Characterization of potassium-solubilizing fungi, Mortierella spp., isolated from a poplar plantation rhizosphere soil.

Potassium-solubilizing microorganisms are capable of secreting acidic chemicals that dissolve and release potassium from soil minerals, thus facilitating potassium uptake by plants. In this study, three potassium-dissolving filamentous fungi were isolated from the rhizosphere soil of a poplar plantation in Jiangsu Province, China. Phylogenetic analyses based on ITS, 18 S, and 28 S showed that these three isolates were most similar to Mortierella. These strains also possessed spherical or ellipsoidal spores, produced sporangia at the hyphal tip, and formed petal-like colonies on PDA media resembling those of Mortierella species. These findings, along with further phenotypic observations, suggest that these isolates were Mortierella species. In addition, the potassium-dissolution experiment showed that strain 2K4 had a relatively high potassium-solubilizing capacity among these isolated fungi. By investigating the influences of different nutrient conditions (carbon source, nitrogen source, and inorganic salt) and initial pH values on the potassium-dissolving ability, the optimal potassium-solubilization conditions of the isolate were determined. When potassium feldspar powder was used as an insoluble potassium source, isolate 2K4 exhibited a significantly better polysaccharide aggregation ability on the formed mycelium-potassium feldspar complex. The composition and content of organic acids secreted by strain 2K4 were further detected, and the potassium-dissolution mechanism of the Mortierella species and its growth promotion effect were discussed, using maize as an example.

Bacterial endosymbionts protect beneficial soil fungus from nematode attack.

Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.

Insecticidal Cyclodepsitetrapeptides from Mortierella alpina.

Early diverging fungi, such as Mortierella alpina, are an emerging source of bioactive peptides. By screening 22 fungal isolates together with precursor-directed biosynthesis, a family of threonine-linked cyclotetradepsipeptides, the cycloacetamides A-F (1-6), was identified. The structure elucidation was conducted using NMR and HR-ESI-MS/MS analyses, and the absolute configuration was determined by Marfey's analysis and total synthesis. Cycloacetamides are not cytotoxic to human cells, while being highly selectively insecticidal against fruit fly larvae.

Potential of Mortierellaceae for polyunsaturated fatty acids production: mini review.

The health benefits of polyunsaturated fatty acids (PUFAs) have encouraged the search for rich sources of these compounds. However, the supply chain of PUFAs from animals and plants presents environmental concerns, such as water pollution, deforestation, animal exploitation and interference in the trophic chain. In this way, a viable alternative has been found in microbial sources, mainly in single cell oil (SCO) production by yeast and filamentous fungi. Mortierellaceae is a filamentous fungal family world-renowned for PUFA-producing strains. For example, Mortierella alpina can be highlighted due to be industrially applied to produce arachidonic acid (20:4 n6), an important component of infant supplement formulas. Thus, the state of the art of strategies to increase PUFAs production by Mortierellaceae strains is presented in this review. Firstly, we have discussed main phylogenetic and biochemical characteristics of these strains for lipid production. Next, strategies based on physiological manipulation, using different carbon and nitrogen sources, temperature, pH and cultivation methods, which can increase PUFA production by optimizing process parameters are presented. Furthermore, it is possible to use metabolic engineering tools, controlling the supply of NADPH and co-factors, and directing the activity of desaturases and elongase to the target PUFA. Thus, this review aims to discuss the functionality and applicability of each of these strategies, in order to support future research for PUFA production by Mortierellaceae species.

The Beneficial Fungus Mortierella hyalina Modulates Amino Acid Homeostasis in Arabidopsis under Nitrogen Starvation.

Non-mycorrhizal but beneficial fungi often mitigate (a)biotic stress-related traits in host plants. The underlying molecular mechanisms are mostly still unknown, as in the interaction between the endophytic growth-promoting soil fungus Mortierella hyalina and Arabidopsis thaliana. Here, abiotic stress in the form of nitrogen (N) deficiency was used to investigate the effects of the fungus on colonized plants. In particular, the hypothesis was investigated that fungal infection could influence N deficiency via an interaction with the high-affinity nitrate transporter NRT2.4, which is induced by N deficiency. For this purpose, Arabidopsis wild-type nrt2.4 knock-out and NRT2.4 reporter lines were grown on media with different nitrate concentrations with or without M. hyalina colonization. We used chemical analysis methods to determine the amino acids and phytohormones. Experimental evidence suggests that the fungus does not modulate NRT2.4 expression under N starvation. Instead, M. hyalina alleviates N starvation in other ways: The fungus supplies nitrogen (15N) to the N-starved plant. The presence of the fungus restores the plants' amino acid homeostasis, which was out of balance due to N deficiency, and causes a strong accumulation of branched-chain amino acids. We conclude that the plant does not need to invest in defense and resources for growth are maintained, which in turn benefits the fungus, suggesting that this interaction should be considered a mutualistic symbiosis.

Functional characterization of two diacylglycerol acyltransferase 1 genes in Mortierella alpina.

Diacylglycerol acyltransferase (DGAT) is a crucial enzyme in the triacylglycerol (TAG) biosynthesis pathway. The oleaginous fungus Mortierella alpina can accumulate large amounts of arachidonic acid (ARA, C20:4) in the form of TAG. Therefore, it is important to study the functional characteristics of its DGAT. Two putative genes MaDGAT1A/1B encoding DGAT1 were identified in M. alpina ATCC 32222 genome by sequence alignment. Sequence alignment with identified DGAT1 homologs showed that MaDGAT1A/1B contain seven conserved motifs that are characteristic of the DGAT1 subfamily. Conserved domain analysis showed that both MaDGAT1A and MaDGAT1B belong to the Membrane-bound O-acyltransferases superfamily. The transforming with MaDGAT1A/1B genes could increase the accumulation of TAG in Saccharomyces cerevisiae to 4·47 and 7·48% of dry cell weight, which was 7·3-fold and 12·3-fold of the control group, respectively, but has no effect on the proportion of fatty acids in TAG. This study showed that MaDGAT1A/1B could effectively promote the accumulation of TAG and therefore may be used in metabolic engineering aimed to increase TAG production of oleaginous fungi.

The role of phenylalanine hydroxylase in lipogenesis in the oleaginous fungus Mortierella alpina.

Phenylalanine hydroxylase (PAH) catalyses the irreversible hydroxylation of phenylalanine to tyrosine, which is the rate-limiting reaction in phenylalanine metabolism in animals. A variety of polyunsaturated fatty acids can be synthesized by the lipid-producing fungus Mortierella alpina, which has a wide range of industrial applications in the production of arachidonic acid. In this study, RNA interference (RNAi) with the gene PAH was used to explore the role of phenylalanine hydroxylation in lipid biosynthesis in M. alpina. Our results indicated that PAH knockdown decreased the PAH transcript level by approximately 55% and attenuated cellular fatty acid biosynthesis. Furthermore, the level of NADPH, which is a critical reducing agent and the limiting factor in lipogenesis, was decreased in response to PAH RNAi, in addition to the downregulated transcription of other genes involved in NADPH production. Our study indicates that PAH is part of an overall enzymatic and regulatory mechanism supplying NADPH required for lipogenesis in M. alpina.

Role of beta-isopropylmalate dehydrogenase in lipid biosynthesis of the oleaginous fungus Mortierella alpina.

Branched-chain amino acids (BCAAs) play an important role in lipid metabolism by serving as signal molecules as well as a potential acetyl-CoA source. Our previous study found that in the oleaginous fungus Mucor circinelloides, beta-isopropylmalate dehydrogenase (IPMDH), an important enzyme participating in the key BCAA leucine biosynthesis, was differentially expressed during lipid accumulation phase and has a positive role on lipogenesis. To further analyze its effects on lipogenesis in another oleaginous fungus Mortierella alpina, the IPMDH-encoding gene MaLeuB was homologously expressed. It was found that the total fatty acid content in the recombinant strain was increased by 20.2% compared with the control strain, which correlated with a 4-fold increase in the MaLeuB transcriptional level. Intracellular metabolites analysis revealed significant changes in amino acid biosynthesis and metabolism, tricarboxylic acid cycle and butanoate metabolism; specifically, leucine and isoleucine levels were upregulated by 6.4-fold and 2.2-fold, respectively. Our genetic engineering approach and metabolomics study demonstrated that MaLeuB is involved in fatty acid metabolism in M. alpina by affecting BCAAs metabolism, and this newly discovered role of IPMDH provides a potential bypass route to increase lipogenesis in oleaginous fungi.

Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina.

Fungi are traditionally considered a reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early-diverging fungi such as Mortierella Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC 32222. The molecular structures of malpicyclins were elucidated by high-resolution tandem mass spectrometry (HR-MS/MS), Marfey's method, and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative quantitative real-time PCR (qRT-PCR) expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPSs), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicated a bacterial origin and a horizontal gene transfer into the fungal genome. We report on the as-yet-unexplored nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as a novel and underrated resource of natural products.IMPORTANCE Fungal natural compounds are industrially produced, with application in antibiotic treatment, cancer medications, and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but reidentification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early-diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, designated malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much more closely related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella Both enzymes were biochemically characterized and are involved in as-yet-unknown biosynthetic pathways of natural products in basal fungi. Hence, this report establishes early-diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

DNA Metabarcoding to Assess the Diversity of Airborne Fungi Present over Keller Peninsula, King George Island, Antarctica.

We assessed fungal diversity present in air samples obtained from King George Island, Antarctica, using DNA metabarcoding through high-throughput sequencing. We detected 186 fungal amplicon sequence variants (ASVs) dominated by the phyla Ascomycota, Basidiomycota, Mortierellomycota, Mucoromycota, and Chytridiomycota. Fungi sp. 1, Agaricomycetes sp. 1, Mortierella parvispora, Mortierella sp. 2, Penicillium sp., Pseudogymnoascus roseus, Microdochium lycopodinum, Mortierella gamsii, Arrhenia sp., Cladosporium sp., Mortierella fimbricystis, Moniliella pollinis, Omphalina sp., Mortierella antarctica, and Pseudogymnoascus appendiculatus were the most dominant ASVs. In addition, several ASVs could only be identified at higher taxonomic levels and may represent previously unknown fungi and/or new records for Antarctica. The fungi detected in the air displayed high indices of diversity, richness, and dominance. The airborne fungal diversity included saprophytic, mutualistic, and plant and animal opportunistic pathogenic taxa. The diversity of taxa detected reinforces the hypothesis that the Antarctic airspora includes fungal propagules of both intra- and inter-continental origin. If regional Antarctic environmental conditions ameliorate further in concert with climate warming, these fungi might be able to reactivate and colonize different Antarctic ecosystems, with as yet unknown consequences for ecosystem function in Antarctica. Further aeromycological studies are necessary to understand how and from where these fungi arrive and move within Antarctica and if environmental changes will encourage the development of non-native fungal species in Antarctica.

Production of arachidonic acid by Mortierella alpina using wastes from potato chips industry.

AIMS: This research aimed to determine the potential use of wastes from the potato chips industry as a carbon source to develop an economical culture medium for the production of biomass, lipids and arachidonic acid (ARA) by Mortierella alpina. METHODS AND RESULTS: A synthetic culture medium was optimized using a Plackett-Burman and central composite rotatable design, and used as a base to evaluate and characterize the potential use of wastes from the potato chips industry as carbon sources for the production of biomass, lipids and ARA by M. alpina. The waste was selected among other solid and liquid hydrolysed residues/by-products, and local low-cost alternatives for nitrogen sources were also evaluated. After 6 days of fermentation, the biomass concentration reached 20 g l-1 with 40% of total lipids, and a 35% ARA content in the lipids fraction. Savings in production were calculated using a sensitivity analysis for the alternative culture medium in different scenarios. CONCLUSIONS: This study showed a 7% savings in culture media expenses in the production of ARA-enriched biomass of M. alpina, compared to the conventional synthetic culture medium, when waste from the potato chips industry was used as an alternative source of carbon and macro/microelements, supplemented with a low-cost yeast extract alternative. SIGNIFICANCE AND IMPACT OF THE STUDY: The demonstration of the use of potato chips wastes as a low-cost carbon source for the biomass, lipids and ARA production, suggesting an eco-friendly alternative for the use of agri-food wastes for valuable metabolites production.

Advances in improving the biotechnological application of oleaginous fungus Mortierella alpina.

Mortierella alpina is an oleaginous filamentous fungus with considerable lipid productivity, and it has been widely used for industrial production of arachidonic acid. The fermentation process of M. alpina is complicated and can be affected by various factors; therefore, a comprehensive knowledge of its metabolic characteristics and key factors governing lipid biosynthesis is required to further improve its industrial performance. In this review, we discuss the metabolic features and extracellular factors that affect lipid biosynthesis in M. alpina. The current progress in fermentation optimisation and metabolic engineering to improve lipid yield are also summarised. Moreover, we review the applications of M. alpina in the food industry and propose fermentation strategies for better utilisation of this genus in the future. In our opinion, the economic performance of M. alpina should be enhanced from multiple levels, including strains with ideal traits, efficient fermentation strategies, controllable fermentation costs, and competitive products of both high value and productivity. By reviewing the peculiarities of M. alpina and current progress to improve its suitability for biotechnological production, we wish to provide more efficient strategies for future development of M. alpina as a high-value lipid cell factory. KEY POINTS: • Understanding M. alpina metabolism is helpful for rational design of its fermentation processes. • Nitrogen source is a key point that affects PUFA's component and fermentation cost in M. alpina. • Dynamic fermentation strategy combined with breeding is needed to increase lipid yield in M. alpina.

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.

Role of the mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina.

OBJECTIVES: The transport of citrate from the mitochondria to the cytoplasm is essential during lipid accumulation. This study aimed to explore the role of mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina. RESULTS: Homologous MaYHM (the gene encoding the mitochondrial citrate-oxoglutarate carrier) was overexpressed in M. alpina. The fatty acid content of MaYHM-overexpressing recombinant strains was increased by up to 30% compared with the control. Moreover, the intracellular α-ketoglutarate level in recombinant strains was increased by 2.2 fold, together with a 23-35% decrease in NAD+-isocitrate dehydrogenase activity compared with the control. The overexpression of MaYHM altered the metabolic flux in the glutamate dehydrogenase shunt and 4-aminobutyric acid shunt during metabolic reprogramming, supplying more carbon to synthesize fatty acids. CONCLUSIONS: Overexpression of MaYHM resulted in more efflux of citrate from mitochondria to the cytoplasm and enhanced lipid accumulation. These findings provide new perspectives for the improvement of industrial lipid production in M. alpina.

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

OBJECTIVES: To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes between carbohydrate and lipid in fermentation process. RESULTS: The extraction of mycelia with HCl following a photometric phenol-sulphuric acid reaction was identified as an optimal method for total carbohydrate analysis in Mortierella alpina, which the extraction efficiency performed 1.1-3.6 fold than other five methods. The total carbohydrate content increased from initial 19.26 to 25.86% during early fermentation process and declined gradually thereafter, while the fatty acid was increasing from 8.47 to 31.03%. For separation and qualitative estimation of intracellular saccharides, the acetonitrile/water freeze-thaw method for extraction and Sugar-Pak I column for separation proved to be possible. With the glucose rapidly decreasing at the beginning of growth, the trehalose accumulated rapidly from 1.63 to 5.04% and then decreased slightly but maintain above 4% of dry biomass. CONCLUSIONS: This work established comprehensive carbohydrate extraction and analysis methods of Mortierella alpina and identified the main saccharide in fermentation process which indicated that the accumulation of fatty acids was related to the change of intracellular carbohydrate content.

Three New Hydroxyphenylacetic Acid Derivatives and A New Alkaloid from Endophytic Fungus Mortierella sp. in Epimedium acuminatum Franch. and Their Antibacterial Activity.

Three new hydroxyphenylacetic acid derivatives, stachylines E-G (1-3), and a new alkaloid, mortieridinone (4), along with six known compounds (5-10), were isolated from endophytic fungus Mortierella sp. in Epimedium acuminatum Franch. Their structures were determined by their spectroscopic analyses and by comparison with the literature data. Compounds 7 and 10 showed selective antibacterial activity against tested multidrug-resistant bacteria with minimum inhibitory concentration (MIC) values ranging from 25 to 3.13 µg/mL.

Identification of degrader bacteria and fungi enriched in rhizosphere soil from a toluene phytoremediation site using DNA stable isotope probing.

Improved knowledge of the ecology of contaminant-degrading organisms is paramount for effective assessment and remediation of aromatic hydrocarbon-impacted sites. DNA stable isotope probing was used herein to identify autochthonous degraders in rhizosphere soil from a hybrid poplar phytoremediation system incubated under semi-field-simulated conditions. High-throughput sequencing of bacterial 16S rRNA and fungal internal transcribed spacer (ITS) rRNA genes in metagenomic samples separated according to nucleic acid buoyant density was used to identify putative toluene degraders. Degrader bacteria were found mainly within the Actinobacteria and Proteobacteria phyla and classified predominantly as Cupriavidus, Rhodococcus, Luteimonas, Burkholderiaceae, Azoarcus, Cellulomonadaceae, and Pseudomonas organisms. Purpureocillium lilacinum and Mortierella alpina fungi were also found to assimilate toluene, while several strains of the fungal poplar endophyte Mortierella elongatus were indirectly implicated as potential degraders. Finally, PICRUSt2 predictive taxonomic functional modeling of 16S rRNA genes was performed to validate successful isolation of stable isotope-labeled DNA in density-resolved samples. Four unique sequences, classified within the Bdellovibrionaceae, Intrasporangiaceae, or Chitinophagaceae families, or within the Sphingobacteriales order were absent from PICRUSt2-generated models and represent potentially novel putative toluene-degrading species. This study illustrates the power of combining stable isotope amendment with advanced metagenomic and bioinformatic techniques to link biodegradation activity with unisolated microorganisms. Novelty statement: This study used emerging molecular biological techniques to identify known and new organisms implicated in aromatic hydrocarbon biodegradation from a field-scale phytoremediation system, including organisms with phyto-specific relevance and having potential for downstream applications (amendment or monitoring) in future and existing systems. Additional novelty in this study comes from the use of taxonomic functional modeling approaches for validation of stable isotope probing techniques. This study provides a basis for expanding existing reference databases of known aromatic hydrocarbon degraders from field-applicable sources and offers technological improvements for future site assessment and management purposes.

Research advances on arachidonic acid production by fermentation and genetic modification of Mortierella alpina.

Arachidonic acid (ARA, 5, 8, 11, 14-cis-eicosatetraenoic acid) is a relevant ω-6 polyunsaturated fatty acid, which plays essential roles in human immune, cardiovascular, and nervous systems. It is widely used in medicine, cosmetics, nutrition, and other fields. Traditionally, ARA is obtained from animal tissues. However, due to the limitation and unsustainability of existing resources, microorganisms are a potential alternative resource for ARA production. In this regard, major efforts have been made on algae and filamentous fungi, among which Mortierella alpina is the most effective strain for industrial ARA production. In this review, we summarized the recent progress in enhancing M. alpina production by optimization of culture medium and fermentation process and genetic modification. In addition, we provided perspectives in synthetic biology methods and technologies to further increase ARA production.

The role of MTHFDL in mediating intracellular lipogenesis in oleaginous Mortierella alpina.

The oleaginous fungus Mortierella alpina can synthesize a variety of polyunsaturated fatty acids, which are used extensively in industry for the production of arachidonic acid (AA). NADPH is the limiting factor and critical reducing agent in lipid biosynthesis. In the folate cycle, methylenetetrahydrofolate dehydrogenase (MTHFDL) catalyzes the conversion of methylene tetrahydrofolate into 10-formyl-tetrahydrofolate with the reduction of NADP+ to NADPH. MTHFDL RNAi was used to investigate the role of the folate cycle in lipogenesis. Gene knockdown decreased the transcript levels of MTHFDL by about 50 % and attenuated cell fatty acid synthesis. The observation of decreased NADPH levels and downregulated NADPH-producing genes in response to MTHFDL RNAi indicates a novel aspect of the NADPH regulatory mechanism. Thus, our study demonstrates that MTHFDL plays key role in the mediation of NADPH in lipogenesis in M. alpina.

Mycoavidus sp. Strain B2-EB: Comparative Genomics Reveals Minimal Genomic Features Required by a Cultivable Burkholderiaceae-Related Endofungal Bacterium.

Obligate bacterial endosymbionts are critical to the existence of many eukaryotes. Such endobacteria are usually characterized by reduced genomes and metabolic dependence on the host, which may cause difficulty in isolating them in pure cultures. Family Burkholderiaceae-related endofungal bacteria affiliated with the Mycoavidus-Glomeribacter clade can be associated with the fungal subphyla Mortierellomycotina and Glomeromycotina. In this study, a cultivable endosymbiotic bacterium, Mycoavidus sp. strain B2-EB, present in the fungal host Mortierella parvispora was obtained successfully. The B2-EB genome (1.88 Mb) represents the smallest genome among the endofungal bacterium Mycoavidus cysteinexigens (2.64-2.80 Mb) of Mortierella elongata and the uncultured endosymbiont "Candidatus Glomeribacter gigasporarum" (1.37 to 2.36 Mb) of arbuscular mycorrhizal fungi. Despite a reduction in genome size, strain B2-EB displays a high genome completeness, suggesting a nondegenerative reduction in the B2-EB genome. Compared with a large proportion of transposable elements (TEs) in other known Mycoavidus genomes (7.2 to 11.5% of the total genome length), TEs accounted for only 2.4% of the B2-EB genome. This pattern, together with a high proportion of single-copy genes in the B2-EB genome, suggests that the B2-EB genome reached a state of relative evolutionary stability. These results represent the most streamlined structure among the cultivable endofungal bacteria and suggest the minimal genome features required by both an endofungal lifestyle and artificial culture. This study allows us to understand the genome evolution of Burkholderiaceae-related endosymbionts and to elucidate microbiological interactions.IMPORTANCE This study attempted the isolation of a novel endobacterium, Mycoavidus sp. B2-EB (JCM 33615), harbored in the fungal host Mortierella parvispora E1425 (JCM 39028). We report the complete genome sequence of this strain, which possesses a reduced genome size with relatively high genome completeness and a streamlined genome structure. The information indicates the minimal genomic features required by both the endofungal lifestyle and artificial cultivation, which furthers our understanding of genome reduction in fungal endosymbionts and extends the culture resources for biotechnological development on engineering synthetic microbiomes.