pTef1 forward primer and a FadX specific reverse primer). As a negative control, we also transformed them with the NotI digested empty YL-pOdd1 vector (BBa_K2983030). A second transformation with a Leu2 plasmid (JMP62-LEU2ex-pTEF [14]) was performed to render the strains prototroph for leucine too.

Experimental Setup

The Y. lipolytica strains expressing the Pg-FadX and Tk-FadX along with the negative control were grown in either rich YPD medium or in minimal glucose medium YNB (containing 1.7 g/L yeast nitrogen base without amino acids and ammonium sulfate, 1.5 g/L NH4Cl, 50 mM KH2PO4-Na2HPO4 buffer pH 6.8 and 60 g/L glucose). The cultivation was performed at 28°C in 500-mL baffled flasks containing 100 mL of liquid media under agitation (180 rmp) as described by [15]. After 72h, cells were pelleted, resuspended in water and frozen at -20°C before lyophilization. Fatty acids contained in about 50 mg of dried yeast were converted to methyl esters (FAMEs) according to the protocol described by Browse et al. [16] and were subsequently analysed by gas chromatography (GC), a technique in which the compounds in a sample are vaporized and migrated with a carrier gas on a stationary phase which is an inert solid support. With such technique it is possible to identify different fatty acids following the length of their carbon chain and the number of unsaturation on those chain, two properties which modify the capacity of fatty acid to migrate with the carrier gas on the column. The GC analysis was carried out with a Varian 3900 instrument equipped with a flame ionization detector and a Varian FactorFour vf-23ms column, where the bleed specification at 260 °C is 3 pA (30 m, 0.25 mm, 0.25 μm). All manipulations were performed taking care of protecting samples from light to avoid UV driven oxidation of punicic acid.

The two standard chemicals, commercial punicic acid methyl ester (Matreya, LLC) and commercial pomegranate’s seeds essential oil (Huiles et Sens, Centiflor Laboratory), were analyzed by GC. From the pure commercial punicic acid methyl eFigurester (Matreya, LLC), a main peak having a retention time of 6.19 minute was observed as shown in 2. Two additional peaks with retention times of 6.30 and 6.39 were also visible, indicating the instability of the punicic acid methyl ester. The presence of punicic acid was also revealed in a commercial pomegranate’s seeds essential oil (Huiles et Sens, Centiflor Laboratory) containing 60% of punicic acid according to the provider’s specifications. As shown in Figure 3, a main peak having a retention time of 6.19 minute was observed. Several other peaks corresponding to the other fatty acids present in the seed preparation are also visible on the GC chromatogram. It is worth highlighting that, compared to commercial punicic acid methyl ester, the main peak has a much higher intensity which helps distinguish it from other minor peaks. This is most probably due to the protective, antioxidant action of the other components of this commercial pomegranate’s seeds essential oil, especially vitamin E.

Result