Production of cis-11-eicosenoic acid by Mortierella fungi.

AIM: To find cis-11-eicosenoic acid (20:1ω9, EA)-producing micro-organisms. METHODS AND RESULTS: We found EA-producing fungi by screening about 300 fungal strains and identified a major fatty acid accumulated in the Mortierella fungi as EA by means of GC-MS analysis. In particular, Mortierella chlamydospora CBS 529.75 produced a high amount of EA (36.3 mg g(-1) of dried cells) on cultivation at 28°C for 4 days and then at 12°C for 3 days. In the result of lipid analysis, most of the EA was a component of triacylglycerols, not phospholipids. CONCLUSION: We found that M. chlamydospora CBS 529.75 was the best producer for the microbial production of EA. SIGNIFICANCE AND IMPACT OF THE STUDY: EA is beneficial as a raw material for medical supplies and a moisturizing component of cosmetic creams. This is the first report of microbial production of EA.

Selective production of cis-9,trans-11 isomer of conjugated linoleic acid from trans-vaccenic acid methyl ester by Delacroixia coronata.

AIMS: Bio-process development for isomer selective and efficient production of cis-9,trans-11-octadecadienoic acid (CLA) from trans-vaccenic acid (t-VA, trans-11-octadecenoic acid) through microbial fatty acid Delta9-desaturation reaction. METHODS AND RESULTS: A total of 550 strains of fungi and yeasts were screened for CLA production from t-VA through Delta9 desaturation. Delacroixia coronata IFO 8586 was selected as a potent producer of CLA from t-VA. Efficient CLA production was observed during cultivation in medium supplemented with the methyl ester of t-VA (t-VAME). Under the optimal conditions with 33.3 mg ml(-1) of t-VAME as substrate, 10.5 mg ml(-1) CLA was produced by D. coronata IFO 8586 after 7 days of cultivation in the medium containing dextrin (5.0%), tryptone (2.0%) and thiourea (0.83 micromol ml(-1)). The strain produced the cis-9,trans-11 isomer of CLA selectively (98% of total CLA), with a small amount of the trans-9,trans-11 isomer (2% of total CLA), mainly in the form of triacylglycerols (69% of total CLA). CONCLUSIONS: A practical bio-process for selective production of cis-9,trans-11 isomer of CLA using filamentous fungus D. coronata IFO 8586 was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: Isomer selective bio-process for the practical production of cis-9,trans-11-CLA was first established. The process is benefitable for expanding the application of CLA for medicinal and nutraceutical purposes.

Cloning and expression of the L-1-amino-2-propanol dehydrogenase gene from Rhodococcus erythropolis, and its application to double chiral compound production.

The gene encoding NADP(+)-dependent L-1-amino-2-propanol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 was cloned and sequenced. A 780-bp nucleotide fragment was confirmed to be the gene encoding AADH by agreement of the N-terminal and internal amino acid sequences of the purified AADH. The gene (aadh) codes a total of 259 amino acid residues, and the deduced amino acid sequence shows similarity to several short-chain dehydrogenase/reductase family proteins. An expression vector, pKKAADH, which contains the full length aadh was constructed. Escherichia coli cells possessing pKKAADH exhibited a 10.4-fold increase in specific activity as to catalysis of the reduction of (S)-1-phenyl-2-methylaminopropan-1-one (MAK), as compared with that of R. erythropolis MAK154 induced by 1-amino-2-propanol (1 mg/ml). Coexpression of aadh with a cofactor regeneration enzyme (glucose dehydrogenase) gene was also performed, and a system for sufficient production of d-pseudoephedrine from racemic MAK was constructed.

Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

Production of 5,8,11-eicosatrienoic acid by a delta5 and delta6 desaturation activity-enhanced mutant derived from a delta12 desaturation activity-defective mutant of Mortierella alpina 1S-4.

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3omega9) was attained with a mutant fungus, Mortierella alpina JT-180, derived from delta12 desaturation activity-defective and delta6 desaturation activity-enhanced M. alpina M209-7. Production of 20:3omega9 by JT-180 was 1.4 times greater than that of the parent strain M209-7. This is thought to be due to its enhanced Delta5 desaturation activity, which was 3.3 times higher than that of M209-7. In both strains, 78.5-80.4% of the total lipids comprised triacylglycerol (TG), and 76.6-79.0% of 20:3omega9 was present in TG. Comparing the fatty acid compositions among various lipid species, the highest percentages (24.1-37.6%) of 20:3omega9 in total lipids were found in phosphatidylcholine. For optimization of 20:3omega9 production by JT-180, a glucose concentration of 4% in the culture medium and shifting of the growth temperature from 28 degrees C to 20 degrees C on the 2nd day were shown to be effective. Under optimal conditions, 20:3omega9 production by JT-180 reached 1.92 g/l culture medium in a 10-l jar fermentor (corresponding to 81.5 mg/g dry mycelia and 18.3% of total fatty acids), which is greater than that reported previously from M209-7 (1.65 g/l).

Delta6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus. Gene cloning and its heterologous expression in a fungus, Aspergillus.

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 delta6-desaturase genes (from borage and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (delta9, delta12-18:2) to gamma-linolenic acid (delta6, delta9, delta12-18:3). This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 457 amino acids from a M. calpina 1S-4 library. The predicted amino-acid sequence showed similarity to those of the above delta6-desaturases, and contained a cytochrome b5-like domain at the N-terminus, being different from the yeast delta9-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 gamma-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 delta6-desaturase activity. The Mortierella delta6-desaturase is the first to be reported in fungi.

Identification of an NADH-cytochrome b(5) reductase gene from an arachidonic acid-producing fungus, Mortierella alpina 1S-4, by sequencing of the encoding cDNA and heterologous expression in a fungus, Aspergillus oryzae.

Based on the sequence information for bovine and yeast NADH-cytochrome b(5) reductases (CbRs), a DNA fragment was cloned from Mortierella alpina 1S-4 after PCR amplification. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 298 amino acid residues which show marked sequence similarity to CbRs from other sources, such as yeast (Saccharomyces cerevisiae), bovine, human, and rat CbRs. These results suggested that this cDNA is a CbR gene. The results of a structural comparison of the flavin-binding beta-barrel domains of CbRs from various species and that of the M. alpina enzyme suggested that the overall barrel-folding patterns are similar to each other and that a specific arrangement of three highly conserved amino acid residues (i.e., arginine, tyrosine, and serine) plays a role in binding with the flavin (another prosthetic group) through hydrogen bonds. The corresponding genomic gene, which was also cloned from M. alpina 1S-4 by means of a hybridization method with the above probe, had four introns of different sizes. These introns had GT at the 5' end and AG at the 3' end, according to a general GT-AG rule. The expression of the full-length cDNA in a filamentous fungus, Aspergillus oryzae, resulted in an increase (4.7 times) in ferricyanide reduction activity involving the use of NADH as an electron donor in the microsomes. The M. alpina CbR was purified by solubilization of microsomes with cholic acid sodium salt, followed by DEAE-Sephacel, Mono-Q HR 5/5, and AMP-Sepharose 4B affinity column chromatographies; there was a 645-fold increase in the NADH-ferricyanide reductase specific activity. The purified CbR preferred NADH over NADPH as an electron donor. This is the first report of an analysis of this enzyme in filamentous fungi.

Genetic analysis of cytochrome b5 from arachidonic acid-producing fungus, Mortierella alpina 1S-4: cloning, RNA editing and expression of the gene in Escherichia coli, and purification and characterization of the gene product.

Information on the amino acid sequences of the internal peptide fragments of cytochrome b5 from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase SalI-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b5. On the basis of nucleotide sequences of both cytochrome b5 genomic gene and cDNA, the genomic cytochrome b5 gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine-->guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of M. alpina 1S-4 cytochrome b5 showed significant similarities with those of cytochrome b5s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b5 gene was expressed to 16% of the total soluble protein in Escherichia coli. The holo-cytochrome b5 accounted for 8% of the total cytochrome b5 in the transformants. The purified cytochrome b5 showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b5.

Identification of Delta12-fatty acid desaturase from arachidonic acid-producing mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae.

Based on the sequence information for the omega3-desaturase genes (from Brassica napus and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (Delta9, Delta12-18 : 2) to alpha-linolenic acid (Delta9, Delta12, Delta15-18 : 3), a cDNA was cloned from the filamentous fungal strain, Mortierella alpina 1S-4, which is used industrially to produce arachidonic acid. Homology analysis with protein databases revealed that the amino acid sequence showed 43.7% identity as the highest match with the microsomal omega6-desaturase (from Glycine max, soybean), whereas it exhibited 38.9% identity with the microsomal omega3-desaturase (from soybean). The evolutionary implications of these enzymes will be discussed. The cloned cDNA was confirmed to encode a Delta12-desaturase, which was involved in the desaturation of oleic acid (Delta9-18 : 1) to linoleic acid, by its expression in both the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Analysis of the fatty acid composition of yeast and fungus transformants demonstrated that linoleic acid (which was not contained in the control strain of S. cerevisiae) was accumulated in the yeast transformant and that the fungal transformant contained a large amount of linoleic acid (71.9%). Genomic Southern blot analysis of the transformants with the Mortierella Delta12-desaturase gene as a probe confirmed integration of this gene into the genome of A. oryzae. The M. alpina 1S-4 Delta12-desaturase is the first example of a cloned nonplant Delta12-desaturase.

Delta 9-fatty acid desaturase from arachidonic acid-producing fungus. Unique gene sequence and its heterologous expression in a fungus, Aspergillus.

Based on the sequence information for delta 9-desaturase genes (from rat, mouse and yeast), which are involved in the desaturation of palmitic acid and stearic acid to palmitoleic acid and oleic acid, respectively, the corresponding cDNA and genomic gene were cloned from the fungal strain, Mortierella alpina 1S-4, which industrially produces arachidonic acid. There was a cytochrome b5-like domain linked to the carboxyl terminus of this Mortierella desaturase, as also seen in the yeast delta 9-desaturase. The Mortierella delta 9-desaturase genomic gene had only one intron, in which a novel phenomenon was observed: there was a GC-end at the 5'-terminus instead of a GT-end that is, in general, found in introns of eukaryotic genes. The full-length cDNA clone was expressed under the control of an amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in drastic changes in the fatty acid composition in the transformant cells; the contents of palmitoleic acid (16:1) and oleic acid (18:1) increased significantly, with accompanying decreases in palmitic acid (16:0) and stearic acid (18:0). These changes were controlled by the addition of maltose as a carbon source to the medium. Also, the expression of the gene caused a significant change in the lipid composition in the Aspergillus transformant. Genomic Southern blot analysis of the transformant with the Mortierella delta 9-desaturase gene as a probe confirmed the integration of this gene into the genome of A. oryzae.

Characterization of the second form of NADH-cytochrome b5 reductase gene from arachidonic acid-producing fungus Mortierella alpina 1S-4.

The second type of cytochrome b5 reductase (Cb5R-II) gene was characterized in the arachidonic acid-producing fungus Mortierella alpina 1S-4. Its cDNA (897 bp) and predicted amino acid (298 aa) sequences show more than 70% similarity to the previously isolated first type of Cb5R. Highly conserved exon-intron organization suggests that the two genes evolved from the duplication of a common ancestral gene. Cb5R-II has a flavin-binding domain at its highly hydrophobic N-terminal and an NADH-binding domain at the C-terminal. In comparison with Cb5R genes from other sources, high homology (46-54%) was found for yeast and plant genes. Phylogenetic analysis revealed that microbial and plant Cb5R genes represent a gene family evolved from one prototype and are different from mammalian Cb5R genes.

A Novel Delta5-Desaturase-Defective Mutant of Mortierella alpina 1S-4 and Its Dihomo-gamma-Linolenic Acid Productivity.

A novel Delta5-desaturase-defective mutant was derived from an arachidonic acid-producing fungus, Mortierella alpina 1S-4, after treating the parental spores with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant produced only a trace (about 1%) amount of arachidonic acid, and the ratio of dihomo-gamma-linolenic acid (DGLA) to total fatty acids in each lipid class was markedly high, accounting for as much as 60% in phosphatidylcholine. Under submerged batch culture conditions, the mutant produced 2.4 g of DGLA per liter (43.3% of total fatty acids) when grown at 28 degrees C for 7 days in a 5-liter jar fermentor. The other major (more that 1%) fatty acids were palmitic acid (21.2%), stearic acid (9.6%), oleic acid (14.3%), linoleic acid (4.4%), and gamma-linolenic acid (5.8%). About 80 mol% of the DGLA produced was found in triacylglycerol.