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.

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.

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.

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 O2 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.

Recent trends in the field of lipid engineering.

Lipid engineering related to biological functions has made remarkable progress in the fields of microbial production of functional lipids, metabolic engineering of microorganisms, elucidation of physiological functions of rare lipids, lipid-related enzyme engineering, and lipid analysis techniques. Various rare lipids are produced by utilizing microorganisms and their enzymes. It is also becoming clear that the rare lipids produced by intestinal bacteria contribute significantly to human health. Technological advances related to identification of lipid structures and quantification of lipids have led to such discoveries in the field of lipid engineering. This article reviews the latest findings that are attracting attention in the field of lipid engineering related to biological functions.

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, and 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.

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 3 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.

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.

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 ß-sitosterol, stigmasterol and campesterol, with ß-sitosterol being the main component. Neurite outgrowth-promoting activities of ß-sitosterol, stigmasterol, campesterol and cholesterol were evaluated in PC12 cells. ß-Sitosterol and stigmasterol showed the strongest activity of the four sterol compounds (ß-sitosterol ≈ stigmasterol > campesterol > cholesterol), and cholesterol did not show any activity. The results indicated that ß-sitosterol was the major component responsible for the neurite outgrowth-promoting activity of sunflower seeds. Results of immunostaining also showed that promotion by ß-sitosterol of neurite formation induced by NGF was accompanied by neurofilament expression. ß-Sitosterol, which showed NGF-enhancing activity, might be a candidate ingredient in food for prevention of AD.

Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4: A review.

The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%-70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4.

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.

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.

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 Δku80 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-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the Δku80 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 Δ5-desaturase gene was succeeded using the Δku80 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.

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 ß-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 ß-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.

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.

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.