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Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.

ABSTRACT: A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast. This ORF (F56H11. 4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation. The substrate specificity of the C. elegans enzyme indicated a preference for Delta(6)-desaturated C18 polyunsaturated fatty acids. Coexpression of this activity with fatty acid desaturases required for the synthesis of C20 polyunsaturated fatty acids resulted in the accumulation of arachidonic acid from linoleic acid and eicosapentaenoic acid from alpha-linolenic acid. These results demonstrate the reconstitution of the n-3 and n-6 polyunsaturated fatty acid biosynthetic pathways. The C. elegans ORF is likely to interact with endogenous components of a yeast elongation system, with the heterologous nematode condensing enzyme F56H11.4 causing a redirection of enzymatic activity toward polyunsaturated C18 fatty acid substrates.

SUBMITTER: Beaudoin F

PROVIDER: S-EPMC18618 | biostudies-literature | 2000 Jun

REPOSITORIES: biostudies-literature

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Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.

Beaudoin F F Michaelson L V LV Hey S J SJ Lewis M J MJ Shewry P R PR Sayanova O O Napier J A JA

Proceedings of the National Academy of Sciences of the United States of America 20000601 12

A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast. This ORF (F56H11. 4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation. The substrate specificity of the C. elegans enzyme indicated a preference for Delta(6)-desaturated C18 polyunsaturated fatty acids. Coexpression of this activity with fatty acid desaturases required for the syn ...[more]

PMID: 10829069

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Project description:BackgroundFlax (Linum usitatissimum L.) is an agriculturally important crop with seed oil enriched in α-linolenic acid (18:3 (cisΔ9, 12, 15); ALA). This polyunsaturated fatty acid (PUFA) is the major determinant for the quality of flax seed oil in food, nutraceuticals and industrial applications. The recently identified enzyme: phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), catalyzes the interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), and has been shown to play an important role in PUFA accumulation in Arabidopsis thaliana seeds.MethodsTwo flax PDCT genes were identified using homology-based approach.ResultsIn this study, we describe the isolation and characterization of two PDCT genes from flax (LuPDCT1 and LuPDCT2) with very high nucleotide sequence identity (97%) whose deduced amino acid sequences exhibited approximately 55% identity with that of A. thaliana PDCT (AtROD1). The genes encoded functionally active enzymes that were strongly expressed in developing embryos. Complementation studies with the A. thaliana rod1 mutant demonstrated that the flax PDCTs were capable of restoring PUFA levels in planta. Furthermore, PUFA levels increased in Saccharomyces cerevisiae when the flax PDCTs were co-expressed with FATTY ACID DESATURASES (FADs), FAD2 and FAD3, while seed-specific expression of LuPDCT1 and LuPDCT2 in A. thaliana resulted in 16.4% and 19.7% increases in C18-PUFAs, respectively, with a concomitant decrease in the proportion of oleic acid (18:1 (cisΔ9); OA).ConclusionsThe two novel PDCT homologs from flax are capable of increasing C18-PUFA levels substantially in metabolically engineered yeast and transgenic A. thaliana seeds. These flax PDCT proteins appear to play an important dual role in the determination of PUFA content by efficiently channelling monounsaturated FAs into PC for desaturation and moving the resulting PUFAs out of PC for subsequent use in TAG synthesis. These results indicate that flax PDCTs would be useful for bioengineering of oil crops to increase PUFA levels for applications in human food and nutritional supplements, animal feed and industrial bioproducts.

Dataset Information

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.

Publications

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.

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