Part:BBa_K2983081
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].
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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 192
Illegal PstI site found at 157 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 192
Illegal PstI site found at 157 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 192
Illegal PstI site found at 157 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 192
Illegal PstI site found at 157 - 1000COMPATIBLE WITH RFC[1000]
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