Molecular evidence that the rate‐limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of an elongase

Abstract

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain for arachidonic acid (AA) production. To determine its physiological properties and to clarify the biosynthetic pathways for PUFA, heterologous and homologous gene expression systems were established in this fungus. The first trial was performed with an enhanced green fluorescent protein gene to assess the transformation efficiency for heterologous gene expression. As a result, strong fluorescence was observed in the spores of the obtained transformant, suggesting that the foreign gene was inherited by the spores. The next trial was performed with a homologous PUFA elongase (GLELOp) gene, this enzyme having been reported to catalyze the elongation of GLA (18:3n-6) to dihomo-gamma-linolenic acid (20:3n-6), and to be the rate-limiting step of AA production. The FA composition of the transformant was different from that of the host strain: The GLA content was decreased whereas that of AA was increased. These data support the hypothesis that the GLELOp enzyme plays an important role in PUFA synthesis, and may indicate how to control PUFA biosynthesis.

Cloning and functional characterisation of polyunsaturated fatty acid elongases of marine and freshwater teleost fish

“…Recently, the production of AA in M. alpina was increased by metabolic engineering through the construction of a strain overexpressing the elongase (GLELO) gene (Takeno et al, 2005). Low C 18-20 elongase activity had previously been observed in a turbot cell line in comparison to an Atlantic salmon cell line, suggesting it may be implicated in the inability of some marine fish to thrive on diets devoid of n-3HUFA (Ghioni et al, 1999).…”

Single cell oil production by Mortierella alpina

“…The stable transformants exhibited OPRTase activity comparable to that of the wild strain and retained the ura5 gene originating from the transformation vector regardless of the culture conditions, while the unstable ones easily lost the marker gene under uracil-containing conditions. By using this transformation system, the expression of the gene encoding EL2, which has been suggested to be the limiting step for AA biosynthesis (Wynn and Ratledge, 2000), constructed in a similar cassette on the expression of the homologous ura5 gene, was successfully performed (Takeno et al, 2005b). The resultant transformants yielded more AA than the wild strain.…”

Cloning and Characterization of the ∆6 Polyunsaturated Fatty Acid Elongase from the Green Microalga Parietochloris incisa

“…Recent studies have identified and characterized PUFA-specific elongases, responsible for the elongation of PUFA in the nematode Caenorhabditis elegans, mammals, fish, algae, lower plants, and fungi [19][20][21][22][23][24][25]. The elongation of 18:3x-6 to 20:3x-6, the immediate precursor of ARA, was shown to be the rate-limiting step in ARA biosynthesis in M. alpina [26,27]. Functional expression of the PUFA elongase condensation component in yeast revealed enzymes of various specificities for C18 and C20 acyl substrates.…”