An isoflavone catabolism gene cluster underlying interkingdom interactions in the soybean rhizosphere.

Plant roots secrete various metabolites, including plant specialized metabolites, into the rhizosphere, and shape the rhizosphere microbiome, which is crucial for the plant health and growth. Isoflavones are major plant specialized metabolites found in legume plants, and are involved in interactions with soil microorganisms as initiation signals in rhizobial symbiosis and as modulators of the legume root microbiota. However, it remains largely unknown the molecular basis underlying the isoflavone-mediated interkingdom interactions in the legume rhizosphere. Here, we isolated Variovorax sp. strain V35, a member of the Comamonadaceae that harbors isoflavone-degrading activity, from soybean roots and discovered a gene cluster responsible for isoflavone degradation named ifc. The characterization of ifc mutants and heterologously expressed Ifc enzymes revealed that isoflavones undergo oxidative catabolism, which is different from the reductive metabolic pathways observed in gut microbiota. We further demonstrated that the ifc genes are frequently found in bacterial strains isolated from legume plants, including mutualistic rhizobia, and contribute to the detoxification of the antibacterial activity of isoflavones. Taken together, our findings reveal an isoflavone catabolism gene cluster in the soybean root microbiota, providing molecular insights into isoflavone-mediated legume-microbiota interactions.

Production of docosahexaenoic acid by a novel isolated Aurantiochytrium sp. 6-2 using fermented defatted soybean as a nitrogen source for sustainable fish feed development.

We focused on the production of docosahexaenoic acid (DHA)-containing microbial lipids by Aurantiochytrium sp. using of defatted soybean (DS) as a nitrogen source. Defatted soybean is a plant biomass that could provide a sustainable supply at a low cost. Results showed that Aurantiochytrium sp. could not directly assimilate the DS as a nitrogen source but could grow well in a medium containing DS fermented with rice malt. When cultivated in a fermented DS (FDS) medium, Aurantiochytrium sp. showed vigorous growth with the addition of sufficient sulfate and chloride ions as inorganic nutrients without seawater salt. A novel isolated Aurantiochytrium sp. 6-2 showed 15.8 ± 3.4 g/L DHA productivity (in 54.8 ± 12.1 g/L total fatty acid production) in 1 L of the FDS medium. Therefore, DHA produced by Aurantiochytrium sp. using FDS enables a stable and sustainable DHA supply and could be an alternative source of natural DHA derived from fish oil.

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.

Characterization of regioselective glycosyltransferase of Rhizobium pusense JCM 16209T useful for resveratrol 4'-O-α-d-glucoside production.

Enzymatic glycosylation is an industrially useful technique for improving the properties of compounds with hydroxy groups, and the biological activities of the resulting glycosides differ depending on the glycosylation position. Therefore, regioselective glycosyltransferases are required for precise synthesis of glycosides. We found that Rhizobium pusense JCM 16209T could catalyze the regioselective glycosylation of resveratrol. To identify the regioselective glycosyltransferase, two α-glucosidases of R. pusense JCM 16209T (RpG I and RpG II) were cloned and expressed in Escherichia coli. The molecular mass of purified recombinant RpG I and II was estimated to be 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RpG I showed strong glycosylation activity toward resveratrol with 4'-selectivity of 98.3%. The enzyme activity was maximized at pH 8.0 and 50 °C, and enhanced in the presence of Cs+ and Li+ ions. The maximum molar yield of resveratrol 4'-O-α-glucoside from resveratrol reached 41.6% at 30 min, and the concentration of the product was 2.08 mmol L-1. Glycosylation activity was observed toward resveratrol as well as toward caffeic acid, ferulic acid, 6-gingerol, flavonoid, and isoflavonoid compounds with high regioselectivity, indicating that RpG I could glycosylate a wide range of substrates. To the best of our knowledge, there are few reports on microbial glycosyltransferases that are useful for regioselective glycosylation. This research could be the first step toward developing technologies for the precise synthesis of glycosides.

Generation of Fusarium oxysporum-suppressive soil with non-soil carriers using a multiple-parallel-mineralization technique.

Disease-suppressive soils exist worldwide. However, the disease-suppression mechanism is unknown, and it's unclear how to produce such soils. The microbiota that develop in a multiple-parallel-mineralization system (MPM) can increase nutrient production efficiency and decrease root disease in hydroponic systems. Artificial media inoculated with MPM microorganisms can degrade organic matter to produce inorganic nutrients similarly to natural soil, but it's unknown whether they can also suppress pathogen growth. Here, we produced an artificial medium that inhibited root disease similarly to disease-suppressive soils. Microbial MPM culture solution was inoculated into non-soil carriers (rockwool, rice husk charcoal, and vermiculite) to test whether it could suppress growth of Fusarium oxysporum f. sp. lactucae J. C. Hubb. & Gerik. We inoculated F. oxysporum f. sp. conglutinans (Wollenweber) Snyder et Hansen strain Cong:11 and F. oxysporum f. sp. lactucae J. C. Hubb. & Gerik into artificial media sown each with Arabidopsis thaliana (L.) Heynh. and Lactuca sativa L. var. capitata supplemented with MPM culture microbes. The MPM microorganisms suppressed F. oxysporum f. sp. lactucae J. C. Hubb. & Gerik growth and prevented plant disease. Thus, MPM-inoculated non-soil carriers that can generate inorganic nutrients from organic matter may also suppress disease in the absence of natural soil. Our study shows novel creation of a disease-suppressive effect in non-soil media using the microbial community from MPM culture solution.

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 the ω3-docosapentaenoic acid-producing microorganism Aurantiochytrium sp. T7.

ω3-Docosapentaenoic acid (ω3-DPA), an ω3-polyunsaturated fatty acid (ω3-PUFA), is expected to have beneficial physiological functions to humans; however, because of its rarity in nature, it has not been fully analyzed. We isolated an ω3-DPA producing microorganism strain T7 from brackish areas in Japan. Although most oleaginous microorganisms rarely accumulate ω3-DPA (<5% of total lipid), strain T7 accumulated ω3-DPA with more than 20% of total fatty acids. The strain T7 was identified as a related species of Aurantiochytrium. In Aurantiochytrium sp. T7, ω3-DPA production reached 164 mg/L culture broth, and the ω3-DPA content reached 23.5% of the total fatty acids when cultivated in a medium containing 2% glucose as the carbon source and 1% yeast extract as the nitrogen source, with a salinity equivalent to 50% of that of seawater and a pH in the acidic range (pH < 5.5). Aurantiochytrium sp. T7 is a promising producer of high-purity ω3-DPA containing-lipid for the functional analysis of ω3-DPA whose physiological function has hardly been elucidated, and a useful strain for investigating the novel metabolic pathway of fatty acids.

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.

16S rRNA Gene Amplicon Sequencing of Microbiota in Polybutylene Succinate Adipate-Packed Denitrification Reactors Used for Water Treatment of Land-Based Recirculating Aquaculture Systems.

Information on the microbiota in polybutylene succinate adipate (PBSA)-packed denitrification reactors is limited. Here, we provide 439,817 high-quality reads of the 16S rRNA gene sequences of microbiota in PBSA-packed denitrification reactors used for land-based recirculating aquaculture. The predominant microorganisms belonged to the following families: Nocardiaceae, Chitinophagaceae, Xanthobacteraceae, Burkholderiaceae, Rhodocyclaceae, Pseudomonadaceae, Rhodanobacteraceae, and Xanthomonadaceae.

Cloning of a novel gene involved in alkane biosynthesis from Klebsiella sp.

Aliphatic medium-chain alkanes, a major component of gasoline, diesel, and jet fuels, are drop-in compatible fuels. Microorganisms with the capacity to produce medium-chain alkanes are promising for the bio-production of drop-in fuel. We found that Klebsiella sp. NBRC100048 has the ability to produce medium-chain alkanes from medium-chain aldehydes. We cloned a gene involved in conversion of aldehydes to alkanes by using a genomic fosmid library derived from Klebsiella sp. NBRC100048. The gene termed orf2991 encodes 506 amino acids and shows 62% sequence homology to the aldehyde dehydrogenase of Escherichia coli, aldB. The finding of orf2991 as a novel alkane-synthesizing enzyme gene similar to E. coli aldehyde dehydrogenase family, which is generally known to catalyze a reaction oxidizing aldehydes to fatty acids, indicated a novel function of aldehyde dehydrogenase. This finding is not only significant academically but allows developing the novel manufacturing methods of alkanes fermentation.

Cobalt-dependent inhibition of nitrite oxidation in Nitrobacter winogradskyi.

Nitrobacter winogradskyi is an abundant, intensively studied autotrophic nitrite-oxidizing bacterium, which is frequently used as a model strain in the two-step nitrification of ammonia (NH3) to nitrate (NO3-) via nitrite (NO2-), either in activated sludge, agricultural field studies or more recently in artificial microbial consortia for organic hydroponics. We observed a hitherto unknown cobalt ion-dependent inhibition of cell growth and NO2- oxidation activity of N. winogradskyi in a mineral medium, which strongly depended on accompanying Ca2+ and Mg2+ concentrations. This inhibition was bacteriostatic, but susceptible to natural chelators. l-Histidine effectively restored cell growth and NO2- oxidation activity of N. winogradskyi in mineral media containing Co2+ with >90% recovery. Our results suggest that Co2+ competed with alkaline earth metals during uptake and that its toxicity was significantly reduced by complexation.

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

Cyclooxygenases are responsible for the production of prostaglandin H2 (PGH2) from arachidonic acid. PGH2 can be converted into some bioactive prostaglandins, including prostaglandin F2α (PGF2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.

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.

Modulation of fatty acid composition and growth in Sporosarcina species in response to temperatures and exogenous branched-chain amino acids.

Psychrotolerant endospore-forming Sporosarcina species have been predominantly isolated from minced fish meat (surimi), which is stored under refrigeration after heat treatment. To develop a better method for preserving surimi-based food products, we studied the growth and fatty acid compositions of the isolated strain S92h as well as Sporosarcina koreensis and Sporosarcina aquimarina at cold and moderate temperatures. The growth rates of strain S92h and S. koreensis were the fastest and slowest at cold temperatures, respectively, although these strains grew at a similar rate at moderate temperatures. In all three strains, the proportions of anteiso-C15:0 and unsaturated fatty acids (UFAs) were significantly higher at cold temperatures than at moderate temperatures. Furthermore, supplementation with valine, leucine, and isoleucine resulted in proportional increases in iso-C16:0, iso-C15:0, and anteiso-C15:0, respectively, among the fatty acid compositions of these strains. The proportions of the UFAs were also altered by the supplementation. At cold temperatures, the growth rates of strain S92h and S. koreensis, but not of S. aquimarina, were affected by supplementation with leucine. Supplementation with isoleucine enhanced the growth of S. koreensis at cold temperatures but not that of the other strains. Valine did not affect the growth of any strain. These results indicate that anteiso-C15:0 and UFAs both play important roles in the cold tolerance of the genus Sporosarcina and that these bacteria modulate their fatty acid compositions in response to the growth environment.

Production of ricinoleic acid-containing monoestolide triacylglycerides in an oleaginous diatom, Chaetoceros gracilis.

Ricinoleic acid (RA), a hydroxyl fatty acid, is suitable for medical and industrial uses and is produced in high-oil-accumulating organisms such as castor bean and the ergot fungus Claviceps. We report here the efficient production of RA in a transgenic diatom Chaetoceros gracilis expressing the fatty acid hydroxylase gene (CpFAH) from Claviceps purpurea. In transgenic C. gracilis, RA content increased at low temperatures, reaching 2.2 pg/cell when cultured for 7 d at 15 °C, without affecting cell growth, and was enhanced (3.3 pg/cell) by the co-expression of a palmitic acid-specific elongase gene. Most of the accumulated RA was linked with monoestolide triacylglycerol (ME TAG), in which one RA molecule was esterified to the α position of the glycerol backbone and was further esterified at its hydroxy group with a fatty acid or second RA moiety, or 1-OH TAG, in which RA was esterified to the glycerol backbone. Overall, 80% of RA was accumulated as ME TAGs. Furthermore, exogenous RA-methyl ester suppressed the growth of wild-type diatoms in a dose-dependent manner and was rapidly converted to ME TAG. These results suggest that C. gracilis masks the hydroxyl group and accumulates RA as the less-toxic ME TAG.

Analysis of microbial community and nitrogen transition with enriched nitrifying soil microbes for organic hydroponics.

Nitrifying microbial consortia were enriched from bark compost in a water system by regulating the amounts of organic nitrogen compounds and by controlling the aeration conditions with addition of CaCO3 for maintaining suitable pH. Repeated enrichment showed reproducible mineralization of organic nitrogen via the conversion of ammonium ions ( ) and nitrite ions ( ) into nitrate ions ( ). The change in microbial composition during the enrichment was investigated by PCR-DGGE analysis with a focus on prokaryote, ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, and eukaryote cell types. The microbial transition had a simple profile and showed clear relation to nitrogen ions transition. Nitrosomonas and Nitrobacter were mainly detected during and oxidation, respectively. These results revealing representative microorganisms acting in each ammonification and nitrification stages will be valuable for the development of artificial simple microbial consortia for organic hydroponics that consisted of identified heterotrophs and autotrophic nitrifying bacteria.

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.