16 research outputs found
Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors.
No full textHistamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to β-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals
Homologous and Heterologous Expression of Delta(12)-Desaturase in Mucor circinelloides Enhanced the Production of Linolenic Acid
No full textLinolenic acid (LA) is gaining more interest within the scientific community. This is because it has a potential medical role in reducing the risk of inflammation, carcinogenesis, atherosclerosis and diabetes and is a valuable nutraceutical for human health. The oleaginous fungus Mucor circinelloides produces a high lipid content (36%), including valuable polyunsaturated fatty acids (PUFAs). However, the critical step in which oleic acid (OA) is converted into LA is not efficient at supplying enough substrates for PUFA synthesis. Hence, we propose a method to increase LA production based on genetic engineering. The overexpression of the Δ12-desaturase gene from M. circinelloides and Mortierella alpina increased the LA content and improved the lipid accumulation (from 14.9% to 21.6% in the Δ12-desaturase gene of the M. circinelloides overexpressing strain (Mc-D12MC) and from 14.9% to 18.7% in the Δ12-desaturase gene of M. alpina overexpressing strain (Mc-D12MA)). Additionally, the up-regulated expression levels of these genes targeted the genes involved in NADPH production, implying that the elevated Δ12-desaturase gene may function as a critical regulator of NADPH and lipid synthesis in M. circinelloides. This study provides the first evidence to support the design of metabolic engineering related to LA and PUFA production in M. circinelloides for potential industrial applications
Altered microbial structure and function after thermokarst formation
No full textPermafrost thaw could induce substantial carbon (C) emissions to the atmosphere, and thus trigger a positive feedback to climate warming. As the engine of biogeochemical cycling, soil microorganisms exert a critical role in mediating the direction and strength of permafrost C-climate feedback. However, our understanding about the impacts of thermokarst (abrupt permafrost thaw) on microbial structure and function remains limited. Here we employed metagenomic sequencing to analyze changes in topsoil (0–15 cm) microbial communities and functional genes along a permafrost thaw sequence (1, 10, and 16 years since permafrost collapse) on the Tibetan Plateau. By combining laboratory incubation and a two-pool model, we then explored changes in soil labile and stable C decomposition along the thaw sequence. Our results showed that topsoil microbial α-diversity decreased, while the community structure and functional gene abundance did not exhibit any significant change at the early stage of collapse (1 year since collapse) relative to non-collapsed control. However, as the time since the collapse increased, both the topsoil microbial community structure and functional genes differed from the control. Abundances of functional genes involved in labile C degradation decreased while those for stable C degradation increased at the late stage of collapse (16 years since collapse), largely driven by changes in substrate properties along the thaw sequence. Accordingly, faster stable C decomposition occurred at the late stage of collapse compared to the control, which was associated with the increase in relative abundance of functional genes for stable C degradation. These results suggest that upland thermokarst alters microbial structure and function, particularly enhances soil stable C decomposition by modulating microbial functional genes, which could reinforce a warmer climate over the decadal timescale
Construction of Eicosatetraenoic Acid Producing Cell Factory by Genetic Engineering of Mucor circinelloides
No full textEicosatetraenoic acid (ETA, 20:4, ω-3) is the desaturation product of dihomo-gamma linolenic acid (DGLA, 20:3, ω-6) catalyzed by delta-17 desaturase, which is considered as a healthy product that helps to lower risk of heart diseases. The oleaginous filamentous fungus, Mucor circinelloides, has been used for a long time as a model micro-organism for GLA production at industrial scales. However, M. circinelloides lacks the key enzymes to synthesize C20 polyunsaturated fatty acids (PUFAs). M. circinelloides could produce DGLA by overexpressing the D6E(GLELO) gene, which could be a useful tool to produce ETA due to the availability of established genetic manipulation tools. Therefore, in this study, delta-17 desaturase (PpD17 and PaD17) genes from Phytophthora parasitica and Pythium aphanidermatum, respectively, were introduced into M. circinelloides to construct an ETA-producing cell factory. Our results showed that the PaD17 and PpD17 overexpression strains’ biomass increased by 25.98 and 23.34 g/L (39.98 and 25.75%), respectively, compared with the control strain. Meanwhile, the lipid contents of the recombinant strains also increased and reached up to 28.88% in Mc-PaD17 and 30.95% in Mc-PpD17, respectively, compared with the control strain (23.38% in Mc-2076). The RT-qPCR results showed that overexpression of delta-17 desaturase genes promoted the expression of cme2, fas2, and D6E, thereby contributing to lipid biosynthesis in M. circinelloides. Meanwhile, the content of ETA reached up to 1.95%, and the yield of ETA was up to 114.69 mg/L in PpD17 overexpression mutants at 96 h. This study provided the first report on the construction of an ETA-producing cell factory by heterologous overexpression of the PpD17 gene in M. circinelloides, which established a new scope for further research in the production of ETA in oleaginous fungi
In Silico Structural and Functional Analysis of the Mitochondrial Malate Transporters in Oleaginous Fungus Mucor circinelloides WJ11
No full textMalate transporter proteins (MTPs) play a pivotal role in regulating flux in the citrate/malate/pyruvate shuttle to deliver acetyl-CoA from the mitochondria to the cytosol and thus regulate lipid biosynthesis in oleaginous fungi. Despite the recent successful exploration of the mitochondrial malate transporters in Mucor circinelloides, research with in silico analyses that include molecular docking and their dynamics, in addition to homology modelling of malate transporters, have not been reported. In this study, the physico-chemical properties and nucleotide sequence analysis of two mitochondrial MTPs (MT and SoDIT-a with Gene/protein ID scafold00018.48 and scafold00239.15, respectively), in M. circinelloides WJ11 were performed. The three-dimensional (3D) model of the mitochondrial MTPs was determined and the best-docked complex stabilities were demonstrated with molecular dynamic (MD) simulations. The activity domain was revealed to form hydrogen bonds and piling interactions with citrate and malate upon docking. Our study showed better binding affinities for the MTPs—reaching up to −3.44 and −7.27 kcal/mol with the MT and SoDIT-a proteins, respectively (compared to the target of −2.85 and −6.00 kcal/mol for citric acid-binding). MD simulations illustrated that the protein complexes demonstrated conformational stability throughout the simulation. This study was the first to elucidate the structural characteristics of mitochondrial MTPs in M. circinelloides WJ11, providing direct evidence regarding the transport mechanism of specific substrates. Furthermore, the current results support ongoing efforts to combine functional and structural data to better understand the MTPs (at the molecular and atomic levels) of an oleaginous fungus such as M. circinelloides
Patterns and drivers of prokaryotic communities in thermokarst lake water across Northern Hemisphere
No full text13 páginas.- 5 figuras.- 81referencias.Aim: The formation of thermokarst lakes could make a large amount of carbon accessible to microbial degradation, potentially intensifying the permafrost carbon-climate feedback via carbon dioxide/methane emissions. Because of their diverse functional roles, prokaryotes could strongly mediate biogeochemical cycles in thermokarst lakes. However, little is known about the large-scale patterns and drivers of these communities. Location: Permafrost regions in the Northern Hemisphere. Time period: Present day. Major taxa studied: Prokaryotes. Methods: Based on a combination of large-scale measurements on the Tibetan Plateau and data syntheses in pan-Arctic regions, we constructed a comprehensive dataset of 16S rRNA sequences from 258 thermokarst lakes across Northern Hemisphere permafrost regions. We also used the local contributions to beta diversity (LCBD) to characterize the variance of prokaryotic species composition and screened underlying drivers by conducting a random forest modelling analysis. Results: Prokaryotes in thermokarst lake water were dominated by the orders Burkholderiales, Micrococcales, Flavobacteriales and Frankiales. The relative abundance of dominant taxa was positively associated with dissolved organic matter (DOM) properties, especially for the chromophoric/aromatic compounds. Microbial structure differed between high-altitude and high-latitude thermokarst lakes, with the dominance of Flavobacterium in high-altitude lakes, and the enrichment of Polynucleobacter in high-latitude lakes. More importantly, climatic variables were among the main drivers shaping the large-scale variation of prokaryotic communities. Specifically, mean annual precipitation was the best predictor for prokaryotic beta diversity across the Northern Hemisphere, as well as in the high-altitude permafrost regions, while mean annual air temperature played a key role in the high-latitude thermokarst lakes. Main conclusions: Our findings demonstrate significant associations between dominant taxa and DOM properties, as well as the important role of climatic factors in affecting prokaryotic communities. These findings suggest that climatic change may alter DOM conditions and induce dynamics in prokaryotic communities of thermokarst lake water in the Northern Hemisphere. © 2023 John Wiley & Sons Ltd.This work was supported by the National Key Research and Development Program of China (2022YFF0801903), National Natural Science Foundation of China (31988102, and 31825006), and Tencent Foundation through the XPLORER PRIZE. M.D‐B. acknowledges support from TED2021‐130908B‐C41/AEI/10.13039/501100011033/Unión Europea NextGenerationEU/PRTR and from the Spanish Ministry of Science and Innovation for the I + D + i project PID2020‐115813RA‐I00 funded by MCIN/AEI/10.13039/501100011033.Peer reviewe
Table_1_Simultaneous overexpression of ∆6-, ∆12- and ∆9-desaturases enhanced the production of γ-linolenic acid in Mucor circinelloides WJ11.DOCX
No full textMucor circinelloides WJ11, an oleaginous filamentous fungus, produces 36% lipid of its cell dry weight when cultured in a high C/N ratio medium, however, the yield of γ-linolenic acid (GLA) is insufficient to make it competitive with other plant sources. To increase the GLA content in M. circinelloides WJ11, this fungus was engineered by overexpression of its key genes such as Δ6-, Δ12-, and Δ9-desaturases involved in GLA production. Firstly, we tried to overexpress two Δ6-desaturase isozymes to determine which one played important role in GLA synthesis. Secondly, Δ6-and Δ12-desaturase were co-overexpressed to check whether linoleic acid (LA), the precursor for GLA synthesis, is a limiting factor or not. Moreover, we tried to explore the effects of simultaneous overexpression of Δ6-, Δ12-, and Δ9-desaturases on GLA production. Our results showed that overexpression (1 gene) of DES61 promoted higher GLA content (21% of total fatty acids) while co-overexpressing (2 genes) DES61 and DES12 and simultaneous overexpressing (3 genes) DES61, DES12, and DES91 increased the GLA production of engineered strains by 1.5 folds and 1.9 folds compared to the control strain, respectively. This study provided more insights into GLA biosynthesis in oleaginous fungi and laid a foundation for further increase in GLA production into fungus such as M. circinelloides.</p
SUCTION MEASUREMENTS AND WATER RETENTION IN UNSATURATED SOILS
No full textTechniques for testing unsaturated soils have been investigated by the author where the measurement and control of parameters were undertaken directly. Suction was measured and controlled with a new high suction tensiometer and water content through mass measurements with a balance. These techniques have been used for the determination of soil water retention curves and for the development of a suction . control system using air circulation and water injection. The techniques allow the soil to be subject to the same drying and wetting conditions that occur in nature and avoid the need for elevated air pressures, as are traditionally involved in testing using the axis translation technique. The performance of the new high suction tensiometer was evaluated, followed by its applications to soil testing. The tensiometer performance focused on the factors controlling cavitation, calibration in the negative pressure range and measurement. It was found that isotropic unloading is the most accurate technique for calibration in the negative range and that axis translation techniques can lead to errors. The research confirms high suction tensiometers are easy to use and versatile devices. Techniques were developed to measure and control suction and water content in unconfined and confined samples. Research on the unconfined samples focused on the procedures to obtain the soil water retention curve: discrete (soil dried or wetted in stages) and continuous (soil drying continuously). While both procedures were found not to influence the curves significantly, it is demonstrated that the continuous procedure is sensitive to factors such as the exposed surface area to drying or wetting and so should be used carefully. For confined conditions, wetting, drying, and water content measurement systems were developed. Wetting was based on the injection of water; drying was based on air circulation through a desiccant within a closed loop system. Water content was determined from the. difference between water injected and that adsorbed by the desiccant. This has been applied as part of a· double cell triaxial testing system that allows continuous determination of suction, water content and volume change. A challenge of such a system was imposing an air tight environment. The suitability of environmental scanning electron microscopy to observe unsaturated soils at the particle level was explored. The imaging of micron-sized materials at different relative humidities allowed a series of observations previously undocumented, among them: water menisci were visible, including their shape and interaction with surfaces; the contact angle between the air-water and water-solid interfaces was measurable.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
