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.

Gene name	FadX genes’ part numbers	FadX transcriptional units’ part numbers	Y. lipolytica genome integration cassettes' part numbers
Punica granatum FadX (Pg-FadX)	BBa_K2983061	BBa_K2983081	BBa_K2983181
Trichosanthes kirilowii FadX (Tk-FadX)	BBa_K2983062	BBa_K2983082	BBa_K2983182

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.

Strain name	Genotype	Reference
JMY195 (Po1d)	MATA ura3-302 leu2-270 xpr2-322	[11]
JMY2159	MATA ura3-302 leu2-270 xpr2-322 pox1-6Δ dga1Δ lro1Δ dga2Δ fad2Δ	[12]
JMY3820	MATα ura3-302 leu2-270 xpr2-322 Δpox1-6 Δtgl4 + pTEF-DGA2 + pTEF-GPD1	[13]

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

However, the amount of punicic acid produced is low and we were only able to detect it when Pg-FadX (BBa_K2983181) and Tk-FadX (BBa_K2983182) were inserted in the genome of Y. lipolytica JMY3820 strain, but not when using as a chassis the wild-type JMY195 or the JMY2159 strains. Also, this production was only deteced when cells were grown in minimal glucose medium YNB.

The production of punicic acid in Y. lipolytica is certainly possible but limited by various factors. It’s rapid degradation, either through the cellular metabolism or by a light induced oxidation may account for the low observed yield. Indeed punicic acid is a very effective anti-oxidant and therefore it is sensitive to oxidation. This oxidation may be responsible for the 3 peaks present in the commercial punicic acid methyl ester (Figure 2) and the protective effect of vitamin E present in the pomegranate oil may account for the stability of punicic acid in this preparation (Figure 3). Also, the production of punicic acid was assessed after 72h of Y. lipolytica culturing, a time inspired by the dynamics of CLA (conjugated linoleic acids) production in similar conditions [15]. This culturing time is in agreement with the observations made when producing punicic acid in other yeast species, Saccharomyces cerevisiae [1,2] or Schizosaccharomyces pombe [10]. A refinement of the culturing conditions thus appears necessary to increase the punicic acid production.
On the other hand, both Pg-FadX and Tk-FadX are expressed under the control of pTef1 promoter (BBa_K2983052), a medium strength constitutive promoter. Increasing the promoter strength is a conceivable alternative for increasing enzyme expression and thus the punicic acid production. Moreover, the use of inducible promoters may allow separating the biomass production from the punicic acid production. This is particularly important when the compound to be produced is toxic.

To rule out the possible toxicity of punicic acid and to evaluate the capacity of Y. lipolytica to store it, and not reduce it (to linoleic or even to oleic acid), we performed a feeding experiment in which cells were grown in the presence of increasing concentrations of pomegranate oil containing 60% punicic acid. The microscopic images presented in Figure 5 show an increase of lipid bodies size when the concentration of punicic acid increases. Extracellular lipids are accumulating inside yeast, albeit at higher pomegranate oil concentration they are also visible in the media. A GC analysis confirmed the presence of punicic acid inside Y. lipolytica (the amount increased at higher pomegranate oil added in the culturing media). To investigate further Y. lipolytica’s ability to store punicic acid, we contacted Dr. Romain Holic, a specialist of punicic acid production in yeast Schizosaccharomyces pombe [10], who kindly analysed our yeast strain by thin-layer chromatography (TLC). The results presented in Figures 6 and 7 show that Y. lipolytica is able to intake punicic acid (contrary to S. pombe). Inside cells, punicic acid is present in its free form, but also accumulates as triacylglycerols with 1, 2 or 3 punicic acid chains per molecule. An unknown compound absorbing in UV (like punicic acid does) is also visible and it may be the punicoyl-CoA. Using TLC, no detectable punicic acid production by the Y. lipolytica JMY3820 strain harboring the Pg-FadX (BBa_K2983181) could be detected, contrary to the S. pombe strain expressing Pg-FadX [10].

Other strain engineering may be envisioned in order to increase punicic acid production, the most obvious being the overexpression, along with the FadX enzymes, of proteins like Fad2 (that converts oleoyl-CoA to linoleoyl-CoA) and Ole1 (that converts stearyl-CoA to oleoyl-CoA) in order to boost CLnA precursors and Ldp1 (lipid droplet protein) and Lro1 (phospholipid:diacylglycerol acyltransferase) in order to increase the storage of CLnA in lipid droplets as triacylglycerols.

Conclusions

We have successfully built two Yarrowia lipolytica strains able to produce limited but detectable amounts of punicic acid, a CLnA with interesting properties. This production is dependent on the expression of a FadX enzyme, either Pg-FadX (BBa_K2983061) or Tk-FadX (BBa_K2983062). Optimizing the culturing conditions and fatty acids preservation, but also performing other strain engineering manipulations may lead to making Yarrowia lipolytica the CLnA production factory we had aimed for.

References

[1]Hornung E, Pernstich C, Feussner I. Formation of conjugated Delta11Delta13-double bonds by Delta12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds. Eur J Biochem (2002) 269, 4852-4859.
[2] Iwabuchi M, Kohno-Murase J, Imamura J. Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds. J Biol Chem (2003) 278, 4603-4610.
[3] Qiu X, Reed DW, Hong H, MacKenzie SL, Covello PS. Identification and analysis of a gene from Calendula officinalis encoding a fatty acid conjugase. Plant Physiol (2001) 125, 847-855.
[4]Cahoon EB, Ripp KG, Hall SE, Kinney AJ. Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid. J Biol Chem (2001) 276, 2637-2643.
[5]Cahoon EB, Carlson TJ, Ripp KG, Schweiger BJ, Cook GA, Hall SE, Kinney AJ. Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos. Proc Natl Acad Sci U S A (1999) 96, 12935-12940.
[6]Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM. Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res (2009) 48, 375-387.
[7]Zhang B, Chen H, Li M, Gu Z, Song Y, Ratledge C, Chen YQ, Zhang H, Chen W. Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid. Microb Cell Fact (2013) 12, 70.
[8] Ledesma-Amaro R, Nicaud JM. Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids. Pasrog Lipid Res (2016) 61, 40-50.
[9] Holic R, Xu Y, Caldo KMP, Singer SD, Field CJ, Weselake RJ, Chen G. Bioactivity and biotechnological production of punicic acid. Appl Microbiol Biotechnol (2018) 102, 3537-3549.
[10] Garaiova M, Mietkiewska E, Weselake RJ, Holic R. Metabolic engineering of Schizosaccharomyces pombe to produce punicic acid, a conjugated fatty acid with nutraceutic properties. Appl Microbiol Biotechnol (2017) 101, 7913-7922.
[11] Barth G, Gaillardin C. Yarrowia lipolytica. In: Wolf K (ed) Non conventional yeasts in biotechnology. Springer, Berlin (1996) 1, 314-388.
[12] Beopoulos A, Verbeke J, Bordes F, Guicherd M, Bressy M, Marty A, Nicaud JM. Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol (2014) 98, 251-262.
[13] Lazar Z, Dulermo T, Neuvéglise C, Crutz-Le Coq AM, Nicaud JM. Hexokinase - A limiting factor in lipid production from fructose in Yarrowia lipolytica. Metab Eng (2014) 26, 89-99.
[14] Dulermo R, Brunel F, Dulermo T, Ledesma-Amaro R, Vion J, Trassaert M, Thomas S, Nicaud JM, Leplat C. Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica. Microb Cell Fact (2017) 16, 31.
[15]Imatoukene N, Verbeke J, Beopoulos A, Idrissi Taghki A, Thomasset B, Sarde CO, Nonus M, Nicaud JM. A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol (2017) 101, 4605-4616.
[16] Browse J, McCourt PJ, Somerville CR. Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem (1986) 152, 141-145.