Team:Evry Paris-Saclay/Demonstrate
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-eleostearic 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 bioproduction 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 five strains JMY1233, JMY1877, JMY2159, JMY3325 and JMY3820 (Table 2). In 4 of these strains the mechanisms of fatty acids’ degradation through the β-oxidation pathway are disrupted (pox1-6Δ). In others, the triacylglycerol synthesis (dga1Δ dga2Δ lro1Δ are1Δ) 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]. However, linoleic acid is the FadX substate and overexpressing Fad2 may lead to inscrease precursor concentration and thus bust punicic acid production. 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 (tgl4Δ), 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. According to it, the best yield should be obtained using the JMY3820 strain.
| Strain name | Genotype | Reference |
| JMY195 (Po1d) | MATA ura3-302 leu2-270 xpr2-322 | [11] |
| JMY1233 | MATA ura3-302 leu2-270 xpr2-322 pox1-6Δ | [12] |
| JMY1877 | MATA ura3-302 leu2-270 xpr2-322 dga1Δ dga2Δ lro1Δ are1Δ | [13] |
| JMY2159 | MATA ura3-302 leu2-270 xpr2-322 pox1-6Δ dga1Δ dga2Δ lro1Δ fad2Δ | [14] |
| JMY3325 | MATA ura3-302 leu2-270 xpr2-322 pox1-6Δ dga1Δ dga2Δ lro1Δ fad2Δ pTEF-FAD2-LEU2 | [15] |
| JMY3820 | MATα ura3-302 leu2-270 xpr2-322 pox1-6Δ tgl4Δ + pTEF-DGA2 + pTEF-GPD1 | [16] |
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 [17]) 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 rpm) 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. [18] 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 ester (Matreya, LLC), a main peak having a retention time of 6.19 minutes was observed as shown in Figure 2. Two additional peaks with retention times of 6.30 minutes and 6.39 minutes 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 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 minutes 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
GC analysis of samples isolated from the fermentation broth of the Y. lipolytica strains harbouring a FadX expression cassette was performed. As shown in Figure 4, the expression cassettes of both Pg-FadX (BBa_K2983181) and Tk-FadX (BBa_K2983182) inserted in the genome of Y. lipolytica JMY3820 strain are able to produce a compound with a retention time of 6.1 minutes. This compound is most likely punicic acid, since its retention time is the same as that of punicic acid from pomegranate seed oil. Also, this peak is absent in the negative control samples which was prepared using an empty YL-pOdd1 (BBa_K2983030). This suggests that our modified yeast are capable of producing punicic acid when expressing either Pg-FadX (BBa_K2983181) or Tk-FadX (BBa_K2983182).
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. This result is in agreement with our computational analysis.
Also, this
production was only detected 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 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
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