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.
We describe the isolation and characterization of a gene (MAELO) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4. Although the homologous MAELO gene had already been isolated from M. alpina ATCC 32221, its function had not yet been identified. The MAELO gene from M. alpina 1S-4 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae. Analysis of the fatty acid composition of the yeast transformant revealed the accumulation of 22-, 24-, and 26-carbon saturated fatty acids. On the other hand, RNA interference of the MAELO gene in M. alpina 1S-4 was carried out. The gene-silenced strain obtained on RNA interference exhibited low contents of 20-, 22-, and 24-carbon saturated fatty acids and a high content of stearic acid (18 carbons), compared with those in the wild strain. The enzyme encoded by the MAELO gene was demonstrated to be involved in the biosynthesis of 20-, 22-, and 24-carbon saturated fatty acids in M. alpina 1S-4.
Increasing the efficiency of hydrolysis of 3-substituted glutaric acid diamides is useful to improve the synthesis of optically active 3-substituted gamma-aminobutyric acid. This is the first report of efficient hydrolysis of CGD using amidase mutant-producing E. coli cells.
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