Mead acid production by disruption of Δ12-desaturase gene in Mortierella alpina 1S-4

Mead acid (MA; 20:3ω9) is one of the ω9 series of polyunsaturated fatty acids (PUFAs). MA is used to inhibit the inflammation of joints and is applied to the medicinal or health food field. We aimed to construct MA-producing strains with disruption of the Δ12-desaturase gene (Δ12ds) via an efficient gene-targeting system using the lig4-disrupted strain of Mortierella alpina 1S-4 as the host. The transformants showed a unique fatty acid composition that only comprised ω9-PUFAs and saturated fatty acids, while ω6-and ω3-PUFAs were not detected, and the total composition of ω9-PUFAs, including oleic acid (18:1ω9), 18:2ω9, 20:1ω9, 20:2ω9, and MA, was up to 68.4% of the total fatty acids. The MA production in the Δ12ds-disruptant reached 0.10 g/L (8.5%), which exceeded 0.050 g/L (4.6%) in the conventional Δ12ds-defective mutant JT-180.

Journal of Bioscience and Bioengineering


Simultaneous Imaging and Characterization of Polyunsaturated Fatty Acids, Carotenoids, and Microcrystalline Guanine in Single Aurantiochytrium limacinum Cells with Linear and Nonlinear Raman Microspectroscopy

Thraustochytrids are heterotrophic marine protists known for their high production capacity of various compounds with health benefits, such as polyunsaturated fatty acids and carotenoids. Although much effort has been focused on developing optimal cultivation methods for efficient microbial production, these high-value compounds and their interrelationships are not well understood at the single-cell level. Here we used spontaneous (linear) Raman and multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy to visualize and characterize lipids (e.g., docosahexaenoic acid) and carotenoids (e.g., astaxanthin) accumulated in single living Aurantiochytrium limacinum cells. Spontaneous Raman imaging with the help of multivariate curve resolution alternating least-squares enabled us to make unambiguous assignments of the molecular components we detected and derive their intracellular distributions separately. Near-IR excited CARS imaging yielded the Raman images at least an order of magnitude faster than spontaneous Raman imaging, with suppressed contributions of carotenoids. As the culture time increased from 2 to 5 days, the lipid amount increased by a factor of 7, whereas the carotenoid amount did not change significantly. Furthermore, we observed a highly localized component in A. limacinum cells. This component was found to be mixed crystals of guanine and other purine derivatives. The present study demonstrates the potential of the linear nonlinear Raman hybrid approach that allows for accurate molecular identification and fast imaging in a label-free manner to link information derived from single cells with strategies for mass culture of useful thraustochytrids.


Ethanol Enhances Astaxanthin Production by Aurantiochytrium sp. O5-1-1

Two-percent ethanol increased the astaxanthin productivity of heterotrophic microalgae Aurantiochytrium sp. O5-1-1 to 2.231 mg/L, 45-fold higher than under ethanol-free condition. Ethanol in the medium decreased at the same rate as spontaneous volatilization, suggesting that it was not a transient signaling factor but a continuous stress on the cells. The triply mutated strain OM3-3 produced 5.075 mg/L astaxanthin under 2% ethanol conditions. Furthermore, the astaxanthin accumulation of the mutant OM3-9 was 0.895 mg/g, which was 150-fold higher than that of strain O5-1-1 in ethanol-free condition. These results are beneficial for the commercial exploitation of carotenoids producing Aurantiochytrium spp.


Quantification of leuco-indigo in indigo-dye-fermenting suspension by normal pulse voltammetry

Quantification of leuco-indigo is most important for Aizome, Japanese indigo-dyeing; however, there has been no convenient quantitative method. This study demonstrated that normal pulse voltammetry under quiescent conditions can be used to detect leuco-indigo. As a result of quantification of leuco-indigo in the depth direction in fermenting suspensions, the steady-state concentrations of leuco-indigo showed sigmoidal profiles in the depth direction. The steady state is caused by competitive reactions of microbial reduction of indigo and autoxidation of leuco-indigo by O dissolved from the air interface of the suspension. In addition, we investigated the effects of stirring the suspension and adding some nutrients to the concentration profile. The weakened activity was partially recovered by the addition of ethanol and remarkably recovered by the addition of hipolypepton or glucose. Knowledge is essential for the proper management of indigo-dye-fermenting suspensions.


Isolation and characterization of indigo-reducing bacteria and analysis of microbiota from indigo fermentation suspensions

In natural indigo dyeing, the water-insoluble indigo included in the composted indigo leaves called sukumo is converted to water-soluble leuco-indigo through the reduction activities of microorganisms under alkaline conditions. To understand the relationship between indigo reduction and microorganisms in indigo-fermentation suspensions, we isolated and identified the microorganisms that reduce indigo and analyzed the microbiota in indigo-fermentation suspensions. Indigo-reducing microorganisms, which were not isolated by means of a conventional indigo carmine-reduction assay method, were isolated by using indigo as a direct substrate and further identified and characterized. We succeeded in isolating bacteria closely related to Corynebacterium glutamicum, Chryseomicrobium aureum, Enterococcus sp. for the first time. Anthraquinone was found to be an effective mediator that facilitated the indigo-reduction activity of the isolated strains. On analysis of the microbiota in indigo-fermentation suspensions, the ratio of indigo-reducing bacteria and others was found to be important for maintaining the indigo-reduction activity.

Bioscience, Biotechnology, and Biochemistry


Microbial production of hydroxy fatty acids utilizing crude glycerol

Biocatalysis and Agricultural Biotechnology


Efficient production of biolipids by crude glycerol-assimilating fungi

The aim of this study was to isolate microorganisms utilizing crude glycerol as a carbon source efficiently and to evaluate their lipid productivity. Fusarium oxysporum W1 grew well on medium containing 20% crude glycerol as well as 50% pure glycerol. The dry cell weights and total fatty acids of F. oxysporum W1 reached 24.5 g/L and 12.4 g/L. Penicillium sp. N1 and P. citrinum N3 were found to accumulate free fatty acids to as much as 56.2% and 48.5% of total fatty acids, respectively, on cultivation in the crude glycerol-containing medium. These strains grew well on medium containing crude glycerol only heat-treated at 80 105 °C without autoclave sterilization.


Indigo-mediated semi-microbial biofuel cell using an indigo-dye fermenting suspension

Aizome (Japanese indigo dyeing) is a unique dyeing method using microbial activity under anaerobic alkaline conditions. In indigo-dye fermenting suspensions; microorganisms reduce indigo into leuco-indigo with acetaldehyde as a reductant. In this study; we constructed a semi-microbial biofuel cell using an indigo-dye fermenting suspension. Carbon fiber and Pt mesh were used as the anode and cathode materials, respectively. The open-circuit voltage (OCV) was 0.6 V, and the maximum output power was 32 µW cm-2 (320 mW m-2). In addition, the continuous stability was evaluated under given conditions starting with the highest power density; the power density rapidly decreased in 0.5 h due to the degradation of the anode. Conversely, at the OCV, the anode potential exhibited high stability for two days. However, the OCV decreased by approximately 80 mV after 2 d compared with the initial value, which was attributed to the performance degradation of the gas-diffusion-cathode system caused by the evaporation of the dispersion solution. This is the first study to construct a semi-microbial biofuel cell using an indigo-dye fermenting suspension.


Voltammetric in-situ monitoring of leuco-indigo in indigo-fermenting suspensions

Cyclic voltammetry was successfully applied to in-vivo monitoring of leuco-indigo in indigo-fermenting suspensions under quiescent conditions without deoxygenation; the working and counter electrodes were kept on the surface of each suspension by a polyethylene vinyl alcohol tube holder. The anodic peak current was used as a measure of the leuco-indigo concentration. The voltammetric wave shape suggested partial solubilization of the indigo with some macromolecules in the fermenting suspensions, which lead to an in-situ method without any electrode surface pretreatment. The anodic peak current well reflected the dyeing activity of a suspensions. The results obtained for laboratory-level fermentation systems clarified the number of days required for dye fermentation, the effectiveness of addition of old suspension as an additive for preparing fresh fermenting suspensions, and the importance of addition of a nitrogen-based nutrient as well as a glucose-based one to recover the indigo-reducing activity. The method can also be applied to determine the amounts of indigo in used dye suspensions and extracts of fermented indigo leaves (sukumo) by adding a chemical reduction pretreatment.

Journal of Bioscience and Bioengineering


Characterization of ω3 fatty acid desaturases from oomycetes and their application toward eicosapentaenoic acid production in Mortierella alpina

ω3 polyunsaturated fatty acids are currently obtained mainly from fisheries, thus sustainable alternative sources such as oleaginous microorganisms are required. Here we describe the isolation, characterization, and application of three novel ω3 desaturases with ω3 polyunsaturated fatty acid-producing activity at ordinary temperatures (28 °C). First, we selected Pythium sulcatum and Plectospira myriandra after screening for oomycetes with high eicosapentaenoic acid/arachidonic acid ratios and isolated the genes psulω3 and pmd17, respectively, which encode ω3 desaturases. Subsequent characterization showed that PSULω3 exhibited ω3 desaturase activity on both C18 and C20 ω6 polyunsaturated fatty acids while PMD17 exhibited ω3 desaturase activity exclusively on C20 ω6 polyunsaturated fatty acids. Expression of psulω3 and pmd17 in the arachidonic acid-producer Mortierella alpina resulted in transformants that produced eicosapentaenoic acid/total fatty acid values of 38% and 40%, respectively, at ordinary temperatures. These ω3 desaturases should facilitate the construction of sustainable ω3 polyunsaturated fatty acid sources.


Mechanistic Insights into Indigo Reduction in Indigo Fermentation: A Voltammetric Study

Indigo is one of the oldest natural blue dyes. Microorganisms and their enzymatic activities are deeply involved in the traditional indigo staining procedure. To elucidate the mechanism of the microbial indigo reduction, we directly performed cyclic voltammetry on alkaline fermenting dye suspensions. A pair of characteristic redox peaks of leuco-indigo was observed in a supernatant fluid of the fermenting dye suspension. On the other hand, it was found that the indigo/leuco-indigo redox couple mediated two-way microbially catalyzed oxidation and reduction in a sediment-rich suspension of the fermenting suspension. Acetaldehyde was supposed to be the electron donor and acceptor of the catalytic reactions. In order to verify the bioelectrocatalytic reaction, we isolated indigo-reducing bacterium K2-3 from the fermenting suspension, and the two-way bioelectrocatalysis was successfully restaged in a model system containing K2-3 and methyl viologen (as a soluble mediator instead of indigo) as well as acetaldehyde at pH 10.


Neurite Outgrowth-Promoting Activity of Compounds in PC12 Cells from Sunflower Seeds

In the current super-aging society, the establishment of methods for prevention and treatment of Alzheimer's disease (AD) is an urgent task. One of the causes of AD is thought to be a decrease in the revel of nerve growth factor (NGF) in the brain. Compounds showing NGF-mimicking activity and NGF-enhancing activity have been examined as possible agents for improving symptoms. In the present study, sunflower seed extract was found to have neurite outgrowth-promoting activity, which is an NGF-enhancing activity, in PC12 cells. To investigate neurite outgrowth-promoting compounds from sunflower seed extract, bioassay-guided purification was carried out. The purified active fraction was obtained by liquid-liquid partition followed by some column chromatographies. Proton nuclear magnetic resonance and gas chromatography-mass spectrometry analyses of the purified active fraction indicated that the fraction was a mixture of β-sitosterol, stigmasterol and campesterol, with β-sitosterol being the main component. Neurite outgrowth-promoting activities of β-sitosterol, stigmasterol, campesterol and cholesterol were evaluated in PC12 cells. β-Sitosterol and stigmasterol showed the strongest activity of the four sterol compounds (β-sitosterol stigmasterol > campesterol > cholesterol), and cholesterol did not show any activity. The results indicated that β-sitosterol was the major component responsible for the neurite outgrowth-promoting activity of sunflower seeds. Results of immunostaining also showed that promotion by β-sitosterol of neurite formation induced by NGF was accompanied by neurofilament expression. β-Sitosterol, which showed NGF-enhancing activity, might be a candidate ingredient in food for prevention of AD.


Metabolic engineering of oleaginous fungus Mortierella alpina for high production of oleic and linoleic acids

The aim of this work was to study the molecular breeding of oleaginous filamentous Mortierella alpina for high production of linoleic (LA) or oleic acid (OA). Heterologous expression of the Δ12-desaturase (DS) gene derived from Coprinopsis cinerea in the Δ6DS activity-defective mutant of M. alpina increased the LA production rate as to total fatty acid to 5 times that in the wild strain. By suppressing the endogenous Δ6I gene expression by RNAi in the Δ12DS activity-defective mutant of M. alpina, the OA accumulation rate as to total fatty acid reached 68.0%. The production of LA and OA in these transformants reached 1.44 and 2.76g/L, respectively, on the 5th day. The Δ6I transcriptional levels of the RNAi-treated strains were suppressed to 1/10th that in the parent strain. The amount of Δ6II RNA in the Δ6I RNAi-treated strain increased to 8 times that in the wild strain.


Microbial production of dihomo-γ-linolenic acid by Δ5-desaturase gene-disruptants of Mortierella alpina 1S-4.

We constructed dihomo-γ-linolenic acid (DGLA)-producing strains with disruption of the Δ5-desaturase (Δ5ds) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Δlig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the Δlig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Δlig4 strain was constructed, and then transformed for disruption of Δ5ds. The isolation of nine Δ5ds-disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Δ5ds-disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4.


Essential fatty acids for oleaginous fungus Mortierella alpina

Biocatalysis and Agricultural Biotechnology


Eicosapentaenoic acid (EPA) production by an oleaginous fungus Mortierella alpina expressing heterologous the Δ17 desaturase gene under ordinary temperature

The oleaginous fungus Mortierella alpina is known to accumulate eicosapentaenoic acid (EPA) only when cultivated at a low temperature (below 15°C). Here, we investigated EPA production at a ordinary temperature (28°C) by expressing the Saprolegnia diclina 17 desaturase gene (sdd17m) in M. alpina ST1358, an 3-desaturation activity-defective mutant derived from M. alpina 1S-4. Expression of the exogenous gene was confirmed by EPA accumulation in transformants at both 28°C and 12°C. The EPA content in total lipids produced by transformants was over 20% at 28°C. Bench-scale fermentation with a 5-L jar fermentor showed that EPA content reached 26.4% of total fatty acids, and final EPA production reached 1.8 g/L. This is the first study to report the accumulation of EPA in M. alpina at a ordinary temperature, and provide a platform technology for the industrial production of EPA using M. alpina as a promising source for EPA.

European Journal of Lipid Science and Technology : EJLST


Disruption of lig4 improves gene targeting efficiency in the oleaginous fungus Mortierella alpina 1S-4.

The oil-producing zygomycete Mortierella alpina 1S-4 is known to accumulate beneficial polyunsaturated fatty acids. We identified the lig4 gene that encodes for a DNA ligase 4 homolog, which functions to repair double strand breaks by non-homologous end joining. We disrupted the lig4 gene to improve the gene targeting efficiency in M. alpina. The M. alpina 1S-4 Δlig4 strains showed no defect in vegetative growth, formation of spores, and fatty acid production, but exhibited high sensitivity to methyl methansulfonate, an agent that causes DNA double-strand breaks. Importantly, gene replacement of ura5 marker by CBXB marker occurred in 67% of Δlig4 strains and the gene targeting efficiency was 21-fold greater than that observed in disruption of the lig4 gene in the M. alpina 1S-4 host strain. Further metabolic engineering of the Δlig4 strains is expected to result in strains that produce higher levels of rare and beneficial polyunsaturated fatty acids and contribute to basic research on the zygomycete.


Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4.

We investigated the omega-3 eicosatetraenoic acid (ETA) production by molecular breeding of the oleaginous fungus Mortierella alpina, which can slightly accumulate ETA only when cultivated at a low temperature. The endogenous ω3-desaturase gene or the heterologous Saprolegnia diclina Δ17 (sdd17m) desaturase gene were overexpressed in M. alpina S14, a Δ5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous ω3-desaturase gene showed ETA at 42.1% content in the total lipids that was 84.2-fold and 3.2-fold higher than that of the wild-type strain 1S-4 and host strain S14, respectively, when cultivated at 12°C. No accumulation of ETA was observed at 28°C. In contrast, transformants with the heterologous sdd17m gene showed 24.9% of the content of total lipids at 28°C. These results indicated that these M. alpina S14 transformants are promising strains for the production of ETA, which is hard to obtain from natural sources.

Journal of Bioscience and Bioengineering


Gene targeting in the oil-producing fungus Mortierella alpina 1S-4 and construction of a strain producing a valuable polyunsaturated fatty acid.

To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed ku80 gene-disrupted strains via single-crossover homologous recombination. The ku80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo--linolenic acid (DGLA)-producing strains were constructed by disruption of the 5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the ku80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the ku80 gene, but the construction of DGLA-producing strain by disruption of the 5-desaturase gene was succeeded using the ku80 strain as a host strain. This report describes the first study on the identification and disruption of the ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4.


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

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.

We found that M. chlamydospora CBS 529.75 was the best producer for the microbial production of EA.


Transformation of zygomycete Mortierella alpina using biolistic particle bombardment

Genetic Transformation Systems in Fungi.


Cryptococcus terricola is a promising oleaginous yeast for biodiesel production from starch through consolidated bioprocessing.

Starch is considered a potential feedstock for biofuel production, particularly in light of the large-scale landfilling of food waste and other starchy materials worldwide. Lipid accumulation by oleaginous yeast is a promising method for biodiesel production from starch. However, most oleaginous yeasts are grown on monosaccharides or oligosaccharides because they cannot directly utilize starch. We therefore investigated the starch-assimilation ability of 1,200 yeasts. We found that Cryptococcus terricola could be used for fuel production through consolidated bioprocessing. C. terricola JCM 24523 exhibited the highest lipid content of 61.96% on medium with 5% starch at 10 days. Fatty acid methyl ester analysis showed that this strain produced high proportions of C16:0 and C18 fatty acids when grown on starch, which are ideal for use in biodiesel. Considering the yield and cost, lipids derived from starch using C. terricola would be a promising alternative source for biodiesel production.


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 -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 -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.


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.


Achlorophyllous alga Prototheca zopfii oxidizes n-alkanes with different carbon-chain lengths through a unique subterminal oxidation pathway.

Some Prototheca spp. were previously reported to convert n-hexadecane to 5-hexadecanol and then to 5-hexadecanone through a unique subterminal oxidation pathway. Further analysis of derivatives derived from n-hexadecane indicated that Prototheca zopfii oxidized n-alkanes with C11 to C17 chain lengths at not only the 5th but also the 4th, 3rd and 2nd positions.

Journal of Bioscience and Bioengineering


Characterization of a trifunctional fatty acid desaturase from oleaginous filamentous fungus Mortierella alpina 1S-4 using a yeast expression system.

A ω3-fatty acid desaturase gene (maw3) which is involved in biosynthesis of n-3 polyunsaturated fatty acids (PUFAs) was previously isolated from Mortierella alpina 1S-4. In this report, we investigated the products of MAW3 catalyzing reaction with endogenous and exogenous fatty acids in the yeast transformant. Two unusual fatty acids de novo synthesized in the yeast transformant expressing maw3 gene were identified as n-4 hexadecadienoic acid (16:2(9cis,12cis)) and n-1 hexadecatrienoic acid (16:3(9cis,12cis,15)) by GC-MS and (1)H NMR analyses. In addition to the desaturation activity at the ω3-position for 18- and 20-carbon PUFAs, MAW3 in the yeast transformant inserted a double bond at Δ12-position of endogenous palmitoleic acid (16:1(9cis)) and further at Δ15-position of the resulting 16:2(9cis,12cis) to result in the formation of 16:3(9cis,12cis,15) leading to a bifunctional Δ12/Δ15-desaturase for 16-carbon fatty acids. Moreover, we evaluated the activity of MAW3 in the yeast transformant under different temperatures. The MAW3 did not have desaturation activities in M. alpina 1S-4 at 28°C but it had in the yeast transformant for various fatty acids. The MAW3 was demonstrated to be a trifunctional Δ12/Δ15/ω3-desaturase, exhibiting Δ12-desaturation for 16:1(9cis), Δ15-desaturation for 16- and 18-carbon fatty acids that had a preexisting cis-double bond at Δ12 position, and ω3-desaturation for 20-carbon fatty acids having that at Δ14-position. It is the first report that the fatty acid desaturase (MAW3) is shown to have Δ12- and Δ15-desaturation activities for a 16-carbon fatty acid, in addition to its major function, ω3-desaturation activity.


Subterminal oxidation of n-alkanes in achlorophyllous alga Prototheca sp.

Some Prototheca sp. are known to be involved in n-hexadecane degradation. Two derivatives derived from n-hexadecane in such Prototheca sp. were identified as 5-hexadecanone and 5-hexadecanol. n-Hexadecane was assumed to be converted to 5-hexadecanol and then to 5-hexadecanone through a unique subterminal oxidation pathway in such Prototheca sp.


The CER22 gene required for the synthesis of cuticular wax alkanes in Arabidopsis thaliana is allelic to CER1.

Cuticular waxes coat the primary aerial tissues of land plants and serve as a protective barrier against non-stomatal water loss and various environmental stresses. Alkanes are the most prominent cuticular wax components and are thought to have an important role in controlling permeability of the cuticle. However, alkane biosynthesis in plants is not well understood. Arabidopsis eceriferum1 (cer1) and cer22 mutants show dramatic reductions in alkane, secondary alcohol, and ketone content, and concomitant increases in aldehyde content, suggesting that one or both of these genes encode an alkane-forming enzyme. To determine the biochemical identity of CER22, and to investigate the relationship between CER1 and CER22 in alkane formation, we mapped the cer22 mutation as a first step to positional cloning. Unexpectedly, mapping revealed linkage of cer22 to markers on chromosome 1 in the vicinity of CER1, and not to markers on chromosome 3 as previously reported. Failure of the cer1-1 and cer22 mutants to complement each other, and the presence of an allele specific mutation in the CER1 gene amplified from cer22 genomic DNA demonstrated that CER22 is identical to CER1. The cer22 mutant was therefore renamed cer1-6. Analyses of CER1 transcript levels, and stem cuticular wax load and composition in the cer1-6 (cer22) line indicated that cer1-6 is a weak mutant allele of CER1. This represents an important step forward in our understanding of alkane synthesis in plants, and will direct future research in the field to focus on the role of CER1 in this process.


Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae.

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.


Purification and cDNA cloning of LaIT2, a novel insecticidal toxin from venom of the scorpion Liocheles australasiae.

The novel insecticidal toxin, LaIT2, was isolated from venom of the scorpion Liocheles australasiae. The amino acid sequence of LaIT2 was determined by an Edman degradation analysis and subsequent cDNA cloning. LaIT2 is composed of 59 amino acids with three disulfide bridges, and shares sequence similarity to the scorpion beta-KTx peptides.

Bioscience, Biotechnology, and Biochemistry


Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae.

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

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 omega3-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/10(8) 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 omega3-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.


Alkaline pH enhances farnesol production by Saccharomyces cerevisiae.

External environments affect prenyl alcohol production by squalene synthetase-deficient mutant Saccharomyces cerevisiae ATCC 64031. Cultivation of the yeast in medium with an initial pH ranging from 7.0 to 8.0 increased the amount of secreted farnesol (FOH). In contrast, acidic medium with a pH below 4.0 increased the intracellular FOH and its isomer nerolidol. These effects of alkaline pH were also observed on constant pH cultivation in a jar fermenter. On cultivation for 133 h, the FOH production reached 102.8 mg/l.

Journal of Bioscience and Bioengineering


Transformation of an oleaginous zygomycete Mortierella alpina 1S-4 with the carboxin resistance gene conferred by mutation of the iron-sulfur subunit of succinate dehydrogenase.

The sdhB gene encoding an iron-sulfur (Ip) subunit of succinate dehydrogenase (SDH, EC 1.3.99.1) complex was cloned from Mortierella alpina 1S-4. The deduced amino acid sequence of SdhB from M. alpina 1S-4 showed high similarity to those of SdhB from other organisms. The mutated sdhB (CBXB) gene encodes a modified SdhB with an amino-acid substitution (a highly conserved histidine residue within the third cysteine-rich cluster of SdhB replaced by a leucine residue) and is known to confer carboxin resistance. We succeeded in transforming M. alpina 1S-4 by using the CBXB gene as a selectable marker gene and expressing the heterologous uidA gene encoding beta-glucuronidase of Escherichia coli. Moreover, transformation efficiency was up to 40-50 transformants per 4.0 x 10(8) spores. This carboxin-transformation system, characterized by marginal background growth and mitotic stability in M. alpina 1S-4, is considered to be widely useful for the wild strain, M. alpina 1S-4, and various derivative mutants without laborious preparation of auxotrophic mutants as a host strain.


Identification of a novel fatty acid elongase with a wide substrate specificity from arachidonic acid-producing fungus Mortierella alpina 1S-4.

The isolation and characterization of a gene (MALCE1) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4 are described. MALCE1 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae, resulting in the accumulation of 18-, 19-, and 20-carbon monounsaturated fatty acids and eicosanoic acid. Furthermore, the MALCE1 yeast transformant efficiently elongated exogenous 9-hexadecenoic acid, 9,12-octadecadienoic acid, and 9,12,15-octadecatrienoic acid. The MALCE1 gene-silenced strain obtained from M. alpina 1S-4 exhibited a low content of octadecanoic acid and a high content of hexadecanoic acid, compared with those in the wild strain. The enzyme encoded by MALCE1 was demonstrated to be involved in the conversion of hexadecanoic acid to octadecanoic acid, its main role in M. alpina 1S-4.


Fatty acid desaturation and elongation reactions of Trichoderma sp. 1-OH-2-3

Journal of the American Oil Chemists' Society


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

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.


Identification of mutation sites on Delta12 desaturase genes from Mortierella alpina 1S-4 mutants.

The mutation sites on the Delta12 desaturase gene in Mortierella alpina Delta12 desaturase-defective mutants SR88, TM912, and Mut48 accumulating Mead acid were identified. Each mutation resulted in an amino acid replacement (H116Y and P166L) in the Delta12 desaturase gene from SR88 and Mut48, respectively.


Prenyl alcohol production by expression of exogenous isopentenyl diphosphate isomerase and farnesyl diphosphate synthase genes in Escherichia coli.

Isopentenyl diphosphate isomerase (idi) and farnesyl diphosphate synthase (ispA) genes were overexpressed in Escherichia coli. The resulting transformant showed 6.8-fold higher production of farnesol (389 microg/l). In a similar manner, overexpression of idi and mutated ispA led to high production of geranylgeraniol (128 microg/l).

Bioscience, Biotechnology, and Biochemistry


Identification of mutation sites on omega3 desaturase genes from Mortierella alpina 1S-4 mutants.

The mutation sites on omega3 desaturase genes in two omega3 desaturase-defective mutants derived from arachidonic acid-producing Mortierella alpina 1S-4 were identified. The mutations each resulted in an amino acid replacement (W232Stop or W386Stop) which caused a lack of omega3 desaturase activity in these mutants.


Overexpression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols.

To develop microbial production method for prenyl alcohols (e.g., (E,E)-farnesol (FOH), (E)-nerolidol (NOH), and (E,E,E)-geranylgeraniol (GGOH)), the genes encoding enzymes in the mevalonate and prenyl diphosphate pathways were overexpressed in Saccharomyces cerevisiae, and the resultant transformants were evaluated as to the production of these alcohols. Overexpression of the gene encoding hydroxymethylglutaryl (HMG)-CoA reductase was most effective among the genes tested. A derivative of S. cerevisiae ATCC 200589, which was selected through screening, was found to be the most suitable host for the production. On cultivation of the resultant transformant, in which the HMG-CoA reductase gene was overexpressed, in a 5-liter bench-scale jar fermenter for 7 d, the production of FOH, NOH, and GGOH reached 145.7, 98.8, and 2.46 mg/l, respectively.


Functional analysis of a fatty acid elongase from arachidonic acid-producing Mortierella alpina 1S-4.

We describe the isolation and characterization of a gene (MAELO) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4. Although the homologous MAELO gene had already been isolated from M. alpina ATCC 32221, its function had not yet been identified. The MAELO gene from M. alpina 1S-4 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae. Analysis of the fatty acid composition of the yeast transformant revealed the accumulation of 22-, 24-, and 26-carbon saturated fatty acids. On the other hand, RNA interference of the MAELO gene in M. alpina 1S-4 was carried out. The gene-silenced strain obtained on RNA interference exhibited low contents of 20-, 22-, and 24-carbon saturated fatty acids and a high content of stearic acid (18 carbons), compared with those in the wild strain. The enzyme encoded by the MAELO gene was demonstrated to be involved in the biosynthesis of 20-, 22-, and 24-carbon saturated fatty acids in M. alpina 1S-4.


Various oils and detergents enhance the microbial production of farnesol and related prenyl alcohols.

The object of this research was improvement of prenyl alcohol production with squalene synthase-deficient mutant Saccharomyces cerevisiae ATCC 64031. On screening of many kinds of additives, we found that oils and detergents significantly enhanced the extracellular production of prenyl alcohols. Soybean oil showed the most prominent effect among the additives tested. Its effect was accelerated by a high concentration of glucose in the medium. The combination of these cultivation conditions led to the production of more than 28 mg/l of farnesol in the soluble fraction of the broth. The addition of these compounds to the medium was an effective method for large-scale production of prenyl alcohols with microorganisms.

Journal of Bioscience and Bioengineering


Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms.

Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol.


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.


Cytological and biochemical analysis of COF1, an Arabidopsis mutant of an ABC transporter gene.

In transposon-tagged lines of Arabidopsis we found two new mutants, cof1-1 and cof1-2 (cuticular defect and organ fusion), that show the phenotype of wilting when grown in soil, organ fusion of rosette leaves and infertility. Toluidine blue testing and scanning electron microscopy observation revealed that these mutants had cuticular defects in the stems and adult leaves, but not in cotyledones. Transmission electron microscopy observation revealed thinner cuticle layers in the mutants, and cuticular materials interspersed between the two fused epidermal layers were observed in the mutant rosette leaves. These two mutants had a transposon insertion in the coding regions of WBC11, which was classified as a member of ABC transporter genes in Arabidopsis. WBC11 showed high sequence similarity to CER5 (also called WBC12), which was involved in cuticular lipid export. Gas chromatographic analysis revealed that C29 alkane extracted from the stem surface of cof1 mutants was reduced whereas C29 ketone was accumulated, which was different from the case of cer5 mutants. While cer5 mutants had fairly normal morphology, cof1 mutants had pleiotropic phenotypes so that COF1/WBC11 could have important roles different from those of CER5/WBC12. We also found that C29 alkane was accumulated in the intracellular extract of cof1 mutants, suggesting a function for WBC11 in the direct transport of lipid molecules. Pollen observation showed that mutant pollen grains were irregularly shaped. The function of COF1/WBC11 in lipid transport for the construction of cuticle layers and pollen coats for normal organ formation is discussed.


Identification of a sterol Delta7 reductase gene involved in desmosterol biosynthesis in Mortierella alpina 1S-4.

Molecular cloning of the gene encoding sterol Delta7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated MoDelta7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of MoDelta7SR showed highest homology of 51% with that of sterol Delta7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the MoDelta7SR gene in yeast Saccharomyces cerevisiae revealed that MoDelta7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of Delta7 reductase. In addition, with gene silencing of MoDelta7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that MoDelta7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol Delta7 reductase from a microorganism.


Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2.

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.


Acidolysis and glyceride synthesis reactions using fatty acids with two Pseudomonas lipases having different substrate specificities.

Enzymatic acidolysis and glyceride synthesis using polyunsaturated fatty acids (PUFAs) with lipases from Pseudomonas fluorescens HU380 (HU-lipase), P. fluorescens AK102 (AK-lipase), and Candida rugosa (CR-lipase) were studied. The acidolysis of triolein with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in n-hexane was evaluated with lipases immobilized on Celite 545. HU-lipase showed the highest incorporation rate at a low temperature (10 degrees C) with either EPA or DHA as the acyl donor, and the rate decreased with increasing reaction temperature. At 45 degrees C, the rates for EPA and DHA were 7.1 and 0.5 relative to those at 10 degrees C, respectively. The EPA incorporation rate was even higher at a low temperature (10 degrees C), and the DHA incorporation rate increased with decreasing temperature. Although AK-lipase showed the reverse tendency for incorporation rate, the DHA incorporation rate increased with increasing reaction temperature with both PUFAs. HU-lipase reacted well with PUFAs such as DHA, EPA, arachidonic acid (AA), mead acid (MA), and dihomo-gamma-linolenic acid (DGLA) on acidolysis and glyceride synthesis. The reactivities of AK-lipase toward these PUFAs except for DGLA, i.e., MA, AA, EPA, and DHA, were low for both reactions. The unique substrate specificities of the lipases from the Pseudomonas strains will enable us to use these lipases for the modification of fats and oils containing PUFAs such as fish oil.


Different specificity of two types of Pseudomonas lipases for C20 fatty acids with a Delta5 unsaturated double bond and their application for selective concentration of fatty acids.

Two kinds of lipases, AK-lipase and HU-lipase, produced by two different Pseudomonas fluorescens strains, AK102 and HU380, respectively, were evaluated as to fatty acid hydrolysis specificity using six types of oil containing higher amounts of C20 fatty acids such as arachidonic acid (5,8,11,14-eicosatetraenoic acid, AA, or 20:4omega6), dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid, DGLA, or 20:3omega6), 5,8,11,14,17-eicosapentaenoic acid (EPA or 20:5omega3), mead acid (5,8,11-eicosatrienoic acid, MA, or 20:3omega9), 8,11-eicosadienoic acid (20:2omega9) and 8,11,14,17-eicosatetraenoic acid (20:4omega3). Although HU-lipase did not show any specificity for C20 fatty acids with respect to the presence or absence of a Delta5 unsaturated bond, it exhibited comparatively low reactivity for 4,7,10,13,16,19-docosahexaenoic acid (DHA or 22:6omega3). In contrast, AK-lipase was less reactive for C20 fatty acids with a Delta5 unsaturated bond. However, the specificity of hydrolysis of AK-lipase gradually decreased as the reaction proceeded. Utilizing this fatty acid specificity, we concentrated either EPA or DHA from fish oils containing both EPA and DHA by means of lipase-catalyzed hydrolysis and urea adduction. Hydrolysis and urea adduction of refined cod oil including 12.2% EPA and 6.9% DHA with HU-lipase provided free fatty acids with 43.1% EPA and 7% DHA, respectively. The resulting yield of concentrated total fatty acids comprised 2.6% of the fatty acids from the cod oil. Thus, EPA was particularly concentrated in the fatty acids derived from refined cod oil on partial hydrolysis with HU-lipase followed by urea adduction. On the other hand, hydrolysis of cuttlefish oil with AK-lipase followed by urea adduction increase slightly the EPA composition from 14.2% to 16.8%, and markedly enhanced the composition of DHA from 16.3% to 44.6% in the hydrolyzed fatty acids. The yield of purified total fatty acids by urea concentrate was 9.4% of the fatty acids from the cuttlefish oil. Thus, DHA was particularly concentrated in the fatty acids derived from on partial hydrolysis with AK-lipase followed by urea adduction. We concluded that EPA and DHA concentrates can be easily and inexpensively obtained using HU-lipase and AK-lipase, respectively. Furthermore, it might be possible to separate and concentrate C20 polyunsaturated fatty acids (PUFAs) with or without a Delta5 double bond from PUFAs rich oils including both fatty acids.


Identification and production of n-8 odd-numbered polyunsaturated fatty acids by a delta12 desaturation-defective mutant of Mortierella alpina 1S-4.

A mutant of Mortierella alpina, JT-180, which is defective in its delta12 desaturase activity but exhibits enhanced activities of delta5 and delta6 desaturases, produced a high proportion (up to 80%) of odd-chain FA when grown on 3% n-heptadecane. Following growth of the mutant on n-heptadecane, three unusual odd-chain fatty acyl residues were identified as 6,9-heptadecadienoic acid (17:2), 8,11 -nonadecadienoic acid (19:2), and 5,8,11-nonadecatrienoic acid (19:3) by means of GC-MS, MS-MS, and NMR analyses. The mycelial contents of these FA reached 20.3, 3.6, and 5.8 mg/g dry mycelia, respectively, when it was cultivated in medium comprising 4% (vol/vol) n-heptadecane and 1% (wt/vol) yeast extract, pH 6.0, at 28 degrees C for 7 d. The biosynthetic route (n-8 route) to 19:3 was presumed to mimic the n-9 route to Mead acid (20:3n-9) in mammals: 17:0 --> 9-17:1 --> 6,9-17:2 --> 8,11-19:2 --> 5,8,11-19:3.


Expression of Δ6, Δ5 desaturase and GLELO elongase genes from Mortierella alpina for production of arachidonic acid in soybean [Glycine max (L.) Merrill] seeds


Changes in composition and content of mycolic acids in glutamate-overproducing Corynebacterium glutamicum.

Overproduction of glutamate by Corynebacterium glutamicum is induced by biotin limitation or by the supplementation of specific detergents, sublethal amounts of penicillin, or cerulenin. But, it remains unclear why these different treatments, which have different sites of primary action, produce similar effects. In this study, it was found that the cellular content of mycolic acids--characteristic constituents of Corynebacterineae that are synthesized from fatty acids and form a cell surface layer--decreased under all conditions that induced glutamate overproduction. Furthermore, short mycolic acids increased under conditions of biotin limitation and cerulenin supplementation. These results suggest that different treatments produce the same effect that causes defects in the mycolic acid layer. This is perhaps one of the key factors in overproduction of glutamate by C. glutamicum.

Bioscience, Biotechnology, and Biochemistry


Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production.

The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production.

Journal of Bioscience and Bioengineering


Production of γ-linolenic acid in Lotus japonicus and Vigna angularis by expression of the Δ6-fatty-acid desaturase gene isolated from Mortierella alpina


Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression.

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-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 Delta12-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 Delta12-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.


Functional characterization of Delta9 and omega9 desaturase genes in Mortierella alpina 1S-4 and its derivative mutants.

Cloning and characterization of the Delta9 desaturase (Delta9I) gene of a fungus, Mortierella alpina 1S-4, was previously reported. In this study, two genes encoding Delta9 desaturase homologs were isolated from this fungus. One is a Delta9 desaturase (Delta9II) that exhibits 86% amino acid sequence similarity to Delta9I. Functional analysis involving expression of the encoding gene in Aspergillus oryzae revealed that Delta9II exhibits Delta9 desaturase activity, 18:0 being converted to 18:1Delta9. However, unlike Delta9I, the Delta9II transformant accumulated a low amount of 16:1Delta9. The other homolog is a omega9 desaturase (omega9) that exhibits 56 and 58% amino acid sequence similarity to Delta9I and Delta9II, respectively. On functional analysis with the Aspergillus transformant, it was found that omega9 does not convert 18:0 to 18:1Delta9, but converts 24:0 and 26:0 to 24:1omega9 and 26:1omega9, respectively. On the other hand, Delta9 desaturation-defective mutants characterized by accumulation of 18:0 were derived from M. alpina 1S-4 with a chemical mutagen, and the mutated sites of the Delta9 desaturase genes were identified. The mutation on the Delta9I gene was assumed to cause an amino acid replacement (W136Stop, G265D, and W360Stop) in the mutants (HR222, T4, and ST56), respectively. In these mutants, there was no mutated site on the Delta9II and omega9 genes. Real-time quantitative PCR (RTQ-PCR) analysis revealed that (1) the transcriptional level of the Delta9I gene in HR222 and T4 was much higher than that in the wild strain until the fifth day of the cultivation periods, (2) the Delta9II gene of the mutants was transcribed until the fourth day at the same level as the Delta9I gene of the wild strain, whereas the Delta9II gene of the wild strain was transcribed at a lower level, and (3) the transcriptional level of the omega9 gene in both the mutants and the wild strain was low, i.e., as low as that of the Delta9II gene of the wild strain. In these Delta9 desaturation-defective mutants, Delta9II is likely to play an important role in Delta9 desaturation.


Identification of mutation sites on delta6 desaturase genes from Mortierella alpina 1S-4 mutants.

Three Delta6 desaturase-defective mutants, designated YB214, HR95, and ST66, were newly isolated from Mortierella alpina 1S-4, after treating wild-type spores with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). These three mutants and Mut49, isolated previously, are capable of accumulating 5,11,14-cis-eicosatrienoic acid (20:3Delta5). Two functional Delta6 desaturases (Delta6I and Delta6II) were found to exist in M. alpina 1S-4. The mutation sites on the Delta6I gene in the Delta6 desaturase-defective mutants were identified. The mutations each resulted in an amino acid replacement (W314Stop, T375K, and G390D) in Delta6I from ST66, HR95, and YB214 respectively, and uncorrected transcription of the Delta6I gene in Mut49 was caused by disappearance of the GT-terminal of the second intron, resulting in low Delta6 desaturation activity in these mutants. On the other hand, there was no mutation site on the Delta6II genes of the mutants.


Identification of mutation sites on delta5 desaturase genes from Mortierella alpina 1S-4 mutants.

The mutation sites on delta5 desaturase genes in delta5 desaturase-defective mutants derived from arachidonic acid-producing Mortierella alpina 1S-4 were identified. The mutations resulted in an amino acid replacement (G189E or W301Stop) and uncorrected transcription caused by recognition of an AG-terminal newly created on C207A gene mutation, resulting in low or no delta5 desaturase activity in these mutants.


Molecular evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of an elongase.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain for arachidonic acid (AA) production. To determine its physiological properties and to clarify the biosynthetic pathways for PUFA, heterologous and homologous gene expression systems were established in this fungus. The first trial was performed with an enhanced green fluorescent protein gene to assess the transformation efficiency for heterologous gene expression. As a result, strong fluorescence was observed in the spores of the obtained transformant, suggesting that the foreign gene was inherited by the spores. The next trial was performed with a homologous PUFA elongase (GLELOp) gene, this enzyme having been reported to catalyze the elongation of GLA (18:3n-6) to dihomo-gamma-linolenic acid (20:3n-6), and to be the rate-limiting step of AA production. The FA composition of the transformant was different from that of the host strain: The GLA content was decreased whereas that of AA was increased. These data support the hypothesis that the GLELOp enzyme plays an important role in PUFA synthesis, and may indicate how to control PUFA biosynthesis.


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.


A novel fungal omega3-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 degrees C. Here, we describe the isolation and characterization of a gene (maw3) that encodes a novel omega3-desaturase from M. alpina 1S-4. Based on the conserved sequence information for M. alpina 1S-4 Delta12-desaturase and Saccharomyces kluyveri omega3-desaturase, the omega3-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 Delta12-desaturase, whereas it exhibited 36% identity with Sac. kluyveri omega3-desaturase. The cloned cDNA was confirmed to encode the omega3-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 omega3-desaturase differs from those of the known fungal omega3-desaturases from Sac. kluyveri and Saprolegnia diclina. Plant, cyanobacterial and Sac. kluyveri omega3-desaturases desaturate 18-carbon n-6 PUFAs, Spr. diclina omega3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans omega3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs. The substrate specificity of M. alpina 1S-4 omega3-desaturase is rather similar to that of C. elegans omega3-desaturase, but the M. alpina omega3-desaturase can more effectively convert AA into EPA when expressed in yeast. The M. alpina 1S-4 omega3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.


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.


Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.

Bioscience, Biotechnology, and Biochemistry


Isolation and characterization of delta(6)-desaturase, an ELO-like enzyme and delta(5)-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris.

The liverwort Marchantia polymorpha contains high proportions of arachidonic and eicosapentaenoic acids. In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and alpha -linolenic acids, respectively, by a series of reactions catalyzed by Delta(6)-desaturase, an ELO-like enzyme involved in Delta(6) elongation and Delta(5)-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 Pichia pastoris. Interestingly, Delta(6) desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Delta(6)-desaturases are known to have a strong preference for glycerolipids.


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.


The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis.

We have identified a new Arabidopsis mutant, yore-yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double-stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre-1 was approximately one-sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome-morphology mutants, glabra2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax-requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.

The Plant Journal : for Cell and Molecular Biology


MpFAE3, a -Ketoacyl-CoA Synthase Gene in the Liverwort Marchantia polymorpha L., Is Preferentially Involved in Elongation of Palmitic Acid to Stearic Acid.

Fatty acid chain elongation is a crucial step in the biosynthesis of long chain fatty acids. An essential reaction in the elongation process is condensation of malonyl-CoA with acyl-CoA, which is catalyzed by beta-ketoacyl-CoA synthase (KCS) in plants. We have isolated and characterized the MpFAE3 gene, one of the KCS gene family in the liverwort Marchantia polymorpha. Transgenic M. polymorpha plants overexpressing MpFAE3 accumulate fatty acids 18:0, 20:0, and 22:0. In these plants, the amount of 16:0 is reduced to 50% of wild type. In a heterologous assay, transgenic methylotrophic yeast expressing the MpFAE3 gene accumulates fatty acid 18:0 and generates several longer fatty acids which are not detectable in the control, accompanied by a decrease of 16:0. These observations indicate that the MpFAE3 protein is preferentially involved in the elongation of 16:0 to 18:0 and also in the subsequent steps of 18:0 to 20:0 and 20:0 to 22:0 in M. polymorpha.


Gene Cloning and Functional Analysis of a Second 6-Fatty Acid Desaturase from an Arachidonic Acid-producing Mortierella Fungus

We demonstrated that Mortierella alpina 1S-4 has two delta 6-desaturases, which are involved in the desaturation of linoleic acid to gamma-linolenic acid. For one of the two delta 6-desaturases, designated as delta 6I, 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 delta 6-desaturases, designated as delta 6II, in M. alpina 1S-4. The predicted amino acid sequences of the Mortierella delta 6-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 delta 9-desaturase, which has the corresponding domain at the C-terminus. The full-length delta 6II cDNA was expressed in A. oryzae, resulting in the accumulation of gamma-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 delta 6I RNA was 2.4-, 9-, and 17-fold higher than that of delta 6II 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 delta 6-desaturase genes.


Functional analysis of a beta-ketoacyl-CoA synthase gene, MpFAE2, by gene silencing in the liverwort Marchantia polymorpha L.

We have isolated a beta-ketoacyl CoA synthase (KCS) gene, MpFAE2, from a liverwort, Marchantia polymorpha, and identified its substrate specificity using the technique of dsRNA-mediated gene silencing and overexpression. KCS catalyzes an essential reaction in the fatty acid elongation process, i.e., condensation of malonyl-CoA with acyl-CoA. By introducing a construct with a hairpin structure containing a partial MpFAE2 gene, the level of the MpFAE2 gene expression was suppressed constitutively. The transgenic plants showed a specific accumulation of fatty acid 18:0. In contrast, in transgenic M. polymorpha plants overexpressing the MpFAE2 gene, fatty acid 22:0 is accumulated. These results indicate that the MpFAE2 gene product catalyzes the elongation steps of 18:0 to 20:0 and possibly also of 20:0 to 22:0.


Role of Acinetobacter calcoaceticus 3,4-dihydrocoumarin hydrolase in oxidative stress defence against peroxoacids.

The physiological role of a bifunctional enzyme, 3,4-dihydrocoumarin hydrolase (DCH), which is capable of both hydrolysis of ester bonds and organic acid-assisted bromination of organic compounds, was investigated. Purified DCH from Acinetobacter calcoaceticus F46 catalysed dose- and time-dependent degradation of peracetic acid. The gene (dch) was cloned from the chromosomal DNA of the bacterium. The dch ORF was 831 bp long, corresponding to a protein of 272 amino acid residues, and the deduced amino acid sequence showed high similarity to those of bacterial serine esterases and perhydrolases. The dch gene was disrupted by homologous recombination on the A. calcoaceticus genome. The dch disruptant strain was more sensitive to growth inhibition by peracetic acid than the parent strain. On the other hand, the recombinant Escherichia coli cells expressing dch were more resistant to peracetic acid. A putative catalase gene was found immediately downstream of dch, and Northern blot hybridization analysis revealed that they are transcribed as part of a polycistronic mRNA. These results suggested that in vivo DCH detoxifies peroxoacids in conjunction with the catalase, i.e. peroxoacids are first hydrolysed to the corresponding acids and hydrogen peroxide by DCH, and then the resulting hydrogen peroxide is degraded by the catalase.


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).


Isolation and characterization of a Δ9-desaturation-defective mutant of an arachidonic acid-producing fungus, Mortierella alpina 1S-4

Journal of the American Oil Chemists' Society


Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism.

Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132. The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily. The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase. A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation. Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains approximately 4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K(m) and V(max) values of 1.0 mm and 2.5 micromol/min/mg of protein, respectively, for barbituric acid. The enzyme specifically acted on barbituric acid, and dihydro-l-orotate, alloxan, and cyanuric acid competitively inhibited its activity. The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli. The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type.


Characterization of the Second Form of NADH-Cytochrome b_5 Reductase Gene from Arachidonic Acid-Producing Fungus Mortierella alpina 1S-4

The second type of cytochrome b_5 reductase (Cb5R-II) gene was characterized in the arachidonic acidproducing 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.

Journal of Bioscience and Bioengineering


Production of 8,11-cis-eicosadienoic acid by a Δ5 and Δ12 desaturase-defective mutant derived from the arachidonic acid-producing fungus Mortierella alpina 1S-4


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.


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.


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 ofthe 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 Sal1-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 adenineguanine 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 Eseherichia 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.


Production of 8,11,14,17-cis-eicosatetraenoic acid (20:4ω3) by a Δ5 and Δ12 desaturase- defective mutant of an arachidonic acid-producing fungus Mortierella alpina 1S-4

Journal of the American Oil Chemists' Society


Production of 8,11,14,17-cis-eicosatetraenoic acid by 5 desaturase-defective mutants of an arachidonic acid-pruducing fungus, Mortierella alpina.


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


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.