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 3-desaturase from M. alpina 1S-4. Based on the conserved sequence information for M. alpina 1S-4 12-desaturase and Saccharomyces kluyveri 3-desaturase, the 3-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 12-desaturase, whereas it exhibited 36% identity with Sac. kluyveri 3-desaturase. The cloned cDNA was confirmed to encode the 3-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 3-desaturase differs from those of the known fungal 3-desaturases from Sac. kluyveri and Saprolegnia diclina. Plant, cyanobacterial and Sac. kluyveri 3-desaturases desaturate 18-carbon n-6 PUFAs, Spr. diclina 3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans 3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs. The substrate specificity of M. alpina 1S-4 3-desaturase is rather similar to that of C. elegans 3-desaturase, but the M. alpina 3-desaturase can more effectively convert AA into EPA when expressed in yeast. The M. alpina 1S-4 3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.     

Production of a novel ω1-eicosapentaenoic acid by Mortierella alpina 1S-4 grown on 1-hexadecene

An arachidonic-producing fungus, Mortierella alpina 1S-4, was found to accumulate -unsaturated fatty acids of C-20 chain length together with 1-hexadecenoic acid, 1-octadecenoic acid and so on, when grown on 1-alkenes, i.e., 1-hexadecene and 1-octadecene. The results of mass spectroscopy and proton NMR showed that a C₂₀ polyunsaturated fatty acid (PUFA) is a novel cis-5,8,11,14,19-eicosapentaenoic acid (20:51). This PUFA was obtained at a yield of 0.13 mg/ml culture broth (2.8% of the fungal total fatty acid content) on cultivation of the fungus in a medium containing 4% (v/v) 1-hexadecene and 1% yeast extract at 28°C for 1 week. Investigation of the distribution of fatty acids showed that about 90% (by mol.) of the PUFA was present in the triglycerides and 10% was in the phospholipid fraction. About 70% of that found in the phospholipids was phosphatidylcholine (PC) and the value accounted for ca. 10% of the total fatty acid content. The formation of these -unsaturated fatty acids was presumed to occur through the arachidonic acid biosynthetic pathway (n-6 route).Abbreviations PUFA polyunsaturated fatty acid - EPA cis-5,8,11,14,17-eicosapentaenoic acid - TG triglycerides - PS phosphatidylserine - PC phosphatidylcholine Present address: Laboratory of Microbial Science, Institute for Fundamental Research, Suntory Ltd., Mishima-gun, Osaka 618, Japan     

Substrate specificity and membrane topologies of the iron-containing ω3 and ω6 desaturases from Mortierella alpina

Applied Microbiology and Biotechnology - Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of...     

Production of prostaglandin F2α by molecular breeding of an oleaginous fungus Mortierella alpina

Cyclooxygenases are responsible for the production of prostaglandin H₂ (PGH₂) from arachidonic acid. PGH₂ can be converted into some bioactive prostaglandins, including prostaglandin F_2α (PGF_2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF_2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF_2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF_2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF_2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.     

Isolation and characterization of wheat ω-gliadin genes

The DNA sequences of two full-length wheat ω-gliadin prolamin genes (ωF20b and ωG3) containing significant 5′ and 3′ flanking DNA sequences are reported. The ωF20b DNA sequence contains an open reading frame encoding a 30,460-Dalton protein, whereas the ωG3 sequence would encode a putative 39,210-Dalton protein except for a stop codon at amino-acid residue position 165. These two ω-gliadin genes are closely related and are of the ARQ-/ARE-variant type as categorized by the derived N-terminal amino-acid sequences and amino-acid compositions. The ω-gliadins were believed be related to the ω-secalins of rye and the C-hordeins of barley, and analyses of these complete ω-gliadin sequences confirm this close relationship. Although the ω-type sequences from all three species are closely related, in this analysis the rye and barley ω-type sequences are the most similar in a pairwise comparison. A comparison of ω-gliadin flanking sequences with respect to that of their orthologs and with respect to wheat gliadin genes suggests the conservation of flanking DNA necessary for gene function. Sequence data for members of all major wheat prolamin families are now available. Received: 24 August 2000 / Accepted: 15 December 2000     

Production of 5,8,11-eicosatrienoic acid by a Δ5 and Δ6 desaturation activity-enhanced mutant derived from a Δ12 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).     

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

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.     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Isolation and physiological and biochemical characterization of the Chlamydomonas reinhardtii C-41 mutant,a superproducer of ζ-carotene

The composition of the carotenes and xanthophylls of Chlamydomonas reinhardtii Dang. C-41, a mutant of a unicellular green alga and a superproducer of ζ-carotene, was studied. The light-harvesting complexes and a complex of the PS-II reaction center were established to be disrupted in the C-41 mutant. However, the mutant retained a high (up to 46%) photosynthetic activity and the capacity to accumulate chlorophylls and carotenoids (up to 50%). The composition of carotenes was studied, and it was shown that, in contrast to wild-type K(+) cells, which accumulate up to 95% of β-carotene and 5% α-carotene, cells of the C-41 mutant contained 43% β-carotene, 19% β-zeacarotene, and 38% ζ-carotene. The high level of C-41 mutant biomass accumulation made it possible to recommend the mutant as a superproducer of ζ-carotene in phytobiotechnology.     

Isolation and characterization of the pMI10 plasmid fromBacillus subtilis,an α-amylase producer

The plasmid DNA pMI10 (5310 bp) was isolated from the alpha-amylase producing strain B. subtilis A18. Thirteen restriction endonucleases were used to digest pMI10 DNA and the restriction map of pMI10 DNA was constructed by mapping PstI (1), HindII (2), BglI (2), BspRI (3) and HindIII (3) sites.     

Isolation and characterization of an antimicrobial peptide from bovine hemoglobin ��-subunit

In this study, a novel 18-residue linear antimicrobial peptide derived from the central part of the bovine hemoglobin ??-subunit was identified. The peptide was purified by a combination of cationic exchange and reversed-phase high-performance liquid chromatography. The sequence was determined to be VNFKLLSHSLLVTLASHL. The theoretical molecular weight of this peptide was calculated to be 1992.38 Da, which is the same as that determined (1992.401 Da) by matrix-assisted laser desorption ionization mass spectrometry. Sequence analysis showed that there is a high degree of homology in this peptide among hemoglobin ??-subunits of bovine, sheep, deer, porcine, and human. In a radial-diffusion plate assay, this purified peptide exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans.     

Isolation and characterization of a sterile-dwarf mutant in Asian cotton （Gossypium arboreum L.）

Plant height is an important trait in cotton. To elucidate the molecular mechanisms of the dwarf phenotype, a sterile-dwarf mutant derived from Gossypium arboreum L. cv. Jinhuazhongmian was developed by ^60Co y-ray irradiation. The results demonstrated that the steriledwarf mutant phenotype was controlled by a pair of recessive gene, which was designated sd^a. Plants carrying the sd^a gene contained lower levels of indole-3-acetic acid （IAA） and abscisic acid （ABA） compared with wild-type （WT） plants. The chlorophyll content and net photosynthetic rate in mutant leaves were markedly decreased. However, it was possible that ABA biosynthesis or signaling was involved in governing the sd^a phenotype. Semi-quantitative RT-PCR analysis detected 13 differentially expressed ESTs, and the steriledwarf mutant exhibited decreased expression levels relative to the WT. The role of nine potential hormone biosynthetic genes in the synthesis of IAA, ABA, polyamines （PAs） and jasmonic acid （JA） were discussed.     

Isolation and characterization of an inter-α-trypsin inhibitor-IgG complex from human serum

Inter-α-trypsin inhibitor is a human serum protease inhibitor of M_r 180 000 which may release physiological derivatives. A complex between IgG and an inter-α-trypsin inhibitor derivative of M_r 30000 has been recently detected in human serum and was found to be inactive against trypsin, in contrast with the known inhibitory activity of the free 30-kDa derivative. The present study deals with detailed characterization of an inter-α-trypsin inhibitor-IgG complex following its purification by affinity chromatography techniques (anti-inter-α-trypsin inhibitor immunoadsorbent and Protein A-Sepharose) in mild conditions. The resulting product reacted simultaneously with anti-IgG and anti-inter-α-trypsin inhibitor antibodies. This complex contained M_r 180 000 inhibitor at least to some extent. It migrated in the β-γ zone in agarose; its molecular weight was estimated to be 1500 000 or more; part of it displayed covalent bonding between inter-α-trypsin inhibitor and IgG; it had a trypsin inhibitor activity. Immunoelectrophoresis allowed one to demonstrate the native complex in serum owing to the use of anti-inter-α-trypsin inhibitor and anti-γ radioactively labelled antibodies. The double immunoreactivity thus evidenced proved to be heterogeneous with respect to its level and location in the native as well as in the purified complex.     

Isolation and characterization of an α-satellite repeated sequence from human chromosome 22

We constructed a library in IL47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag⁺/Cs₂SO₄ gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of -R1-DNA and the X specific -satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived -satellite sequences analogous to other chromosome-specific satellite sequences described previously.     

Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1 (EF-1), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1. An intron is located in the 5 untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1tef-c (97%) than to the P. purpurea EF-1tef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.     

Production and characterization of extracellular α-amylases from the thermophilic fungus Thermomyces lanuginosus (wild and mutant strains)

-Amylases from the thermophilic fungus, Thermomyces lanuginosus ATCC 34626 (wild and mutant strains), were purified to homogeneity by a simple procedure including, consecutively, precipitation with ice-cold 2-propanol, anion-exchange and molecular-sieve chromatographic methods. The molecular masses of the purified -amylases (both with pI values of 3.0) were 58 kDa by SDS-PAGE. The optimal pH of -amylase activity was 5.0 for the wild enzyme and 4.5 for the mutant one. 1-Cyclohexyl-3-(2-morpholinyl-4-ethyl)-carbodiimide (40–100 mM) and N-bromosuccinimide (0.1–1 mM) inhibited the enzymes, suggesting the involvement of carboxylic groups and tryptophan residues in the catalytic process.     

Isolation and Characterization of an Unusual Nucleoside, 1-α-D-Ribofuranosyl-4-pyridone-3-carboxamide,from the Urines of Normal Human Individuals and Leukemic Patients

Abstract

A novel modified nucleoside, 1-α-D-Ribofuranosyl-4-pyridone-3-carboxamide, has been isolated from the urines of one normal human individual and two CML patients. The structure was assigned on the basis of UV, NMR and mass spectral data and confirmed by comparison with an authentic sample synthesized in our laboratory. This constitutes the first example of the occurrence of a nucleoside with an α-riboside linkage in human urine.     

Production and Isolation of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

A new inhibitor of dopamine β-hydroxylase, dopastin, has been isolated. The dopastin-producing strain was found in a mushroom culture, and after being separated, it was confirmed to be a member of Pseudomonas. Dopastin was obtained as colorless needles, mp 116~119°C, (c=0.5, С₂Н₅ОН), C₉H₁₇N₃O₃. The catalytic hydrogenation afforded dihydro-dopastin which also inhibits dopamine β-hydroxylase.