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     

Isolation and characterization of an ω3-desaturation-defective mutant of an arachidonic acid-producing fungus,Mortierella alpina 1S-4

A mutant considered to be defective in the conversion of n-6 to n-3 fatty acids (3-desaturation) was derived from a 5-desaturation-defective mutant (Mut44) of Mortierella alpina 1S-4, after treating its spores with N-methyl-N-nitro-N-nitrosoguanidine. This mutant cannot produce 8(Z),11(Z),14(Z),17(Z)-eicosatetraenoic acid or any other n-3 fatty acids, of which about 10% was found in its parental strain upon cultivation at 12°C. The mutant's growth rate was comparable to that of the parental strain when grown at 28°C, but it became much slower when the mutant grew at 12°C, at which the lag phase for Mut44 was about 2 d but 5 d for the mutant.Abbreviations 18:33 9(Z),12(Z),15(Z)-octadecatrienoic acid - 18:43 6(Z),9(Z),12(Z),15(Z)-octadecatetraenoic acid - 20:43 8(Z),11(Z),14(Z),17(Z)-eicosatetraenoic acid - AA arachidonic acid - DHGA dihomo--linolenic acid - EPA 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid - GLC gas-liquid chromatography - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PC phosphatidylcholine     

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.     

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.     

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.     

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 arachidonic acid and dihomo-γ-linolenic acid from glycerol by oil-producing filamentous fungi, Mortierella in the ARS culture collection

The filamentous fungi of the genus Mortierella are known to produce arachidonic acid from glucose, and the species alpina is currently used in industrial production of arachidonic acid in Japan. In anticipation of a large excess of the co-product glycerol from the national biodiesel program, we are trying to find new uses for bioglycerin. We screened 12 Mortierella species: M. alpina NRRL 6302, M. claussenii NRRL 2760, M. elongata NRRL 5246, M. epigama NRRL 5512, M. humilis NRRL 6369, M. hygrophila NRRL 2591, M. minutissima NRRL 6462, M. multidivaricata NRRL 6456, M. nantahalensis NRRL 5216, M. parvispora NRRL 2941, M. sepedonioides NRRL 6425, and M. zychae NRRL 2592 for their production of arachidonic acid (AA) and dihomo-γ-linolenic acid (DGLA) from glycerol. With glucose as substrate all of the strains tested produced AA and DGLA. The total fatty acid content of 125 mg/g cell dry weight (CDW) and fatty acid composition for AA (19.63%) and DGLA (5.95%) in the mycelia of M. alpina grown on glucose were comparable with those reported by Takeno et al. (Appl Environ Microbiol 71:5124–5128, 2005). With glycerol as substrate all species tested grew on glycerol and produced AA and DGLA except M. nantahalensis NRRL 5216, which could not grow on glycerol. The amount of AA and DGLA produced were comparable with those obtained with glucose-grown mycelia. The top five AA producers (mg AA/CDW) from glycerol were in the following order: M. parvispora > M. claussenii > M. alpina > M. zychae > M. minutissima. The top five dry mycelia weights were: M. zychae > M. epigama > M. hygrophila > M. humilis > M. minutissima. The top five species for total fatty acids production (mg /g CDW) were: M. claussenii > M. parvispora > M. minutissima > M. hygrophila > M. maltidivaricata. We selected two species, M. alpina and M. zychae for further studies with glycerol substrate. Their optimum production conditions were determined. Time course studies showed that the maximum cell growth and AA production for both species were at 6 days of incubation. Therefore, glycerol can be considered for industrial use in the production of AA and DGLA.     

Adrenic acid Δ4 desaturation and fatty acid composition in the liver of marine-oil fed streptozotocin diabetic rats

The aim of the present study was to assess the effect of streptozotocin diabetes and insulin treatment on adrenic acid Δ4 desaturation and fatty acid composition of liver microsomes in Wistar rats fed a fat free semi-synthetic basal diet supplemented with 10% EPA-rich marine oil. Results showed that, in liver microsomes of hyperglycemic rats, the ratio in total lipids was elevated and desaturation of adrenic acid to n-6 docosapentaenoic acid was enhanced. Insulin treatment with 2.0 I.U./100 g body weight⁻¹ twice a day for 3 days resulted in hypoglycemia and suppressed both the increased Δ4 n-6 desaturation and ratio. It is concluded that the Δ4 desaturation enzyme system, which is activated by experimental diabetes, is regulated by mechanisms different from those regulating Δ6 and Δ5 desaturations.     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

Formation and degradation of Δ9-1,18-octadecenedioic acid from oleic acid by Candida tropicalis

Summary The formation and degradation of ⁹-1,18-octadecenedioic acid from oleic acid by mutant S ₇₆ of Candida tropicalis was studied. It was found that the mutant could convert oleic acid to ⁹-1,18-octadecenedioic acid by -oxidation of the terminal methyl group. The substance was identified by various analytical methods and could be degraded to unsaturated dioic acids of shorter chain length with even numbers of carbon atoms from 6 to 16, and saturated dioic acids with 6, 8 and 10 carbon atoms. Neither unsaturated nor saturated dioic acids with odd numbers of carbon atoms were found in the media. From these results some metabolic pathways of the degradation of this unsaturated dioic acid are proposed. Some mass spectra of unsaturated dioic acids are presented.     

Production of ω-hydroxyundec-9-enoic acid and n-heptanoic acid from ricinoleic acid by recombinant Escherichia coli-based biocatalyst

ω-Hydroxyundec-9-enoic acid and n-heptanoic acid are valuable building blocks for the production of flavors and antifungal agents as well as bioplastics such as polyamides and polyesters. However, a biosynthetic process to allow high productivity and product yield has not been reported. In the present study, we engineered an Escherichia coli-based biocatalytic process to efficiently produce ω-hydroxyundec-9-enoic acid and n-heptanoic acid from a renewable fatty acid (i.e., ricinoleic acid). Expression systems for catalytic enzymes (i.e., an alcohol dehydrogenase of Micrococcus luteus, a Baeyer–Villiger monooxygenase of Pseudomonas putida KT2440, an esterase of Pseudomonas fluorescens SIK WI) and biotransformation conditions were investigated. Biotransformation during stationary growth phase of recombinant E. coli in a bioreactor allowed to produce ω-hydroxyundec-9-enoic acid and n-heptanoic acid at a rate of 3.2 mM/h resulting in a final product concentration of ca. 20 mM. The total amount of ω-hydroxyundec-9-enoic acid and n-heptanoic acid produced reached 6.5 g/L (4.0 g/L of ω-hydroxyundec-9-enoic acid and 2.5 g/L of n-heptanoic acid). These results indicate that the high value carboxylic acids ω-hydroxyundec-9-enoic acid and n-heptanoic acid can be produced from a renewable fatty acid via whole-cell biotransformation.     

The characteristics of desaturation of Trichoderma sp. AM076 and formation of 9,12,15-hexadecatrienoic acid (16 : 3ω1) through Δ15 desaturation of 9,12-hexadecadienoic acid

We investigated the characteristics of desaturation in Trichoderma sp. AM076. Although 6,9,12-octadecatrienoic acid (18 : 3ω6) was detected when Trichoderma sp. AM076 was cultivated in the presence of 6,9-octadecadienoic acid (18 : 2ω9), the desaturation products of 6,9,12-octadecatrienoic acid (18 : 3ω6) and 6-octadecenoic acid (18 : 1Δ6) were not detected. These results suggest that the double bonds at the Δ6 position of 18 : 3ω6 and 18 : 1Δ6 disturb their Δ15 and Δ9 desaturation, respectively. This fungus also introduced a double bond at the Δ15 position of 9,12-hexadecadienoic acid (16 : 2ω4), thereby yielding a novel C16 polyunsaturated fatty acid (PUFA) identified as 9,12,15-hexadecatrienoic acid (16 : 3ω1). Further investigations revealed that the mutant having enhanced accumulation of linolenic aid (18 : 3ω3) accumulates 16 : 3ω1 as one of the major PUFAs, together with 9,12-octadecadienoic acid (16 : 2ω4), when grown with palmitoleic acid (16 : 1ω7). These results suggest that, in this strain, the reaction that catalyzes the conversion of linoleic acid to linolenic acid, similar to the conversion of 16 : 2ω4 to 16 : 3ω1, is not ω3 desaturation but Δ15 desaturation.     

Coexpression of Elo-like enzyme and Δ5, Δ4-desaturases derived from Thraustochytrium aureum ATCC 34304 and the production of DHA and DPA in Pichia pastoris

Thraustochytrium aureum ATCC 34304 produces a high level of polyunsaturated fatty acids (PUFAs), which are typically synthesized by strings of reactions catalyzed by desaturase and elongase enzymes. In this study, the genes related to the biosynthesis of PUFAs were investigated and targeted to enable optimization of the production of PUFAs. To the best of our knowledge, this is first study to evaluate the co-expression of genes TaElo, Tad5, and Tad4genes derived from T. aureum. We found that C22 PUFAs such as docosapentaenoic acid (DPA, C22:5n–6) and docosahexaenoic acid (DHA, 22:6n–3) were synthesized from γ-linolenic acid (GLA, C18:3n–6) and stearidonic acid (SDA, C18:4n–3), respectively, as exogenous substrates via a series of reactions catalyzed by an Elo-like enzyme and Δ5, Δ4-desaturase enzymes. In addition, the results of this study revealed that the TaElo gene could synthesize the Δ6-and Δ5-elongation products. Taken together, these results confirmed that the Elo-like enzyme was involved in multiple reactions leading to the production of PUFAs and that the TaElo, Tad5, and Tad4 genes were capable of functioning together to produce DPA and DHA using GLA and SDA.     

Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC–ELSD to reveal accumulation differences of sugar and lipid

Biotechnology Letters - To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes...     

Production of a benzylated flavonoid from 5,7,3′,4′,5′-pentamethoxyflavanone by Penicillium griseoroseum

Penicillium griseoroseum, isolated as an endophytic microorganism from Coffeea arabica seeds, was grown in Czapeck's medium containing 5,7,3′,4′,5′-pentamethoxyflavanone. The fungus incorporated a dimethylated tetraketide, clavatol, a typical fungal secondary metabolite, into the flavanone structure at C-6, resulting in a novel benzylated flavonoid. Clavatol was also found free in the fungus extract. The compounds were isolated by chromatographic procedures and identified by extensive spectroscopic studies, including MS/MS and 1D and 2D NMR. The process probably involves enzymes catalyzing C–C bound formation, which is uncommon in fungi. The possibility of fungi participation in the biosynthesis of plant benzylated flavonoids is discussed.     

Isolation and Characterization of a Δ5-Desaturase from Oblongichytrium sp.

We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Δ5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Δ5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms.     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.