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The widespread attention towards 1,4-butanediol (BDO) as a key chemical raw material stems from its potential in producing biodegradable plastics. However, the efficiency of its biosynthesis via current bioprocesses is limited. In this study, a dual-pathway approach for 1,4-BDO production from succinic acid was developed. Specifically, a double-enzyme catalytic pathway involving carboxylic acid reductase and ethanol dehydrogenase was proposed. Optimization of the expression levels of the pathway enzymes led to a significant 318 % increase in 1,4-BDO titer. Additionally, the rate-limiting enzyme MmCAR was engineered to enhance the kcat/KM values by 50 % and increase 1,4-BDO titer by 46.7 %. To address cofactor supply limitations, an NADPH and ATP cycling system was established, resulting in a 48.9 % increase in 1,4-BDO production. Ultimately, after 48â hours, 1,4-BDO titers reached 201â mg/L and 1555â mg/L in shake flask and 5â L fermenter, respectively. This work represents a significant advancement in 1,4-BDO synthesis from succinic acid, with potential applications in the organic chemical and food industries.
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Butileno Glicóis , Escherichia coli , Ácido Succínico , Butileno Glicóis/metabolismo , Butileno Glicóis/química , Ácido Succínico/metabolismo , Ácido Succínico/química , Escherichia coli/metabolismo , Escherichia coli/genética , Biocatálise , Álcool Desidrogenase/metabolismo , Oxirredutases/metabolismo , Oxirredutases/genética , FermentaçãoRESUMO
BACKGROUND: The production of succinic acid (SA) from biomass has attracted worldwide interest. Saccharomyces cerevisiae is preferred for SA production due to its strong tolerance to low pH conditions, ease of genetic manipulation, and extensive application in industrial processes. However, when compared with bacterial producers, the SA titers and productivities achieved by engineered S. cerevisiae strains were relatively low. To develop efficient SA-producing strains, it's necessary to clearly understand how S. cerevisiae cells respond to SA. RESULTS: In this study, we cultivated five S. cerevisiae strains with different genetic backgrounds under different concentrations of SA. Among them, KF7 and NBRC1958 demonstrated high tolerance to SA, whereas NBRC2018 displayed the least tolerance. Therefore, these three strains were chosen to study how S. cerevisiae responds to SA. Under a concentration of 20 g/L SA, only a few differentially expressed genes were observed in three strains. At the higher concentration of 60 g/L SA, the response mechanisms of the three strains diverged notably. For KF7, genes involved in the glyoxylate cycle were significantly downregulated, whereas genes involved in gluconeogenesis, the pentose phosphate pathway, protein folding, and meiosis were significantly upregulated. For NBRC1958, genes related to the biosynthesis of vitamin B6, thiamin, and purine were significantly downregulated, whereas genes related to protein folding, toxin efflux, and cell wall remodeling were significantly upregulated. For NBRC2018, there was a significant upregulation of genes connected to the pentose phosphate pathway, gluconeogenesis, fatty acid utilization, and protein folding, except for the small heat shock protein gene HSP26. Overexpression of HSP26 and HSP42 notably enhanced the cell growth of NBRC1958 both in the presence and absence of SA. CONCLUSIONS: The inherent activities of small heat shock proteins, the levels of acetyl-CoA and the strains' potential capacity to consume SA all seem to affect the responses and tolerances of S. cerevisiae strains to SA. These factors should be taken into consideration when choosing host strains for SA production. This study provides a theoretical basis and identifies potential host strains for the development of robust and efficient SA-producing strains.
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Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae , Ácido Succínico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , FermentaçãoRESUMO
As a consequence of alcoholic fermentation (AF) in wine, several compounds are released by yeasts, and some of them are linked to the general quality and mouthfeel perceptions in wine. However, others, such as succinic acid, act as inhibitors, mainly of malolactic fermentation. Succinic acid is produced by non-Saccharomyces and Saccharomyces yeasts during the initial stages of AF, and the presence of some amino acids such as γ-aminobutyric acid (GABA) and glutamic acid can increase the concentration of succinic acid. However, the influence of these amino acids on succinic acid production has been studied very little to date. In this work, we studied the production of succinic acid by different strains of non-Saccharomyces and Saccharomyces yeasts during AF in synthetic must, and the influence of the addition of GABA or glutamic acid or a combination of both. The results showed that succinic acid can be produced by non-Saccharomyces yeasts with values in the range of 0.2-0.4 g/L. Moreover, the addition of GABA or glutamic acid can increase the concentration of succinic acid produced by some strains to almost 100 mg/L more than the control, while other strains produce less. Consequently, higher succinic acid production by non-Saccharomyces yeast in coinoculated fermentations with S. cerevisiae strains could represent a risk of inhibiting Oenococcus oeni and therefore the MLF.
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Oenococcus , Vinho , Vinho/análise , Vinho/microbiologia , Saccharomyces cerevisiae/metabolismo , Ácido Glutâmico/metabolismo , Ácido Succínico/metabolismo , Leveduras/metabolismo , Aminoácidos , Ácido gama-Aminobutírico/metabolismo , Oenococcus/metabolismo , FermentaçãoRESUMO
Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. KEY POINTS: ⢠Global gene transcription of K. marxianus is changed by succinic acid (SA) ⢠Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA ⢠Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance.
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Kluyveromyces , Ácido Succínico , Kluyveromyces/genética , Perfilação da Expressão Gênica , TranscriptomaRESUMO
The production of succinic acid from corn stover is a promising and sustainable route; however, during the pretreatment stage, byproducts such as organic acids, furan-based compounds, and phenolic compounds generated from corn stover inhibit the microbial fermentation process. Selecting strains that are resistant to stress and utilizing nondetoxified corn stover hydrolysate as a feedstock for succinic acid production could be effective. In this study, A. succinogenes CICC11014 was selected as the original strain, and the stress-resistant strain A. succinogenes M4 was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis and further screening. Compared to the original strain, A. succinogenes M4 exhibited a twofold increase in stress resistance and a 113% increase in succinic acid production when hydrolysate was used as the substrate. By conducting whole-genome resequencing of A. succinogenes M4 and comparing it with the original strain, four nonsynonymous gene mutations and two upstream regions with base losses were identified. KEY POINTS: ⢠A high-stress-resistant strain A. succinogenes M4 was obtained by ARTP mutation ⢠The production of succinic acid increased by 113% ⢠The mutated genes of A. succinogenes M4 were detected and analyzed.
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Actinobacillus , Zea mays , Zea mays/química , Ácido Succínico , Melhoramento Vegetal , Fermentação , MutaçãoRESUMO
BACKGROUND: There is limited evidence of succinic acid release from amber necklace that justifies its biological plausibility. AIM: This study aimed to evaluate the release of succinic acid from Baltic amber beads in the presence of Staphylococcus epidermidis. DESIGN: The Baltic amber beads from the necklace were stratified according to their weight (average 0.05 g ± 0.067). Subsequently, the beads (n = 8) were submerged in 0.9% buffered saline (Control) or brain-heart infusion culture medium in the presence of a commercial strain of S. epidermidis, a resident skin bacterium incubated at 37°C for 24 h or 7 days. The samples were centrifuged, and the supernatants were analyzed by 1H Nuclear Magnetic Resonance. Multivariate analyses were adopted using the sparse partial least squares discriminant analysis method (p < .05). RESULTS: The group incubated with saline solution showed small release of succinic acid only after 7 days. In the groups with S. epidermidis, the release of succinic acid was observed in the both presence and absence of amber beads, indicating that succinic acid is a product released by bacteria. CONCLUSIONS: It was found that amber beads do not exhibit the ability to release expressive succinic acid, especially in a short period of time, which does not justify their use in infants. The most production of succinic acid is tributed to S. epidermidis.
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Mortierella alpina is popular for lipid production, but the low carbon conversion rate and lipid yield are major obstacles for its economic performance. Here, external addition of organic acids involved in tricarboxylic acid cycle was used to tune carbon flux and improve lipid production. Citrate was determined to be the best organic acid that can be used for enhancing lipid production. By the addition of citrate, the lipid titer and content were approximately 1.24 and 1.34 times higher, respectively. Meanwhile, citrate supplement also promoted the accumulation of succinate, an important value-added platform chemical. Owing to the improved lipid and succinate production through adding citrate, the carbon conversion rate of M. alpina reached up to 52.17%, much higher than that of the control group (14.11%). The addition of citrate could redistribute carbon flux by regulating the expression level of genes related to tricarboxylic acid cycle metabolism. More carbon fluxes flow to lipid and succinate synthesis, which greatly improved the carbon conversion efficiency of M. alpina. This study provides an effective and straightforward strategy with potential economic benefits to improve carbon conversion efficiency in M. alpina.
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Carbono , Ciclo do Ácido Cítrico , Ácido Cítrico , Mortierella , Ácido Succínico , Mortierella/metabolismo , Mortierella/genética , Ácido Succínico/metabolismo , Carbono/metabolismo , Ácido Cítrico/metabolismo , Lipídeos/biossíntese , Metabolismo dos Lipídeos , Regulação Fúngica da Expressão Gênica , FermentaçãoRESUMO
Utilizing CO2 for bio-succinic acid production is an attractive approach to achieve carbon capture and recycling (CCR) with simultaneous production of a useful platform chemical. Actinobacillus succinogenes and Basfia succiniciproducens were selected and investigated as microbial catalysts. Firstly, the type and concentration of inorganic carbon concentration and glucose concentration were evaluated. 6 g C/L MgCO3 and 24 g C/L glucose were found to be the optimal basic operational conditions, with succinic acid production and carbon yield of over 30 g/L and over 40%, respectively. Then, for maximum gaseous CO2 fixation, carbonate was replaced with CO2 at different ratios. The "less carbonate more CO2" condition of the inorganic carbon source was set as carbonate: CO2 = 1:9 (based on the mass of carbon). This condition presented the highest availability of CO2 by well-balanced chemical reaction equilibrium and phase equilibrium, showing the best performance with regarding CO2 fixation (about 15 mg C/(L·hr)), with suppressed lactic acid accumulation. According to key enzymes analysis, the ratio of phosphoenolpyruvate carboxykinase to lactic dehydrogenase was enhanced at high ratios of gaseous CO2, which could promote glucose conversion through the succinic acid path. To further increase gaseous CO2 fixation and succinic acid production and selectivity, stepwise CO2 addition was evaluated. 50%-65% increase in inorganic carbon utilization was obtained coupled with 20%-30% increase in succinic acid selectivity. This was due to the promotion of the succinic acid branch of the glucose metabolism, while suppressing the pyruvate branch, along with the inhibition on the conversion from glucose to lactic acid.
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Dióxido de Carbono , Ácido Succínico , Dióxido de Carbono/metabolismo , Ácido Succínico/metabolismo , Actinobacillus/metabolismo , Glucose/metabolismoRESUMO
Plastic pollution, an increasingly serious global problem, can be addressed through the full lifecycle management of plastics, including plastics recycling as one of the most promising approaches. System design, catalyst development, and product separation are the keys in improving the economics of electrocatalytic plastics recycling. Here, a membrane-free co-production system was devised to produce succinic acid (SA) at both anode and cathode respectively by the co-electrolysis of polybutylene succinate (PBS) waste plastics and biomass-derived maleic acid (MA) for the first time. To this end, Cr3+-Ni(OH)2 electrocatalyst featuring much enhanced 1,4-butanediol (BDO) oxidation reaction (BOR) activity has been synthesized and the role of doped Cr has been revealed as an "electron puller" to accelerate the rate-determining step (RDS) in the Ni2+/Ni3+ cycling. Impressively, an extra-high SA production rate of 3.02 g h-1 and ultra-high apparent Faraday efficiency towards SA (FEapparent=181.5%) have been obtained. A carbon dioxide-assisted sequential precipitation approach has been developed to produce high-purity SA and byproduct NaHCO3 solids. Preliminary techno-economic analysis demonstrates that the reported system is economically profitable and promising for future industrial applications.
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The accumulation of cadmium (Cd) in plants is strongly impacted by soil microbes, but its mechanism remains poorly understood. Here, we report the mechanism of reduced Cd accumulation in rice by coculture of Enterobacter and Comamonas species. In pot experiments, inoculation with the coculture decreased Cd content in rice grain and increased the amount of nonbioavailable Cd in Cd-spiked soils. Fluorescence in situ hybridization and scanning electron microscopy detection showed that the coculture colonized in the rhizosphere and rice root vascular tissue and intercellular space. Soil metagenomics data showed that the coculture increased the abundance of sulfate reduction and biofilm formation genes and related bacterial species. Moreover, the coculture increased the content of organic matter, available nitrogen, and potassium and increased the activities of arylsulfatase, ß-galactosidase, phenoloxidase, arylamidase, urease, dehydrogenase, and peroxidase in soils. In subsequent rice transcriptomics assays, we found that the inoculation with coculture activated a hypersensitive response, defense-related induction, and mitogen-activated protein kinase signaling pathway in rice. Heterologous protein expression in yeast confirmed the function of four Cd-binding proteins (HIP28-1, HIP28-4, BCP2, and CID8), a Cd efflux protein (BCP1), and three Cd uptake proteins (COPT4, NRAM5, and HKT6) in rice. Succinic acid and phenylalanine were subsequently proved to inhibit rice divalent Cd [Cd(II)] uptake and activate Cd(II) efflux in rice roots. Thus, we propose a model that the coculture protects rice against Cd stress via Cd immobilization in soils and reducing Cd uptake in rice. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Comamonas , Oryza , Poluentes do Solo , Cádmio/análise , Cádmio/metabolismo , Oryza/metabolismo , Enterobacter/genética , Comamonas/metabolismo , Técnicas de Cocultura , Hibridização in Situ Fluorescente , Solo/química , Poluentes do Solo/análise , Poluentes do Solo/metabolismoRESUMO
Metabolic reprogramming plays a pivotal role in the differentiation and function of immune cells including dendritic cells (DCs). Regulatory DCs can be generated in regional tissue niches like splenic stroma and act as an important part of stromal control of immune response for the maintenance of immune tolerance. However, the metabolic alterations during splenic stroma-driven regulatory DCs differentiation and the metabolic enzyme involved in regulatory DCs function remain poorly understood. By combining metabolomic, transcriptomic, and functional investigations of mature DCs (maDCs) and diffDCs (regulatory DCs differentiated from activated mature DCs through coculturing with splenic stroma), here we identified succinate-CoA ligase subunit beta Suclg2 as a key metabolic enzyme that reprograms the proinflammatory status of mature DCs into a tolerogenic phenotype via preventing NF-κB signaling activation. diffDCs downregulate succinic acid levels and increase the Suclg2 expression along with their differentiation from mature DCs. Suclg2-interference impaired the tolerogenic function of diffDCs in inducing T cell apoptosis and enhanced activation of NF-κB signaling and expression of inflammatory genes CD40, Ccl5, and Il12b in diffDCs. Furthermore, we identified Lactb as a new positive regulator of NF-κB signaling in diffDCs whose succinylation at the lysine 288 residue was inhibited by Suclg2. Our study reveals that the metabolic enzyme Suclg2 is required to maintain the immunoregulatory function of diffDCs, adding mechanistic insights into the metabolic regulation of DC-based immunity and tolerance.
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Células Dendríticas , NF-kappa B , Diferenciação Celular , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Tolerância Imunológica , NF-kappa B/metabolismo , Transdução de Sinais , Succinato-CoA Ligases/imunologia , beta-Lactamases/imunologiaRESUMO
This study involves the isolation of succinic acid (SA)-producing microorganisms from different samples, including the rumen, sludge, soil, and wastewater. For primary screening, 29 isolates exhibited a zone of clearance around the colony, indicating acid production. For secondary screening using thin-layer chromatography, only two isolates symbolized SA production according to their Rf values. These two isolates were further identified as Bacillus velezensis and Enterococcus gallinarum by phylogenetic analysis using the neighbor-joining method. The high SA concentrations of 50.2 and 66.9 g/L were produced by B. velezensis and E. gallinarum with an SA yield of 0.836 and 1.12 g/g glucose, respectively. The high SA concentration from these newly isolated strains was achieved with a low formation of unwanted acids compared with those from Actinobacillus succinogenes ATCC 55618. Moreover, E. gallinarum was cultured in palm oil mill wastewater (POMW) and molasses, which were cheap substrates. The high SA production of 73.9 g/L with low other acids (the ratio of SA to total acids = 0.917) was achieved using POMW and molasses (80:20) as substrates.
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Ácido Succínico , Águas Residuárias , Filogenia , Fermentação , MelaçoRESUMO
Metabolic engineering has shown that optimizing metabolic pathways' fluxes for industrial purposes requires a methodical approach. Accordingly, in this study, in silico metabolic modeling was utilized to characterize the lesser-known strain Basfia succiniciproducens under different environmental conditions, followed by the use of industrially relevant substrates for succinic acid synthesis. Based on RT-qPCR carried out in flask experiments, we discovered a relatively large difference in the expression levels of ldhA gene compared to glucose in both xylose and glycerol cultures. In bioreactor-scale fermentations, the impact of different gas phases (CO2, CO2/AIR) on biomass yield, substrate consumption, and metabolite profiles was also investigated. In the case of glycerol, the addition of CO2 increased biomass as well as target product formation, while using CO2/AIR gas phase resulted in higher target product yield (0.184 mMâ mM-1). In case of xylose, using CO2 alone would result in higher succinic acid production (0.277 mMâ mM-1). The promising rumen bacteria, B. succiniciproducens, has shown to be suitable for succinic acid production from both xylose and glycerol. As a result, our findings present new opportunities for broadening the range of raw materials used in this significant biochemical process. Our study also sheds light on fermentation parameter optimization for this strain, namely that, CO2/AIR supply has a positive effect on target product formation.
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Dióxido de Carbono , Ácido Succínico , Animais , Ácido Succínico/metabolismo , Glicerol , Xilose , FermentaçãoRESUMO
This work aimed to study the efficiency of nano- and micro- fiber membranes in immobilizing Actinobacillus succinogenes CCTCC M2012036 for succinic acid production. Among the four kinds of electrospun nanofiber membranes of cellulose acetate, chitosan, poly(vinyl alcohol) (PVA) and chitosan-PVA, the cellulose acetate nanofiber membrane-immobilized cells performed the best with a succinic acid concentration and yield to be 27.3 ± 3.5 g/L and 70.9 ± 5.8%. The cell-immobilized viscose microfiber membrane presented good reuse stability, and 17 batches of fermentation without activity loss were realized with the highest succinic acid yield of 83.20%. A microfiber membrane bioreactor was further constructed with the cell-immobilized viscose microfiber membrane to perform fermentation on a larger scale, and the concentration, yield and productivity of succinic acid were 73.20 g/L, 86.50% and 1.49 g/(Lâ h) using a fed-batch strategy, which were 124.30%, 127.60% and 124.2% of those obtained in the traditional fermenter. This study provided an approach for improving the practicality of biological succinic acid production.
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Actinobacillus , Quitosana , Ácido Succínico , Reatores Biológicos , FermentaçãoRESUMO
As a common harmful pollutant, cadmium (Cd) can easily enter the human body through the food chain, posing a major threat to human health. Gut microbiota play a key role in Cd absorption. Docosahexaenoic acid (DHA) is thought to have a potential role in the treatment of Cd poisoning. This study investigated the therapeutic effect and mechanism of DHA in Cd-exposed mice from the perspective of the gut microbiota. The results showed that DHA significantly increased the Cd content in feces and decreased the Cd accumulation in the organs of mice. The gut microbiota results showed that DHA significantly restored the abundance of Parabacteroides in the gut microbiota of Cd-exposed mice. Parabacteroides distasonis (P. distasonis), a representative strain of the Parabacteroides, also showed Cd- and toxicity-reduction capabilities. P. distasonis significantly restored the gut damage caused by Cd exposure. At the same time, P. distasonis reduced the Cd content in the liver, spleen, lung, kidneys, gut, and blood to varying degrees and significantly increased the Cd content in feces. The succinic acid produced by P. distasonis plays an important role in promoting Cd excretion in Cd-exposed mice. Therefore, these results suggest that P. distasonis may have a potential role in DHA-mediated Cd excretion in Cd-exposed mice.
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Líquidos Corporais , Microbioma Gastrointestinal , Humanos , Animais , Camundongos , Cádmio/toxicidade , Ácidos Docosa-Hexaenoicos/farmacologia , FezesRESUMO
BACKGROUND: Klebsiella pneumoniae is widely distributed in water and plays a major role in both human and poultry infections. Many K. pneumoniae strains form biofilms on various surfaces, enhancing their pathogenicity and resistance to antibiotics. The water supply pipeline of chicken farms has become a hotbed for the growth of K pneumoniae biofilm because of its humid environment, and because the chicken drinking water pipeline is thin, it is easily blocked by the biofilm, and the diffused cells can cause repeated and persistent infections. Iron is vital to the growth of microorganisms and the formation of biofilms. Therefore, the aim of this study was to examine the effects of iron on K. pneumoniae biofilm formation and any associated metabolic changes to provide a rationale for reducing the formation of biofilms. RESULTS: Biofilm formation was enhanced to the greatest extent by the presence of 0.16 mM FeCl2, producing a denser structure under electron microscopy. The number of biofilm-forming and planktonic bacteria did not change, but protein and polysaccharide concentrations in the bacterial extracellular polymeric substances (EPS) were significantly increased by iron supplementation. To clarify this mechanism, intracellular metabolomic analysis was carried out, showing that the differential, down-regulated metabolites included succinic acid. The addition of 1.7 mM succinic acid counteracted the biofilm-forming effect of iron, with no bactericidal side effects. CONCLUSION: This study demonstrates the importance of succinic acid and iron in K. pneumoniae biofilms, and provides insight into the formation of K. pneumoniae biofilms and direction for the development of new antibacterial agents.
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Klebsiella pneumoniae , Ácido Succínico , Antibacterianos/farmacologia , Biofilmes , Ferro/farmacologia , Ácido Succínico/farmacologiaRESUMO
INTRODUCTION: Glycerol is a byproduct from the biodiesel industry that can be biotransformed by Escherichia coli to high added-value products such as succinate under aerobic conditions. The main genetic engineering strategies to achieve this aim involve the mutation of succinate dehydrogenase (sdhA) gene and also those responsible for acetate synthesis including acetate kinase, phosphate acetyl transferase and pyruvate oxidase encoded by ackA, pta and pox genes respectively in the ΔsdhAΔack-ptaΔpox (M4) mutant. Other genetic manipulations to rewire the metabolism toward succinate consist on the activation of the glyoxylate shunt or blockage the pentose phosphate pathway (PPP) by deletion of isocitrate lyase repressor (iclR) or gluconate dehydrogenase (gnd) genes on M4-ΔiclR and M4-Δgnd mutants respectively. OBJECTIVE: To deeply understand the effect of the blocking of the pentose phosphate pathway (PPP) or the activation of the glyoxylate shunt, metabolite profiles were analyzed on M4-Δgnd, M4-ΔiclR and M4 mutants. METHODS: Metabolomics was performed by FT-IR and GC-MS for metabolite fingerprinting and HPLC for quantification of succinate and glycerol. RESULTS: Most of the 65 identified metabolites showed lower relative levels in the M4-ΔiclR and M4-Δgnd mutants than those of the M4. However, fructose 1,6-biphosphate, trehalose, isovaleric acid and mannitol relative concentrations were increased in M4-ΔiclR and M4-Δgnd mutants. To further improve succinate production, the synthesis of mannitol was suppressed by deletion of mannitol dehydrogenase (mtlD) on M4-ΔgndΔmtlD mutant that increase ~ 20% respect to M4-Δgnd. CONCLUSION: Metabolomics can serve as a holistic tool to identify bottlenecks in metabolic pathways by a non-rational design. Genetic manipulation to release these restrictions could increase the production of succinate.
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Escherichia coli , Ácido Succínico , Escherichia coli/genética , Escherichia coli/metabolismo , Glicerol/metabolismo , Glioxilatos/metabolismo , Manitol/metabolismo , Engenharia Metabólica , Metabolômica , Espectroscopia de Infravermelho com Transformada de Fourier , Ácido Succínico/metabolismoRESUMO
BACKGROUND: Polyamide (nylon) is an important material, which has aroused plenty of attention from all aspects. PA 5.4 is one kind of nylon with excellent property, which consists of cadaverine and succinic acid. Due to the environmental pollution, bio-production of cadaverine and succinic acid has been more attractive due to the less pollution and environmental friendliness. Microbes, like Escherichia coli, has been employed as cell factory to produce cadaverine and succinic acid. However, the accumulation of cadaverine will cause severe damage on cells resulting in inhibition on cell growth and cadaverine production. Herein, a novel two stage co-production of succinic acid and cadaverine was designed based on an efficient thermos-regulated switch to avoid the inhibitory brought by cadaverine. RESULTS: The fermentation process was divided into two phase, one for cell growth and lysine production and the other for cadaverine and succinic acid synthesis. The genes of ldhA and ackA were deleted to construct succinic acid pathway in cadaverine producer strain. Then, a thermal switch system based on pR/pL promoter and CI857 was established and optimized. The fermentation conditions were investigated that the optimal temperature for the first stage was determined as 33 â and the optimal temperature for the second stage was 39 â. Additionally, the time to shifting temperature was identified as the fermentation anaphase. For further enhance cadaverine and succinic acid production, a scale-up fermentation in 5 L bioreactor was operated. As a result, the titer, yield and productivity of cadaverine was 55.58 g/L, 0.38 g/g glucose and 1.74 g/(L·h), respectively. 28.39 g/L of succinic acid was also obtained with yield of 0.19 g/g glucose. CONCLUSION: The succinic acid metabolic pathway was constructed into cadaverine producer strain to realize the co-production of succinic acid and cadaverine. This study provided a novel craft for industrial co-production of cadaverine and succinic acid.
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Escherichia coli , Ácido Succínico , Cadaverina , Escherichia coli/genética , Nylons , GlucoseRESUMO
BACKGROUND: The microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO2). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO2-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.23 Cmol Cmol-1. The results of that previous study suggested that the glyoxylate cycle considerably contributed to SA accumulation in the respective strain. The current study aimed at improving the flux into the rTCA pathway accompanied by a higher CO2-fixation and SA yield. RESULTS: By changing the design of the expression cassettes for the rTCA pathway, overexpressing PYC2, and adding CaCO3 to the batch fermentations, an SA yield on glycerol of 0.63 Cmol Cmol-1 was achieved (i.e. 47.1% of the theoretical maximum). The modifications in this 2nd-generation SA producer improved the maximum biomass-specific glycerol consumption rate by a factor of nearly four compared to the isogenic baseline strain solely equipped with the dihydroxyacetone (DHA) pathway for glycerol catabolism. The data also suggest that the glyoxylate cycle did not contribute to the SA production in the new strain. Cultivation conditions which directly or indirectly increased the concentration of bicarbonate, led to an accumulation of malate in addition to the predominant product SA (ca. 0.1 Cmol Cmol-1 at the time point when SA yield was highest). Off-gas analysis in controlled bioreactors with CO2-enriched gas-phase indicated that CO2 was fixed during the SA production phase. CONCLUSIONS: The data strongly suggest that a major part of dicarboxylic acids in our 2nd-generation SA-producer was formed via the rTCA pathway enabling a net fixation of CO2. The greatly increased capacity of the rTCA pathway obviously allowed successful competition with other pathways for the common precursor pyruvate. The overexpression of PYC2 and the increased availability of bicarbonate, the co-substrate for the PYC reaction, further strengthened this capacity. The achievements are encouraging to invest in future efforts establishing a process for SA production from (crude) glycerol and CO2.
Assuntos
Saccharomyces cerevisiae , Ácido Succínico , Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Meios de Cultura/metabolismo , Glicerol/metabolismo , Glioxilatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/metabolismoRESUMO
BACKGROUND/AIMS: Although the adriamycin-induced nephropathy model is frequently employed in the study of nephrotic syndrome and focal segmental glomerulosclerosis, the accompanying myocardial damage has always been a cause for concern. Therefore, there is a great need to study cardiorenal communication in this model. METHODS: An adriamycin-induced nephropathy model was established via tail vein injection. The levels of the biochemical indicators serum albumin, serum globulin, serum total protein, serum cholesterol, serum creatinine (SCr), urinary protein, and urinary creatinine (UCr) were measured, and histopathological changes in the heart and kidneys were assessed using hematoxylin-eosin staining. Metabolomic changes in the heart, blood, and kidneys were analyzed using the metabolomics method based on ultra-performance liquid chromatography Q-Exactive Orbitrap mass spectrometry. RESULTS: Compared with the control group, the model group showed significant decreases in serum protein and total protein levels, albumin/globulin ratio, and creatinine clearance rate as well as significant increases in serum cholesterol, SCr, urinary protein, and UCr levels. Significant pathological changes were observed in the renal pathology sections in the model group, including diffusely merged glomerular epithelial cells, inflammatory infiltration, and vacuolated glomerular cells. Additionally, thickened myocardial fibers, swollen nuclei, inflammatory infiltration, and partial myocardial necrosis could be seen in the cardiac pathology sections in the model group. Based on multivariate statistical analysis, a total of 20 differential metabolites associated with 15 metabolic pathways were identified in the heart, 7 differential metabolites with 7 metabolic pathways were identified in the blood, and 16 differential metabolites with 21 metabolic pathways were identified in the kidney. Moreover, 6 common metabolic pathways shared by the heart and kidney were identified: arginine and proline metabolism; arginine biosynthesis; glutathione metabolism; alanine, aspartate, and glutamate metabolism; beta-alanine metabolism; and histidine metabolism. Among these metabolic pathways, alanine, aspartate, and glutamate metabolism was shared by the heart, blood, and kidney. Succinic acid was found to be the key regulatory metabolite in cardiorenal metabolic communication. CONCLUSION: Six metabolic pathways were found to be involved in cardiorenal metabolic communication in an adriamycin-induced nephropathy model, in which alanine, aspartate, and glutamate metabolism may be the metabolic link between the heart and kidney in the development and maintenance of oxidative stress and inflammation. Succinic acid may serve as a key regulatory metabolic switch or marker of cardiac and renal co-injury, as shown in an adriamycin-induced nephropathy model.