Selection and characterization of promoters based on genomic approach for the molecular breeding of oleaginous fungus Mortierella alpina 1S-4.

To express a foreign gene effectively, a good expression system is required. In this study, we investigated various promoters as useful tools for gene manipulation in oleaginous fungus Mortierella alpina 1S-4. We selected and cloned the promoter regions of 28 genes in M. alpina 1S-4 on the basis of expression sequence tag abundance data. The activity of each promoter was evaluated using the ß-glucuronidase (GUS) reporter gene. Eight of these promoters were shown to enhance GUS expression more efficiently than a histone promoter, which is conventionally used for the gene manipulation in M. alpina. Especially, the predicted protein 3 and the predicted protein 6 promoters demonstrated approximately fivefold higher activity than the histone promoter. The activity of some promoters changed along with the cultivation phase of M. alpina 1S-4. Seven promoters with constitutive or time-dependent, high-level expression activity were selected, and deletion analysis was carried out to determine the promoter regions required to retain activity. This is the first report of comprehensive promoter analysis based on a genomic approach for M. alpina. The promoters described here will be useful tools for gene manipulation in this strain.

Characterization of galactose-dependent promoters from an oleaginous fungus Mortierella alpina 1S-4.

An inducible promoter is a useful tool for the controlled expression of a given gene. In this report, we describe galactose-dependent promoters for potential use in an oleaginous fungus Mortierella alpina. We cloned the putative promoter regions of two genes encoding galactose metabolic enzymes, GAL1 and GAL10, from the genome of M. alpina 1S-4. The ß-glucuronidase (GUS) reporter gene assay in M. alpina 1S-4 revealed that regulation of these promoters was dependent on the presence of galactose in the medium both with and without other sugars. With the GAL10 promoter, an approximately 50-fold increase of GUS activity was demonstrated by addition of galactose into the culture media at any cultivation phase. The 5' deletion analysis of the GAL10 promoter revealed that a promoter region of over 2,000 bp length was required for its high-level activity and sufficient inducible response. Significantly, this is the first report of inducible promoters of zygomycetes. The GAL10 promoter will be a valuable tool for gene manipulation in M. alpina 1S-4.

Improvement of arachidonic acid production by mutants with lower n-3 desaturation activity derived from Mortierella alpina.

Five mutants were obtained, Y11, Y135, Y164, Y180 and Y61, capable of accumulating higher amounts of arachidonic acid (AA) than Mortierella alpina 1S-4, an industrial strain for the production of AA-rich triacylglycerol (TG). This is thought to be due to low or no activity of n-3 desaturation with conversion of AA to eicosapentaenoic acid, which functions at a cultural temperature below 20 degrees C. In small-scale cultivation under optimum conditions, Y11 and Y61 respectively accumulated 4.97 mg/ml and 4.11 mg/ml of AA, using a high concentration of glucose at 20 degrees C, compared with 3.74 mg/ml for M. alpina 1S-4. In a 5-1 jar fermentor, the AA content in Y11 and Y61 kept increasing during cultivation, with consumption of the glucose in the medium; and this reached 1.48 mg/ml and 1.77 mg/ml (118 mg/g, 120 mg/g of dry mycelia) at day 10, respectively, compared with 0.95 mg/ml (86 mg/g of dry mycelia) for M. alpina 1S-4. From the results of lipid analysis, the TG contents of Y11 and Y61 in the major lipids were significantly higher than that of M. alpina 1S-4; and the AA percentages in TG of Y11 and Y61 were also higher. Both Y11 and Y61 are potential producers of TG rich in AA.