Part:BBa_K2983181
FadX of Punica granatum expression cassette (BBa_K2983080) in YL-pOdd1
This part is an Y. lipolytica genome integration cassettes containing the FadX of Punica granatum (Pg-FadX, BBa_K2983061) expression cassette under the control of pTef1e (BBa_K2983052) promoter and Lip2 terminator (BBa_K2983055) inserted by Golden Gate with BsaI in YL-pOdd1 (BBa_K2983030).
FadX of Punica granatum is a bifunctional fatty acid conjugase / desaturase (EC: 1.14.19.16) enzyme [1,2] that converts linoleic acid (incorporated into phosphatidylcholine) into punicic acid, C18:3 (9Z, 11E, 13Z) (Figure 1).
pTef1e (BBa_K2983052) is a constitutive promoter active in the oleaginous yeast Yarrowia lipolytica.
This part is functional in Y. lipolytica and behaves as predicted: it's expression leads to the production of punicic acid.
Usage and Biology
Punicic acid, C18:3 (9Z, 11E, 13Z) is a conjugated linolenic acid (CLnA) and has interesting properties such as anti-obesity, anti-inflammatory, anti-cancer, anti-diabetes activities [3]. To achieve a sustainable bioproduction of punicic acid 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 [4-8].
Linoleic acid, C18:2 (9Z,12Z), the substrate of FadX enzymes, is a natural metabolite of Y. 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) [1] and another one from the chinese cucumber / chinese snake gourd / Trichosanthes kirilowii (Tk-FadX, BBa_K2983062) [2].
To express the Pg-FadX enzyme in Y. lipolytica, we codon optimized the sequences for Y. lipolytica and placed it under the control of the pTef1 promoter (BBa_K2983052) and of the Lip2 terminator (BBa_K2983055). The resulting FadX transcriptional unit (BBa_K2983081) was assembled into our YL-pOdd1 plasmid (BBa_K2983030) which is part of our Loop assembly system dedicated to Y. lipolytica. Thus, we generated a FadX expression plasmids (BBa_K2983181) able to integrate upon transformation, into an Y. lipolytica Po1d stain.
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 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 (fad2'Δ) is disrupted, which was shown to favor punicic acid production in yeast Schizosaccharomyces pombe [7]. 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 our wiki. According to it, the best yield should be obtained using the JMY3820 strain.
| Table 1. Yarrowia lipolytica strains used as chassis for fatty acids’ production. | ||
|---|---|---|
| Strain name | Genotype | Reference |
| JMY195 (Po1d) | MATA ura3-302 leu2-270 xpr2-322 | [8] |
| JMY1233 | MATA ura3-302 leu2-270 xpr2-322 pox1-6Δ | [9] |
| JMY1877 | MATA ura3-302 leu2-270 xpr2-322 dga1Δ dga2Δ lro1Δ are1Δ | [10] |
| JMY2159 | MATA ura3-302 leu2-270 xpr2-322 pox1-6Δ dga1Δ dga2Δ lro1Δ fad2Δ | [11] |
| JMY3325 | MATA ura3-302 leu2-270 xpr2-322 pox1-6Δ dga1Δ dga2Δ lro1Δ fad2Δ pTEF-FAD2-LEU2 | [12] |
| JMY3820 | MATα ura3-302 leu2-270 xpr2-322 pox1-6Δ tgl4 + pTEF-DGA2 + pTEF-GPD1 | [13] |
All these Y. lipolytica strains were transformed with the NotI digested Pg-FadX expression plasmid (BBa_K2983181) 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 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 [12]. 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. [15] 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.
Results
GC analysis of samples isolated from the fermentation broth of the Y. lipolytica strains harbouring the Pg-FadX expression cassette (BBa_K2983081) was performed. As shown in Figure 4, the expression cassettes of Pg-FadX (BBa_K2983181) inserted in the genome of Y. lipolytica JMY3820 strain is 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 Pg-FadX.
However, the amount of punicic acid produced is low and we were only able to detect it when Pg-FadX (BBa_K2983181) was 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 [12]. 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 [7]. A refinement of the culturing conditions thus appears necessary to increase the punicic acid production.
On the other hand, Pg-FadX is expressed under the control of pTef1 promoter (BBa_K2983052), a strong 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, the lipids present in Y. lipolytica JMY3820 expressing Pg-FadX (BBa_K2983181) were analysed by thin-layer chromatography (TLC) (in the lab of Dr. Romain Holic, a specialist of punicic acid production in yeast Schizosaccharomyces pombe [7]).
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 [7].
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.
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] 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.
[4] 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.
[5] 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.
[6] 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.
[7] 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.
[8] Barth G, Gaillardin C. Yarrowia lipolytica. In: Wolf K (ed) Non conventional yeasts in biotechnology. Springer, Berlin (1996) 1, 314-388.
[9] Beopoulos A, Mrozova Z, Thevenieau F, Le Dall MT, Hapala I, Papanikolaou S, Chardot T, Nicaud JM. Control of lipid accumulation in the yeast Yarrowia lipolytica. Appl Environ Microbiol (2008) 74, 7779-7789.
[10] Beopoulos A, Haddouche R, Kabran P, Dulermo T, Chardot T, Nicaud JM. Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts. Appl Microbiol Biotechnol (2012) 93, 1523-1537.
[11] 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.
[12] 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.
[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] 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 1727
Illegal PstI site found at 1692 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3212
Illegal SpeI site found at 1727
Illegal PstI site found at 1692 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 304
Illegal XhoI site found at 1281
Illegal XhoI site found at 1314 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 1727
Illegal PstI site found at 1692 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 1727
Illegal PstI site found at 1692
Illegal NgoMIV site found at 1126
Illegal AgeI site found at 1023 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1520
Illegal SapI.rc site found at 3220
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