Punicic acid ameliorates obesity-related hyperlipidemia and fatty liver in mice via regulation of intestinal flora and lipopolysaccharide-related signaling pathways.

Punicic acid (PA), mainly found in pomegranate seed oil (PSO), has attracted increasing attention due to its potential to mitigate obesity. The regulation of intestinal microflora was identified as a crucial factor and an effective strategy to reverse obesity-related hyperlipidemia and non-alcoholic fatty liver disease (NAFLD). To assess the impact of PSO on hyperlipidemia related to obesity, we investigated the hepatic lipid status and gut microbiota regulation in mice over 13 weeks of feeding a high-fructose high-fat diet (HFHFD). Serum lipid markers, including TG, TC and LDL-C, were markedly reduced in hyperlipidemic mice. PSO supplementation reduced hepatic lipid accumulation and steatosis, inhibited the expression of pro-inflammatory mediators (including IL-6 and IL-1ß), and restored the normal levels of the anti-inflammatory cytokine IL-10. In addition, PSO also alleviated oxidative stress and increased T-AOC and SOD activities, as well as GSH levels, while reducing the MDA content in the liver of HFHFD-fed mice. The activation of TLR4/MyD88/NF-κB and TLR4/IL-22/STAT3 signaling pathways in the liver due to the HFHFD was also evidently inhibited by PSO. Furthermore, supplementation of PSO ameliorated the HFHFD-induced dysbiosis of intestinal microflora, resulting in a markedly increased proportion of Muribaculaceae, a decreased ratio of Blautia, and elevated levels of microbiota-derived short-chain fatty acids (SCFAs). Moreover, the expression of tight junction proteins correlated with intestinal barrier function was notably restored in the colon. The collected results indicate that PSO may be an effective nutraceutical ingredient for attenuating lipid metabolic disorders.

Siderophore-synthesizing NRPS reprogram lipid metabolic profiles for phenotype and function changes of Arthrobotrys oligospora.

The objective of this paper is to explore the function of the AOL-s00215g415 (Aog415) gene, which encodes for the synthesis of siderophore in the nematode trapping fungal model strain A. oligospora, in order to understand the relationship between siderophore biosynthesis and nematode trapping activity. After a through sequence analysis, it was determined that Aog415 is a siderophore-synthesizing NRPS. The product of this gene was then identified to be the hydroxamate siderophore desferriferrichrome, using mass spectrometry analysis. When compared to the WT strains, the Aog415 knockout strain exhibited a 60% decrease in siderophore content in fermentation broth. Additionally, the number of predatory rings of decreased by 23.21%, while the spore yield increased by 37.34%. The deletion of Aog415 did not affect the growth of A. oligospora in diverse nutrient medium. Lipid metabolism-related pathways were the primary targets of Aog415 disruption as revealed by the metabolomic analysis. In comparison to the WT, a significant reduction in the levels of glycerophospholipids, and glycolipids was observed in the mutation. The metabolic alteration in fatty acyls and amino acid-like molecules were significantly disrupted. The knockout of Aog415 impaired the biosynthesis of the hydroxamate siderophore desferriferrichrome, remodeled the flow of fatty acid in A. oligospora, and mainly reprogrammed the membrane lipid metabolism in cells. Desferriferrichrome, a hydroxamate siderophore affects the growth, metabolism and nematode trapping ability of A. oligospora by regulating iron intake and cell membrane homeostasis. Our study uncovered the significant contribution of siderophores to the growth and nematode trapping ability and constructed the relationship among siderophores biosynthesis, lipid metabolism and nematode trapping activity of A. oligospora, which provides a new insight for the development of nematode biocontrol agents based on nematode trapping fungi.

Latilactobacillus curvatus FFZZH5L isolated from pickled cowpea enhanced antioxidant activity in Caenorhabditis elegans by upregulating the level of glutathione S-transferase.

Latilactobacillus curvatus is a potential probiotic that possesses beneficial health properties and fermentation traits; however, the extent of understanding of the antioxidant activities of L. curvatus is limited. This study investigates the antioxidant activities of a new L. curvatus FFZZH5L strain. The strain exhibits broad tolerance to acids, bases and salts and demonstrated good adaption to the gastrointestinal environment, with a survival rate of 45% after 24 h of treatment in artificial gastrointestinal juice. Moreover, L. curvatus FFZZH5L exhibits inhibitory effects on Staphylococcus aureus, with a self-aggregation rate of 34.8% and a co-aggregation rate of 82.2%. In vitro, the DPPH radical scavenging ability and GSH-px enzyme activity of L. curvatus FFZZH5L reach 64.27% and 15.95 U mL-1, respectively. Treatment of C. elegans with L. curvatus FFZZH5L in vivo significantly extended the organism's lifespan. Furthermore, the activity of SOD, GSH-px and T-AOC was increased by 33.6%, 43.4% and 58.3%, respectively. Feeding C. elegans with L. curvatus FFZZH5L decreased the MDA, lipofuscin and ROS levels by 9%-36.4%. L. curvatus FFZZH5L effectively protected C. elegans against juglone-induced oxidative stress damage and led to a significant increase in the organism's survival under heat stress. The RT-qPCR analysis suggests that feeding C. elegans with L. curvatus FFZZH5L upregulates the expression levels of antioxidant-related genes including glutathione S-transferase 4 (gst-4), gst-1, gst-10, sod-3, sod-5, and sod-10 in C. elegans. Our investigation confirms the probiotic and antioxidant properties of L. curvatus, indicating its potential application in functional foods and the pharmaceutical industry.

Function discovery of a non-ribosomal peptide synthetase-like encoding gene in the nematode-trapping fungus Arthrobotrys oligospora.

In this study, the function of a non-ribosomal peptide synthetase-like (NRPS-like) encoding gene AOL_s00188g306 (g306) was investigated to reveal the association between NRPS and nematocidal activity in the nematode-trapping fungus Arthrobotrys oligospora. Sequence analysis indicated that the encoded product of g306 is an adenylation domain of non-ribosomal peptide synthetases and extended short-chain dehydrogenase/reductase domain-containing proteins, and displays a wide substrate spectrum. The Δg306 mutants were more sensitive to chemical stressors than the wild type. Disruption of g306 impeded the nematocidal efficiency of A. oligospora. Metabolomics analysis showed that secondary metabolite biosynthesis and lipid metabolism were altered in the mutants. The phenotypic changes in the mutants can be attributed to the down-regulation of various metabolites, including fatty acyls, prenol lipids, steroidsand steroid derivative, and amino acid derivatives, identified in the present study. This study investigated the association between the non-ribosomal polypeptide-encoding gene g306 and nematicidal activity in A. oligospora, providing a reference for resolving the predation mechanism of nematode-trapping fungus.

Roles of the Fungal-Specific Lysine Biosynthetic Pathway in the Nematode-Trapping Fungus Arthrobotrys oligospora Identified through Metabolomics Analyses.

In higher fungi, lysine is biosynthesized via the α-aminoadipate (AAA) pathway, which differs from plants, bacteria, and lower fungi. The differences offer a unique opportunity to develop a molecular regulatory strategy for the biological control of plant parasitic nematodes, based on nematode-trapping fungi. In this study, in the nematode-trapping fungus model Arthrobotrys oligospora, we characterized the core gene in the AAA pathway, encoding α-aminoadipate reductase (Aoaar), via sequence analyses and through comparing the growth, and biochemical and global metabolic profiles of the wild-type and Aoaar knockout strains. Aoaar not only has α-aminoadipic acid reductase activity, which serves fungal L-lysine biosynthesis, but it also is a core gene of the non-ribosomal peptides biosynthetic gene cluster. Compared with WT, the growth rate, conidial production, number of predation rings formed, and nematode feeding rate of the ΔAoaar strain were decreased by 40-60%, 36%, 32%, and 52%, respectively. Amino acid metabolism, the biosynthesis of peptides and analogues, phenylpropanoid and polyketide biosynthesis, and lipid metabolism and carbon metabolism were metabolically reprogrammed in the ΔAoaar strains. The disruption of Aoaar perturbed the biosynthesis of intermediates in the lysine metabolism pathway, then reprogrammed amino acid and amino acid-related secondary metabolism, and finally, it impeded the growth and nematocidal ability of A. oligospora. This study provides an important reference for uncovering the role of amino acid-related primary and secondary metabolism in nematode capture by nematode-trapping fungi, and confirms the feasibility of Aoarr as a molecular target to regulate nematode-trapping fungi to biocontrol nematodes.

Gut bacterial extracellular vesicles: important players in regulating intestinal microenvironment.

Intestinal microenvironment dysbiosis is one of the major causes of diseases, such as obesity, diabetes, inflammatory bowel disease, and colon cancer. Microbiota-based strategies have excellent clinical potential in the treatment of repetitive and refractory diseases; however, the underlying regulatory mechanisms remain elusive. Identification of the internal regulatory mechanism of the gut microbiome and the interaction mechanisms involving bacteria-host is essential to achieve precise control of the gut microbiome and obtain effective clinical data. Gut bacteria-derived extracellular vesicles (GBEVs) are lipid bilayer nanoparticles secreted by the gut microbiota and are considered key players in bacteria-bacteria and bacteria-host communication. This review focusses on the role of GBEVs in gut microbiota interactions and bacteria-host communication, and the potential clinical applications of GBEVs.

Autophagy Improves ARA-Rich TAG Accumulation in Mortierella alpina by Regulating Resource Allocation.

The present study was designed to explore the possibility of improving lipid production in oleaginous filamentous fungus Mortierella alpina based on an autophagy regulation strategy. According to multiomics information, vacuolate-centered macroautophagy was identified as the main type of autophagy in M. alpina under nitrogen-limited conditions. Mutation of autophagy-related gene MAatg8 led to impaired fatty acid synthesis, while overexpression of both MAatg8 and phosphatidylserine decarboxylases (MApsd2) showed promoting effects on fatty acid synthesis. MAatg8 overexpression strain with external supply of ethanolamine significantly increased arachidonic acid (ARA)-rich triacylglycerol (TAG) and biomass synthesis in M. alpina, and the final fatty acid content increased by approximately 110% compared with that in the wild-type strain. Metabolomics and lipidomics analyses revealed that cell autophagy enhanced the recycling of preformed carbon, nitrogen, and lipid in mycelium, and the released carbon skeleton and energy were contributed to the accumulation of TAG in M. alpina. This study suggests that regulation of autophagy-related MAatg8-phosphatidylethanolamine (MAatg8-PE) conjugation system could be a promising strategy for attaining higher lipid production and biomass growth. The mechanism of autophagy in regulating nitrogen limitation-induced lipid accumulation elucidated in this study provides a reference for development of autophagy-based strategies for improving nutrient use efficiency and high value-added lipid production by oleaginous microorganism. IMPORTANCE Studies have indicated that functional oil accumulation occurs in oleaginous microorganisms under nitrogen limitation. However, until now, large-scale application of nitrogen-deficiency strategies was limited by low biomass. Therefore, the identification of the critical nodes of nitrogen deficiency-induced lipid accumulation is urgently needed to further guide functional oil production. The significance of our research is in uncovering the function of cell autophagy in the ARA-rich TAG accumulation of oleaginous fungus M. alpina and demonstrating the feasibility of improving lipid production based on an autophagy regulation strategy at the molecular and omics levels. Our study proves that regulation of cell autophagy through the MAatg8-PE conjugation system-related gene overexpression or exogenous supply of ethanolamine would be an efficient strategy to increase and maintain biomass productivity when high TAG content is obtained under nitrogen deficiency, which could be useful for the development of new strategies that will achieve more biomass and maximal lipid productivity.

The fucose-specific lectin gene AOL_s00054g276 affects trap formation and nematocidal activity of the nematophagous fungus Arthrobotrys oligospora.

Nematode-trapping fungi are natural enemies of nematodes in nature. Arthrobotrys oligospora, a typical nematode-trapping fungus with a clear genetic background, can capture and infect nematodes by forming adhesive three-dimensional networks. Lectins, a class of glycoproteins containing glycosyl-specific recognition domains, play an important role in biological recognition. However, the fucose-specific lectins have rarely been studied regarding the process of preying on nematodes. In this study, we characterized the biological role of the fucose-specific lectin-encoding gene AOL_s00054g276 (g276) in A. oligospora. The gene g276 was first deleted based on homologous recombination, then the phenotype and nematocidal activity of the Δg276 mutant was evaluated. The results showed that the deletion of gene g276 delayed trap formation and weakened its nematocidal activity; however, mycelial growth, conidia production, conidial germination rates and adaption to environmental stresses were not affected. Our results suggest that the fucose-specific lectin-encoding gene g276 might be associated with the morphogenesis of this fungus, and its deletion resulted in a significantly low density of three-dimensional traps (P < 0.05) and a significantly low nematode-trapping efficiency (P < 0.001). These findings provide a basis for further elucidating the mechanism of A. oligospora preying on nematodes and lay a foundation for the development and utilization of fungal-derived lectins for nematode control in the future.

A putative F-box-domain-encoding gene AOL_s00076g207 regulates the development and pathogenicity of Arthrobotrys oligospora.

F-box protein is a key component of the Skp1-cullin-F-box-type ubiquitin ligase complex (SCF-ULC) that marks its target proteins with ubiquitin for proteasomal degradation. In this study, we explored the potential role of AOL_s00076g207 (Aog207) in Arthrobotrys oligospora, a model fungus for studying nematodes-fungi interactions. The Aog207 gene encodes a putative F-box protein of the SCF-ULC. Deletion of Aog207 could inhibit mycelial growth in TYGA and PDA media. More importantly, the conidial germination rate of ΔAog207 mutants was remarkably declined compared to that of wild-type (WT) strain, and the mutant strains were more sensitive toward chemical stressors than the WT strain. In addition, ΔAog207 mutants generated fewer traps and captured fewer nematodes than WT strain. In summary, Aog207 disruption significantly affected the pathogenicity, mycelial growth, conidial germination, environmental adaptation and trap formation of A. oligospora. These findings may facilitate a better understanding of the nematode predation mechanism of A. oligospora and provide an experimental basis for developing biological control agents against nematodes.

Lipid metabolism research in oleaginous fungus Mortierella alpina: Current progress and future prospects.

The oleaginous fungus Mortierella alpina has distinct advantages in long-chain PUFAs production, and it is the only source for dietary arachidonic acid (ARA) certificated by FDA and European Commission. This review provides an overall introduction to M. alpina, including its major research methods, key factors governing lipid biosynthesis, metabolic engineering and omics studies. Currently, the research interests in M. alpina focus on improving lipid yield and fatty acid desaturation degree by enhancing fatty acid precursors and the reducing power NADPH, and genetic manipulation on PUFAs synthetic pathways is carried to optimise fatty acid composition. Besides, multi-omics studies have been applied to elucidate the global regulatory mechanism of lipogenesis in M. alpina. However, research challenges towards achieving a lipid cell factory lie in strain breeding and cost control due to the coenocytic mycelium, long fermentation period and insufficient conversion rate from carbon to lipid. We also proposed future research goals based on a multilevel regulating strategy: obtaining ideal chassis by directional evolution and high-throughput screening; rewiring central carbon metabolism and inhibiting competitive pathways by multi-gene manipulation system to enhance carbon to lipid conversion rate; optimisation of protein function based on post-translational modification; application of dynamic fermentation strategies suitable for different fermentation phases. By reviewing the comprehensive research progress of this oleaginous fungus, we aim to further comprehend the fungal lipid metabolism and provide reference information and guidelines for the exploration of microbial oils from the perspectives of fundamental research to industrial application.

Untargeted and targeted metabolomics reveal changes in the chemical constituents of instant dark tea during liquid-state fermentation by Eurotium cristatum.

Instant green tea powder was used as raw material to prepare an instant dark tea via liquid-state fermentation by Eurotium cristatum. To understand how the chemical constituents present in fermented green tea develop during fermentation, samples were collected on different days during fermentation for qualitative analyses by ultra-performance liquid chromatography-Q Exactive Orbitrap/Mass spectrometry. Untargeted metabolomics analyses revealed that the levels of original secondary metabolites in the instant green tea changed significantly from day 3 to day 5 during fermentation. Targeted metabolomics indicated that the levels of galloylated catechins (GCs) and free amino acids (FAAs) significantly decreased, but the nongalloylated catechins (NGCs), alkaloids, thearubigins and theabrownins increased dramatically after fermentation. The changes in the contents of catechins, gallic acid and free amino acids in the instant dark tea samples were positively related to the DPPH radical scavenging activities in vitro, and the phenolic acids and FAAs were positively related to the inhibitory effects towards α-glucosidase. These results showed that fermentation by Eurotium cristatum is critical to the formation of certain qualities of instant dark tea.

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.

[Application of omics technology in oleaginous microorganisms].

Microbial oils are potential resources of fuels and food oils in the future. In recent years, with the rapid development of systems biology technology, understanding the physiological metabolism and lipid accumulation characteristics of oleaginous microorganisms from a global perspective has become a research focus. As an important tool for systems biology research, omics technology has been widely used to reveal the mechanism of high-efficiency production of oils by oleaginous microorganisms. This provides a basis for rational genetic modification and fermentation process control of oleaginous microorganisms. In this article, we summarize the application of omics technology in oleaginous microorganisms, introduced the commonly used sample pre-processing and data analysis methods for omics analysis of oleaginous microorganisms, reviewe the researches for revealing the mechanism of efficient lipid production by oleaginous microorganisms based on omics technologies including genomics, transcriptomics, proteomics (modification) and metabolomics (lipidomics), as well as mathematical models based on omics data. The future development and application of omics technology for microbial oil production are also proposed.

Metabolomics analysis reveals the role of oxygen control in the nitrogen limitation induced lipid accumulation in Mortierella alpina.

Lipid hyperaccumulation in oleaginous microorganisms is generally induced by nitrogen limitation, while oxygen supply can influence biomass growth and cell metabolism. Although strategies based on nitrogen limitation or oxygen control have been extensively explored and applied in various oleaginous microorganisms, the role of oxygen supply in nitrogen limitation induced lipid hyperaccumulation still remains unclear. Here, we systematically surveyed the effects of oxygen supply on the oleaginous fungus M. alpina cultured in nitrogen limited conditions through integration of physiochemical parameters and metabolomics analysis. Our results indicated that a high oxygen supply promoted carbon/nitrogen consumption and was used for rapid biomass synthesis, while either high or low oxygen supply conditions were adverse to lipid and ARA accumulation. Different oxygen supply level significantly affected the balance between fermentation for lipid synthesis and respiration for energy generation. Under nitrogen limitation, a suitable oxygen supply promoted the recycling of preformed nitrogen and increased the redirection of carbon towards fatty acid synthesis through the hub centred around glutamic acid coupled to the intermediate metabolism of carbon in the TCA cycle, while a high oxygen supply favored the respiration process and enhanced the degradation of LC-PUFAs, rather than fermentation for fatty acid synthesis. This system-level insight reveals the underlying metabolic mechanism of oxygen control in nitrogen limitation induced lipid accumulation, and provides theoretical support for the integration of oxygen control with nutrient supply for efficient microbial oil production.

Time-resolved multi-omics analysis reveals the role of nutrient stress-induced resource reallocation for TAG accumulation in oleaginous fungus Mortierella alpina.

BACKGROUND: Global resource reallocation is an established critical strategy through which organisms deal with environmental stress. The regulation of intracellular lipid storage or utilization is one of the most important strategies for maintaining energy homeostasis and optimizing growth. Oleaginous microorganisms respond to nitrogen deprivation by inducing lipid hyper accumulation; however, the associations between resource allocation and lipid accumulation are poorly understood. RESULTS: Here, the time-resolved metabolomics, lipidomics, and proteomics data were generated in response to nutrient availability to examine how metabolic alternations induced by nitrogen deprivation drive the triacylglycerols (TAG) accumulation in M. alpina. The subsequent accumulation of TAG under nitrogen deprivation was a consequence of the reallocation of carbon, nitrogen sources, and lipids, rather than an up-regulation of TAG biosynthesis genes. On one hand, nitrogen deprivation induced the down-regulation of isocitrate dehydrogenase level in TCA cycle and redirected glycolytic flux of carbon from amino acid biosynthesis into fatty acids' synthesis; on the other hand, nitrogen deprivation induced the up-regulation of cell autophagy and ubiquitin-mediated protein proteolysis which resulted in a recycling of preformed protein nitrogen and carbon. Combining with the up-regulation of glutamate decarboxylase and succinic semialdehyde dehydrogenase in GABA shunt, and the phosphoenolpyruvate carboxykinase in the central hub involving pyruvate/phosphoenolpyruvate/oxaloacetate, the products from nitrogen-containing compounds degradation were recycled to be intermediates of TCA cycle and be shunted toward de novo biosynthesis of fatty acids. We found that nitrogen deprivation increased the protein level of phospholipase C/D that contributes to degradation of phosphatidylcholine and phosphatidylethanolamine, and supplied acyl chains for TAG biosynthesis pathway. In addition, ATP from substrate phosphorylation was presumed to be a critical factor regulation of the global resource allocation and fatty acids' synthesis rate. CONCLUSIONS: The present findings offer a panoramic view of resource allocation by M. alpina in response to nutrient stress and revealed a set of intriguing associations between resource reallocation and TAG accumulation. This system-level insight provides a rich resource with which to explore in-depth functional characterization and gain information about the strategic combination of strain development and process integration to achieve optimal lipid productivity under nutrient stress.

Two-stage pH control combined with oxygen-enriched air strategies for the highly efficient production of EPA by Mortierella alpina CCFM698 with fed-batch fermentation.

Dissolved oxygen and pH are critical factors influencing cell growth and metabolism. In our previous work, we constructed the recombinant strain Mortierella alpina CCFM698, which has the ability to produce EPA at room temperature. However, our experiments showed that the dissolved oxygen produced by the aeration and agitation of the fermenter was insufficient for cell growth and EPA synthesis by this recombinant strain. Moreover, the optimum pH for cell growth was incompatible with that of EPA accumulation. This study introduced a combined strategy of two-stage pH control with oxygen-enriched air in fed-batch fermentation to facilitate both cell growth and EPA production in M. alpina CCFM698. After 10 days of fermentation in a 7.5 L tank, the biomass production reached 41.2 g/L, with a lipid content of 31.5%, and EPA accounting for 26.7% of total lipids. The final EPA production reached 3.47 g/L, which is the highest yet achieved by M. alpina. This study reveals the critical role of dissolved oxygen and pH control for EPA production of M. alpina, and provides an easy and efficient strategy for industrial production of EPA.

Ultra Performance Liquid Chromatography-Q Exactive Orbitrap/Mass Spectrometry-Based Lipidomics Reveals the Influence of Nitrogen Sources on Lipid Biosynthesis of Mortierella alpina.

The objective of the present study was to reveal the effects of four types of nitrogen sources (soymeal, yeast extract, KNO3, and ammonium tartrate) on the lipid metabolism of the oleaginous fungus Mortierella alpina using untargeted lipidomics, targeted fatty acid, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. Our results showed clear differences in the contents and compositions of lipids between four types of nitrogen sources. Soymeal and ammonium tartrate supplementation favored the accumulation of triglycerides with arachidonic acid (ARA) and C16-18 fatty acids, respectively. These results were further validated by our targeted fatty acid analysis. RT-qPCR analysis of related genes in M. alpina between the four nitrogen source conditions found that soymeal supplementation dramatically increased the expression of GPAT, ELOVL, and Δ12/Δ6 desaturase. Our findings provided new insights into the regulation of lipid biosynthesis in M. alpina and potential avenues for genetic manipulation and highlighted the importance of an optimal nitrogen source for ARA-rich oil production.

Role of Adenosine Monophosphate Deaminase during Fatty Acid Accumulation in Oleaginous Fungus Mortierella alpina.

In oleaginous micro-organisms, nitrogen limitation activates adenosine monophosphate deaminase (AMPD) and promotes lipogenesis via the inhibition of isocitrate dehydrogenase. We found that the overexpression of homologous AMPD in Mortierella alpina favored lipid synthesis over cell growth. Total fatty acid content in the recombinant strain was 15.0-34.3% higher than that in the control, even though their biomass was similar. During the early fermentation stage, the intracellular AMP level reduced by 40-60%, together with a 1.9-2.7-fold increase in citrate content compared with the control, therefore provided more precursors for fatty acid synthesis. Moreover, the decreased AMP level resulted in metabolic reprogramming, reflected by the blocked TCA cycle and reduction of amino acids, distributing more carbon to lipid synthesis pathways. By coupling the energy balance with lipogenesis, this study provides new insights into cell metabolism under nitrogen-limited conditions and targets the regulation of fatty acid accumulation in oleaginous micro-organisms.

Evaluation of metabolome sample preparation and extraction methodologies for oleaginous filamentous fungi Mortierella alpina.

INTRODUCTION: Metabolomics has been successfully applied to guide the rational engineering of industrial strains and improve the performance of bioprocesses. Mortierella alpina has traditionally been one of the most popular industrial strains for the production of polyunsaturated fatty acids. However, a systematic comparison and optimisation of the metabolomic analysis methods of M. alpina has not yet been reported. OBJECTIVE: We sought to identify potential weaknesses that are important for accurate metabolomic analysis. We also aimed to determine an efficient sample preparation protocol for metabolomics studies in the oleaginous filamentous fungus M. alpina. METHODS: In this study, using GC-MS, we evaluated three sample preparation protocols and five solvent mixtures by assessment of the metabolite profile differences, the sum of peak intensities and the reproducibility of metabolite quantification. RESULTS: The freeze-dried biomass had better reproducibility and recovery than fresh biomass for metabolite extraction and data normalisation that is part of a metabolomics analysis of filamentous fungi M. alpina. Methanol:water (1:1) was superior for the profiling of metabolites in oleaginous fungi M. alpina. The unbiased metabolite profiling difference between the growth phase and lipids synthesis phase revealed that the degradation of amino acids were critical nodes for the efficient synthesis of lipids in M. alpina. CONCLUSION: The use of freeze-dried biomass for metabolite extraction and data normalisation was more efficient at measuring the active state of the intracellular metabolites in M. alpina. We recommend extracting the intracellular metabolites with methanol:water (1:1). An important role of amino acid oxidation in the nitrogen limitation-mediated lipid accumulation was found.

High-theabrownins instant dark tea product by Aspergillus niger via submerged fermentation: α-glucosidase and pancreatic lipase inhibition and antioxidant activity.

BACKGROUND: Theabrownins (TB) are bioactive components that are usually extracted from Chinese dark tea, in which they are present at low concentrations. The present study aimed to produce an instant dark tea high in theabrownins via submerged fermentation by the fungus Aspergillus niger. Three fermentation parameters that affect theabrownins content (i.e. inoculum size, liquid-solid ratio and rotation speed) were optimized using response surface methodology. RESULT: Optimum fermentation conditions were modeled to be an inoculum of 5.40% (v/v), a liquid-solid ratio of 27.45 mL g-1 and a rotation speed of 184 rpm and were predicted to yield 292.99 g kg-1 TB. Under these experimentally conditions, the TB content of the instant dark tea was 291.93 g kg-1 . The antioxidant capacity and α-glucosidase and pancreatic lipase inhibitory activities of the high-TB instant black tea were higher than four other typical instant dark tea products. CONCLUSION: The results of the present study show that careful management of culture conditions can produce a dark tea high in theabrownins. Furthermore, high-theabrownins instant dark tea could serve as a source of bioactive products and be used in functional foods as an ingredient imparting antioxidant properties and the ability to inhibit pancreatic lipase and α-glucosidase. © 2017 Society of Chemical Industry.