Difference between revisions of "Part:BBa K2983081"

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FadX of Punica granatum expression cassette under the control of pTef1 (BBa_K2983052) promoter

FadX of Punica granatum expression cassette under the control of pTef1 (BBa_K2983052) promoter

Usage and Biology

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].

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 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].

Figure 1:Conversion of linoleic acid to punicic acid (both incorporated into phosphatidylcholine)

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 (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 (trouver comment insérer le lien)

Sequence and Features