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Isocitrate dehydrogenase (IDH) is a key enzyme in the TCA cycle, which can provide reducing power for cell growth, and participate in algal energy metabolism and anabolism[1]. IDH2 is the gene encoded in Y.lipolytica. It was registered in 2019. In order to characterize the effect of the IDH2 gene in lipid synthesis and growth, we constructed a knockout strain po1f ΔylIDH2 using homologous recombination. The YNB medium have been chosen for testing Results showed that the strain po1f ΔylIDH2 can effectively increase the accumulation of biomass and fatty acid content. Among them, the strain po1f ΔylIDH2 biomass and fatty acid content increased by 5.63% and 10.10%, respectively. These results provide references for future iGEM teams to improve biomass and lipid accumulation in Y.lipolytica. | Isocitrate dehydrogenase (IDH) is a key enzyme in the TCA cycle, which can provide reducing power for cell growth, and participate in algal energy metabolism and anabolism[1]. IDH2 is the gene encoded in Y.lipolytica. It was registered in 2019. In order to characterize the effect of the IDH2 gene in lipid synthesis and growth, we constructed a knockout strain po1f ΔylIDH2 using homologous recombination. The YNB medium have been chosen for testing Results showed that the strain po1f ΔylIDH2 can effectively increase the accumulation of biomass and fatty acid content. Among them, the strain po1f ΔylIDH2 biomass and fatty acid content increased by 5.63% and 10.10%, respectively. These results provide references for future iGEM teams to improve biomass and lipid accumulation in Y.lipolytica. | ||
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Revision as of 13:49, 10 October 2022
Delta-12 desaturase
Contents
Short description
Delta-12 desaturase involved in an Omega-3 biosynthetic pathway.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 426
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Description
This part encodes a Δ12 desaturase derived from Synechocystis sp PCC 6803. It is a membrane-bound enzyme able to introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (18:1, Δ9). This converts the substrate into linoleic acid (18:2 Δ9,12), a polyunsaturated fatty acid (PUFA). Mass spectrometry results show that feeding oleic acid to transformed E. coli, the bacterial membrane composition changes. Linoleic acid is observed.
Characterisation
In order to show desaturase activity of this enzyme, we performed a lipid analysis on fatty acid methyl esters (FAME) by gas chromatography–mass spectrometry GC-MS. Our samples were membrane assays and lipid extracts from E. coli expressing this desaturase.
As a control, the same FAME-GC analysis was performed on unmodified cells, and the lipid profiles were compared. FAME 18:2 (Δ9,12) standard was also run to compare the desaturation pattern to the expected 18:2 of the transformed cells.
Importantly, the cells were grown in the presence of manually added substrate 18:1 (Δ9). These 18:1 fatty acids are not present in unmodified E. coli BL21.
Results
Our results indicate that C18:2 is present in both the lipid extracts and membrane assays from cells transformed with the Δ-12 desaturase, unlike in untransformed cells. Moreover, this 18:2 has the same desaturation pattern as the control, meaning the 18:2 found in the transformed cells is the expected 18:2 (Δ9,12).
Conclusion
Lipid profiles of E. coli transformed with our construct show that this Δ-12 desaturase is able to catalyze the desaturation of oleic acid to give linoleic acid. In this way, this BioBrickTM can be used to build a biosynthetic pathway for PUFAs and Omega-3 fatty acids, along with other desaturases and elongases.
References
LIVORE V., TRIPODI K., UTARRO A., 2007. Elongation of polyunsaturated fatty acids in trypanosomatids. FEBS Journal, 274: 264–274.
Contribution From NNU-China 2022
Group: [ https://2022.igem.wiki/nnu-china/]
Author: Yaru Jiang
Characterization from iGEM22-NNU-China
Delta-12 desaturase
Isocitrate dehydrogenase (IDH) is a key enzyme in the TCA cycle, which can provide reducing power for cell growth, and participate in algal energy metabolism and anabolism[1]. IDH2 is the gene encoded in Y.lipolytica. It was registered in 2019. In order to characterize the effect of the IDH2 gene in lipid synthesis and growth, we constructed a knockout strain po1f ΔylIDH2 using homologous recombination. The YNB medium have been chosen for testing Results showed that the strain po1f ΔylIDH2 can effectively increase the accumulation of biomass and fatty acid content. Among them, the strain po1f ΔylIDH2 biomass and fatty acid content increased by 5.63% and 10.10%, respectively. These results provide references for future iGEM teams to improve biomass and lipid accumulation in Y.lipolytica.
- Fig. 1. The Growth and lipid synthesis of strain po1f ΔylIDH2 under different culture conditions. A. Changes of growth curves in two fermentation mediums. B. Changes of fatty acid content in the two fermentation mediums at 120h
Reference
[1]Gombos, Z., Wada, H., Varkonyi, Z., Los, D. A., & Murata, N. (1996). Characterization of the Fad12 mutant of Synechocystis that is defective in Δ12 acyl-lipid desaturase activity. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism, 1299(1), 117-123.