Eiji Sakuradani

Δ6-Fatty acid desaturase from an arachidonic acid-producing Mortierella fungus: Gene cloning and its heterologous expression in a fungus, Aspergillus

Abstract A DNA fragment was cloned from the fungal strain, Mortierella alpina 1S-4 (which is used industrially to produce arachidonic acid), after PCR amplification with oligonucleotide primers designed based on the sequence information for Δ6-desaturase genes (from borage and Caenorhabditis elegans ), which are involved in the desaturation of linoleic acid (Δ9, Δ12–18:2) to γ-linolenic acid (Δ6, Δ9, Δ12–18:3). This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 457 amino acids from a M. alpina 1S-4 library. The predicted amino-acid sequence showed similarity to those of the above Δ6-desaturases, and contained a cytochrome b 5 -like domain at the N-terminus, being different from the yeast Δ9-desaturase which has the corresponding domain at the C-terminus. The full-length cDNA clone was expressed under the control of the amyB promoter in a filamentous fungus, Aspergillus oryzae , resulting in the accumulation of γ-linolenic acid (which was not detected in the control Aspergillus ) to the level of 25.2% of the total fatty acids. These findings revealed that the recombinant product has Δ6-desaturase activity.

A novel fungal ω3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4

A filamentous fungus, Mortierella alpina 1S-4, is capable of producing not only arachidonic acid (AA; 20:4n-6) but also eicosapentaenoic acid (EPA; 20:5n-3) below a cultural temperature of 20°C. Here, we describe the isolation and characterization of a gene (maw3) that encodes a novel ω3-desaturase from M. alpina 1S-4. Based on the conserved sequence information for M. alpina 1S-4 Δ12-desaturase and Saccharomyces kluyveri ω3-desaturase, the ω3-desaturase gene from M. alpina 1S-4 was cloned. Homology analysis of protein databases revealed that the amino acid sequence showed 51% identity, at the highest, with M. alpina 1S-4 Δ12-desaturase, whereas it exhibited 36% identity with Sac. kluyveri ω3-desaturase. The cloned cDNA was confirmed to encode the ω3-desaturase by its expression in the yeast Sac. cerevisiae. Analysis of the fatty acid composition of the yeast transformant demonstrated that 18-carbon and 20-carbon n-3 polyunsaturated fatty acids (PUFAs) were accumulated through conversion of exogenous 18-carbon and 20-carbon n-6 PUFAs. The substrate specificity of the M. alpina 1S-4 ω3-desaturase differs from those of the known fungal ω3-desaturases from Sac. kluyveri and Saprolegnia diclina. Plant, cyanobacterial and Sac. kluyveri ω3-desaturases desaturate 18-carbon n-6 PUFAs, Spr. diclina ω3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans ω3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs. The substrate specificity of M. alpina 1S-4 ω3-desaturase is rather similar to that of C. elegans ω3-desaturase, but the M. alpina ω3-desaturase can more effectively convert AA into EPA when expressed in yeast. The M. alpina 1S-4 ω3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.

Desaturase-defective fungal mutants: useful tools for the regulation and overproduction of polyunsaturated fatty acids

Increasing demand for biologically important polyunsaturated fatty acids has led to the search for alternative sources, especially fungi. Although successes in this area have induced interest in developing fungal fermentation processes, manipulation of their lipid composition requires new biotechnological strategies to achieve high yields of the desired polyunsaturated fatty acids. Fungal desaturase mutants with unique enzyme systems are useful not only for the regulation and overproduction of valuable polyunsaturated fatty acids but also because they are excellent models for the elucidation of fungal lipogenesis.

Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding.

ABSTRACT Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5− strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for ω3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/108 spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal ω3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.

Isolation and characterization of Δ6-desaturase, an ELO-like enzyme and Δ5-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris

The liverwort Marchantiapolymorpha contains high proportions of arachidonic and eicosapentaenoic acids. In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and α-linolenic acids, respectively, by a series of reactions catalyzed by Δ6-desaturase, an ELO-like enzyme involved in Δ6 elongation and Δ5-desaturase. Here we report the isolation and characterization of the cDNAs, MpDES6, MpELO1 and MpDES5, coding for the respective enzymes from M.polymorpha. Co-expression of the MpDES6, MpELO1 and MpDES5 cDNAs resulted in the accumulation of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichiapastoris. Interestingly, Δ6 desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Δ6-desaturases are known to have a strong preference for glycerolipids.

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

ABSTRACT (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4

Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5− mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.

Gene Cloning and Functional Analysis of a Second Δ6-Fatty Acid Desaturase from an Arachidonic Acid-producing Mortierella Fungus

We demonstrated that Mortierella alpina 1S-4 has two Δ6-desaturases, which are involved in the desaturation of linoleic acid to γ-linolenic acid. For one of the two Δ6-desaturases, designated as Δ6I, gene cloning and its heterologous expression in a fungus, Aspergillus oryzae, has previously been reported. In addition, we indicated in this paper that there is an isozyme of the two Δ6-desaturases, designated as Δ6II, in M. alpina 1S-4. The predicted amino acid sequences of the Mortierella Δ6-desaturases were similar to those of ones from other organisms, i.e. borage and Caenorhabditis elegans, and had a cytochrome b5-like domain at the N-terminus, being different from the yeast Δ9-desaturase, which has the corresponding domain at the C-terminus. The full-length Δ6II cDNA was expressed in A. oryzae, resulting in the accumulation of γ-linolenic acid (which was not detected in the control Aspergillus) up to 37% of the total fatty acids. The analysis of real-time quantitative PCR (RTQ-PCR) showed that the quantity of Δ6I RNA was 2.4-, 9-, and 17-fold higher than that of Δ6II RNA on 2, 3, and 4 days in M. alpina 1S-4, respectively. M. alpina 1S-4 is the first fungus to be confirmed to have two functional Δ6-desaturase genes.

Improvement of the Fatty Acid Composition of an Oil-Producing Filamentous Fungus, Mortierella alpina 1S-4, through RNA Interference with Δ12-Desaturase Gene Expression

ABSTRACT An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Δ12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Δ12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Δ12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.

Selection of oleaginous yeasts with high lipid productivity for practical biodiesel production.

The lipid-accumulating ability of 500 yeast strains isolated in Japan was evaluated. Primary screening revealed that 31 strains were identified as potential lipid producers, from which 12 strains were cultivated in a medium containing 3% glucose. It was found that JCM 24511 accumulated the highest lipid content, up to 61.53%, while JCM 24512 grew the fastest. They were tentatively identified as Cryptococcus sp. and Cryptococcus musci, respectively. The maximum lipid concentration of 1.49g/L was achieved by JCM 24512. Similarly, JCM 24511 also achieved a high lipid production of 1.37g/L. High lipid productivity is the most important characteristic of oleaginous yeasts from the viewpoint of practical production. Among the strains tested here, JCM 24512 had the best lipid productivity, 0.37g/L/day. The results show that the isolated yeasts could be promising candidates for biodiesel production.