Identification of a novel D6‐acyl‐group desaturase by targeted gene disruption in Physcomitrella patens

Abstract

SummaryThe moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel ∆5-and ∆6-desaturases are required. To isolate one of these, PCR-based cloning was used, and resulted in the isolation of a full-length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N-terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b 5 -related region and a C-terminal sequence with low similarity (27% identity) to acyl-lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of γ-linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a ∆6-desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC-MS. Only the transformed yeast cells were able to introduce a further double bond into the ∆6-position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.

Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula

“…The moss P. patens contains not only plant typical C18-fatty acids like LA or α-LeA but also γ-LeA and C20-fatty acids like AA and eicosapentaenoic acid (EPA) (Girke et al, 1998). These fatty acids may all serve as source for the formation of oxylipins.…”

“…These organisms contain C18, C20, and longer chain length fatty acids (Dembitsky, 1993). In the moss Physcomitrella patens for example the main fatty acids are palmitic acid, LA and arachidonic acid (AA) (Girke et al, 1998). Certain mosses are known to emit aldehydes and their corresponding alcohols, like octenal, octenol but also hexanal and hexenal (Dembitsky, 1993).…”

Targeted knockout in Physcomitrella reveals direct actions of phytochrome in the cytoplasm

“…PP1 was targeted with a circular insertional knockout construct (7) based on a 1.2-kb HindIII fragment cloned into the unique HindIII site of pGL2 (11); in this case, an additional NcoI site was inserted into the recombinant gene to provide additional proof of targeted knockout in Southern analysis. PP2-4 were targeted with replacement knockout constructs (13) in which the resistance cassette is flanked on both sides by Ϸ1 kb of gene-specific DNA. For PP2 knockout, a 2.1-kb genomic PCR product obtained with primers 5Ј-GGGAAGATGTCGACTCCCAAGAAG-3Ј (binding despite mismatches) and 5Ј-GCTGAGCTCATCCAT-GTC-3Ј was cloned into the EcoRV site of pBluescript II (KS Ϫ ) (Stratagene).…”

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway

“…1. Although the two desaturases (⌬ 6 -and ⌬ 5 -fatty acid desaturases) required for this pathway have been cloned recently from a number of different sources (7)(8)(9)(10)(11)(12)(13)(14), no genes have been characterized for the C2 elongation of the C18 PUFA. Biochemical characterization of mammalian elongation systems (most notably from liver microsomes) has indicated that the ''elongase'' actually consists of four enzyme activities, being made up of a condensing enzyme, a ␤-ketoreductase, a dehydrase, and an enoyl reductase (reviewed in ref.…”