Functional characterization of Δ9 and ω9 desaturase genes in Mortierella alpina 1S-4 and its derivative mutants

Abstract

Cloning and characterization of the Delta9 desaturase (Delta9I) gene of a fungus, Mortierella alpina 1S-4, was previously reported. In this study, two genes encoding Delta9 desaturase homologs were isolated from this fungus. One is a Delta9 desaturase (Delta9II) that exhibits 86% amino acid sequence similarity to Delta9I. Functional analysis involving expression of the encoding gene in Aspergillus oryzae revealed that Delta9II exhibits Delta9 desaturase activity, 18:0 being converted to 18:1Delta9. However, unlike Delta9I, the Delta9II transformant accumulated a low amount of 16:1Delta9. The other homolog is a omega9 desaturase (omega9) that exhibits 56 and 58% amino acid sequence similarity to Delta9I and Delta9II, respectively. On functional analysis with the Aspergillus transformant, it was found that omega9 does not convert 18:0 to 18:1Delta9, but converts 24:0 and 26:0 to 24:1omega9 and 26:1omega9, respectively. On the other hand, Delta9 desaturation-defective mutants characterized by accumulation of 18:0 were derived from M. alpina 1S-4 with a chemical mutagen, and the mutated sites of the Delta9 desaturase genes were identified. The mutation on the Delta9I gene was assumed to cause an amino acid replacement (W136Stop, G265D, and W360Stop) in the mutants (HR222, T4, and ST56), respectively. In these mutants, there was no mutated site on the Delta9II and omega9 genes. Real-time quantitative PCR (RTQ-PCR) analysis revealed that (1) the transcriptional level of the Delta9I gene in HR222 and T4 was much higher than that in the wild strain until the fifth day of the cultivation periods, (2) the Delta9II gene of the mutants was transcribed until the fourth day at the same level as the Delta9I gene of the wild strain, whereas the Delta9II gene of the wild strain was transcribed at a lower level, and (3) the transcriptional level of the omega9 gene in both the mutants and the wild strain was low, i.e., as low as that of the Delta9II gene of the wild strain. In these Delta9 desaturation-defective mutants, Delta9II is likely to play an important role in Delta9 desaturation.

Fermentative Production of Nervonic Acid by Mortierella capitata RD000969

“…In Mortierella alpina 1S-4, ω9 desaturase, which converts C 24:0 and C 26:0 fatty acids to C 24:1 n-9 and C 26:1 n-9 fatty acids, has been identified 28 . It is likely that ω9 desaturase is involved in the synthesis of C 23:1 n-9 fatty acid produced by strain RD000969.…”

Advances in the Production of Various Polyunsaturated Fatty Acids through Oleaginous FungusMortierella alpinaBreeding

“…Transcriptional analysis revealed that the genes encoding Á9-2 and !9 are not transcribed as much as that encoding Á9-1 in the wild strain, whereas the Á9-2 gene is transcribed effectively at the same level as or a higher level than the Á9-1 gene in mutants defective in Á9-1. 61) On the other hand, !9 mRNA is hardly synthesized in either the wild strain or the mutants. These results suggest that Á9-2 plays a major role in Á9 desaturation in these mutants with a Á9-1 desaturase deficiency.…”

“…61) The G nucleotides at the 561st, 947th, and 1,233rd positions from the start codon in the Á9-1 genes of HR222, T4, and ST56 were replaced by an A nucleotide, causing amino acid replacement of W136Stop, G265D, and W360Stop respectively. Transcriptional analysis revealed that the genes encoding Á9-2 and !9 are not transcribed as much as that encoding Á9-1 in the wild strain, whereas the Á9-2 gene is transcribed effectively at the same level as or a higher level than the Á9-1 gene in mutants defective in Á9-1.…”

Biosynthesis of polyunsaturated fatty acids in lower eukaryotes

“…It has been used for years in the industry to produce AA-rich oils (9,10,30). All enzymes for synthesizing AA and EPA have been isolated and characterized, including the stearoyl CoA D9 desaturases (19), D12 and o3 methyl-end desaturases, D6 and D5 front-end desaturases and D6 elongase (10,13,22,30). Elongation appears to be the rate limiting step and overexpression of D6 elongase in M. alpina has been recently reported to improve the AA production (43).…”

“…The resultant g-linolenic acid (18:3D 6,9,12 ) and stearidonic acid (18:4D 6,9,12,15 ) are elongated to the corresponding C20 D8 PUFAs and further desaturated by a D5 desaturase to AA (20:4D 5,8,11,14 ) and eicosapentaenoic acid (EPA, 20:5D 5,8,11,14,17 ), respectively. The production of DHA (22:6D 4,7,10,13,16,19 ) would theoretically imply the elongation of EPA to 22:5D 7,10,13,16,19 and desaturation by a D4 desaturase (Fig. 3).…”