Construction of engineered yeast producing ammonia from glutamine and soybean residues (okara).
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ABSTRACT:
SUBMITTER: Watanabe Y
PROVIDER: S-EPMC7158961 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.
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Project description:In this study, a method for preparing low molecular weight peptides (HPH-VAP) from okara using high-pressure homogenization assisted double enzymes was proposed. In order to explore its advantages, the effects of various methods on protein extraction rate and on the structure, antioxidant and immune properties of peptides were compared. The results showed that the protein extraction rate of this method was increased by 69% and 51% compared with other methods, and the structure only led to changes in the hydrogen bonds between peptide chains. HPH-VAP was screened out through functional characteristics, its structure was identified by HPLC-MS/MS, and further immunological activity analysis was carried out. The results showed that it promoted cell phagocytic ability, NO level and release of cytokines IL-6, IFN- γ, TNF-α. Therefore, this method is an effective and applicable method for industrial preparation of okara peptides, and has a positive effect on the reuse of okara resources.
| S-EPMC8645904 | biostudies-literature
Project description:NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS). Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine) in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown) glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine) is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS structural elements associated with glutamine-utilizing capabilities.
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Project description:The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.
| S-EPMC2843202 | biostudies-literature
Project description:BACKGROUND:A microorganism engineered for non-native tasks may suffer stresses it never met before. Therefore, we examined whether a Kluyveromyces marxianus strain engineered with a carotenoid biosynthesis pathway can serve as an anti-stress chassis for building cell factories. RESULTS:Carotenoids, a family of antioxidants, are valuable natural products with high commercial potential. We showed that the free radical removal ability of carotenoids can confer the engineered host with a higher tolerance to ethanol, so that it can produce more bio-ethanol than the wild type. Moreover, we found that this engineered strain has improved tolerance to other toxic effects including furfurals, heavy metals such as arsenate (biomass contaminant) and isobutanol (end product). Furthermore, the enhanced ethanol tolerance of the host can be applied to bioconversion of a natural medicine that needs to use ethanol as the delivery solvent of hydrophobic precursors. The result suggested that the engineered yeast showed enhanced tolerance to ethanol-dissolved hydrophobic 10-deacetylbaccatin III, which is considered a sustainable precursor for paclitaxel (taxol) bioconversion. CONCLUSIONS:The stress tolerances of the engineered yeast strain showed tolerance to several toxins, so it may serve as a chassis for cell factories to produce target products, and the co-production of carotenoids may make the biorefinary more cost-effective.
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Project description:The protrotophic laboratory strain CEN.PK113-7D (MAT a) was grown in laboratory fermentors with a working volume of 1 litre at dilution rate (D) of 0.20 per hour (in duplicate for each nitrogen (glutamine and ammonium) limited condition). After cultivation for few hundred generations in ammonium, samples from 2 continuous cultures were taken for array analysis. After cultivation for few hundred generations in glutamine, one evolved strain was picked and cultivated in 2 biological replicate chemostats until steady state was reached, and samples for array analysis were collected. All collected cell samples were cooled below 2 degree C within ten seconds by mixing 40% sample and 60% crushed ice.
2008-06-01 | E-MEXP-1498 | biostudies-arrayexpress
Project description:This study was aimed at the valorization of the okara byproduct deriving form soy food manufacturing, by using ultrasound at different temperatures for extracting the residual proteins. The physicochemical and conformational changes of the extracted proteins were investigated in order to optimize the procedure. Increasing the temperature from 20 up to 80 °C greatly enhanced the yields and the protein solubility without affecting the viscosity. The protein secondary and tertiary structures were also gradually modified in a significant way. After the ultrasonication at the highest temperature, a significant morphological transition from well-defined single round structures to highly aggregated ones was observed, which was confirmed by measuring the water contact angles and wettability. After the ultrasound process, the improvement of peptides generation and the different amino acid exposition within the protein led to an increase of the antioxidant properties. The integrated strategy applied in this study allows to foster the okara protein obtained after ultrasound extraction as valuable materials for new applications.
| S-EPMC7998950 | biostudies-literature
Project description:Industrial fermentation provides a wide variety of bioproducts, such as food, biofuels and pharmaceuticals. Self-cycling fermentation (SCF), an advanced automated semi-continuous fermentation approach, has shown significant advantages over batch reactors (BR); including cell synchrony and improved production. Here, Saccharomyces cerevisiae engineered to overproduce shikimic acid was grown under SCF operation. This led to four-fold increases in product yield and volumetric productivity compared to BR. Transcriptomic analyses were performed to understand the cellular mechanisms leading to these increases. Results indicate an up-regulation of a large number of genes related to the cell cycle and DNA replication in the early stages of SCF cycles, inferring substantial synchronization. Moreover, numerous genes related to gluconeogenesis, the citrate cycle and oxidative phosphorylation were significantly up-regulated in the late stages of SCF cycles, consistent with significant increases in shikimic acid yield and productivity.
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Project description:SLC4A11 is a NH3 sensitive membrane transporter with H+ channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown. Here we demonstrate that SLC4A11 is localized to the inner mitochondrial membrane of CE and SLC4A11 transfected PS120 fibroblasts, where it acts as an NH3-sensitive mitochondrial uncoupler that enhances glutamine-dependent oxygen consumption, electron transport chain activity, and ATP levels by suppressing damaging Reactive Oxygen Species (ROS) production. In the presence of glutamine, Slc4a11-/- (KO) mouse CE generate significantly greater mitochondrial superoxide, a greater proportion of damaged depolarized mitochondria, and more apoptotic cells than WT. KO CE can be rescued by MitoQ, reducing NH3 production by GLS1 inhibition or dimethyl αKetoglutarate supplementation, or by BAM15 mitochondrial uncoupling. Slc4a11 KO mouse corneal edema can be partially reversed by αKetoglutarate eye drops. Moreover, we demonstrate that this role for SLC4A11 is not specific to CE cells, as SLC4A11 knockdown in glutamine-addicted colon carcinoma cells reduced glutamine catabolism, increased ROS production, and inhibited cell proliferation. Overall, our studies reveal a unique metabolic mechanism that reduces mitochondrial oxidative stress while promoting glutamine catabolism.
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Project description:The microbiota-gut-brain axis has attracted increasing attention in the last decade. Here, we investigated whether okara, a soybean by-product rich in dietary fiber, can attenuate cognitive impairment in senescence-accelerated mouse prone 8 (SAMP8) mice by altering gut microbial composition. Mice were fed either a standard diet, or a diet containing okara (7.5% or 15%, w/w) for 26 weeks. In the memory test, the 7.5% okara-fed mice showed a longer step-through latency and the 15% okara-fed mice had a short escape latency compared with control mice. The 15% okara-fed mice displayed decreased body weight, increased fecal weight, and altered cecal microbiota composition compared with the control group; however, there was no significant difference in the serum lactic acid and butyric acid levels among these mice groups. The 7.5% okara-fed mice had significantly higher NeuN intensity in the hippocampus compared with control mice. Furthermore, a decrease in inflammatory cytokine TNF- and an increase in brain-derived neurotrophic factor (BDNF) was observed in the 7.5% okara-fed group. The expression of synthesizing enzyme of acetylcholine was increased by the okara diets, and the acetylcholine level in the brain was higher in the 7.5% okara-fed group than in the control. These suggest that oral administration of okara could delay cognitive decline without drastically changing gut microbiota.
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