Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro-aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild-type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L-1) corresponded to the lower kLa value of 5 h-1. Above 5 h-1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L-1. Taking into account this threshold, a fed-batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild-type strain was able to produce 93.6 g L-1 of succinate, which is one of the highest concentration reported in the literature.

Succinic acid, extracted from amber, is widely used in cardiovascular therapy.

Enterobacter sp. LU1 could efficiently convert glycerol to succinic acid under anaerobic conditions after the addition of lactose. In this study, media constituents affecting both Enterobacter sp. LU1 biomass and succinic acid production were investigated employing response surface methodology (RSM) with central composite design. Statistical methods led to the development of an efficient and inexpensive microbiological media based on crude glycerol, whey permeate as carbon sources and urea as a nitrogen source. The optimized production of bacterial biomass in aerobic conditions was predicted and the interactive effects between crude glycerol, urea and magnesium sulfate were investigated. As a result, a model for predicting the concentration of bacterial biocatalyst biomass was developed with crude glycerol as a sole carbon source. In addition, it was observed that the interactive effect between crude glycerol and urea was statistically significant. Response surface methodology was also employed to develop the model for predicting the concentration of succinic acid produced. Validity of the model was confirmed during verification experiments wherein actual results differed from predicted values by 0.77%. The applied statistical methods proved the feasibility for anaerobic succinic acid production on crude glycerol without expensive yeast extract addition. In conclusion, the RSM method can provide valuable information for succinic acid scale-up fermentation using Enterobacter sp. LU1.

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 μM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L-1 of SA with the maximum productivity of 21.3 g L-1 h-1, demonstrating the importance of enzyme optimization in strain development.

The present study hypothesized that fumaric acid and succinic acid may exhibit therapeutic effects on gestational hypertension. During pregnancy, estrogen upregulates ten-eleven translocation 1 (TET1) expression, which subsequently increases calcium-activated potassium channel subunit β1 (KCNMB1) expression. KCNMB1 is associated with hypertension. Fumaric acid and succinic acid are understood to inhibit TET. Therefore, the present study investigated whether fumaric acid and succinic acid exhibit therapeutic effects on gestational hypertension and whether these effects are mediated by TET1 and KCNMB1. Nω-Nitro-L-arginine methyl ester hydrochloride was injected into rats to establish a gestational hypertension model. Dimethyl fumarate (DMF) and succinic acid were administrated into rats to treat gestational hypertension. Rats were divided into five groups: i) Control; ii) model; iii) DMF; iv) succinic acid; and v) DMF + succinic acid. Blood pressure was monitored by a noninvasive meter and urinary protein was determined using a urinary protein kit. Placenta pathology was examined by hematoxylin-eosin staining. Compared with the control group, urinary protein and blood pressure in the model group increased significantly. The placental cells in the control group were arranged orderly. However, in the model group, decidual cellular edema of placenta and vacuolar degeneration were observed, and the intervascular membrane was markedly thicker with plenty of fibrin deposition. These results indicate successful establishment of a gestational hypertension model. However, compared with the model group, urinary protein, blood pressure, edema, vacuoles and fibrin deposition were markedly reduced in the DMF, succinic acid and DMF + succinic acid groups. mRNA and protein levels of TET1 and KCNMB1 in placenta were evaluated by immunohistochemical analysis, reverse transcription-quantitative polymerase chain reaction and western blotting. The TET1 and KCNMB1 levels in the model group were markedly increased compared with those in the control group. However, compared with the model group, the expression levels were markedly downregulated in the DMF, succinic acid and DMF + succinic acid groups. In conclusion, fumaric acid and succinic acid may treat gestational hypertension by downregulating the expression of KCNMB1 and TET1.

Microbial carbon dioxide assimilation and conversion to chemical platform molecules has the potential to be developed as economic, sustainable processes. The carbon dioxide assimilation can proceed by a variety of natural pathways and recently even synthetic CO2 fixation routes have been designed. Early assessment of the performance of the different carbon fixation alternatives within biotechnological processes is desirable to evaluate their potential. Here we applied stoichiometric metabolic modeling based on physiological and process data to evaluate different process variants for the conversion of C1 carbon compounds to the industrial relevant platform chemical succinic acid. We computationally analyzed the performance of cyanobacteria, acetogens, methylotrophs, and synthetic CO2 fixation pathways in Saccharomyces cerevisiae in terms of production rates, product yields, and the optimization potential. This analysis provided insight into the economic feasibility and allowed to estimate the future industrial applicability by estimating overall production costs. With reported, or estimated data of engineered or wild type strains, none of the simulated microbial succinate production processes showed a performance allowing competitive production. The main limiting factors were identified as gas and photon transfer and metabolic activities whereas metabolic network structure was not restricting. In simulations with optimized parameters most process alternatives reached economically interesting values, hence, represent promising alternatives to sugar-based fermentations.

Succinic acid is one of the most interesting platform chemicals that can be produced in a biorefinery approach. In this study, continuous succinic acid production by Actinobacillus succinogenes fermentation in a packed-bed biofilm reactor (PBBR) was investigated.

Renewable chemicals have attracted attention due to increasing interest in environmental concerns and resource utilization. Biobased production of industrial compounds from nonfood biomass has become increasingly important as a sustainable replacement for traditional petroleum-based production processes depending on fossil resources. Therefore, we engineered an Enterobacter cloacae budC and ldhA double-deletion strain (namely, EC∆budC∆ldhA) to redirect carbon fluxes and optimized the culture conditions to co-produce succinic acid and acetoin.

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 mM⋅mM-1). In case of xylose, using CO2 alone would result in higher succinic acid production (0.277 mM⋅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.

Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the α-keto-glutarate dehydrogenase catalyzed conversion of α-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2(nd)-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we demonstrate how systems biology tools coupled with directed evolution and selection allows non-intuitive, rapid and substantial re-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals.

The metabolic engineering of Saccharomyces cerevisiae for the production of succinic acid has progressed dramatically, and a series of high-producing hosts are available. At low cultivation pH and high titers, the product transport can become bidirectional, i.e. the acid is reentering the cell and is again exported or even catabolized. Here, a quantitative approach for the identification of product recycling fluxes is developed.

Previously, our lab replaced the endogenous FAD-dependent pathway for glycerol catabolism in S. cerevisiae by the synthetic NAD-dependent dihydroxyacetone (DHA) pathway. The respective modifications allow the full exploitation of glycerol's higher reducing power (compared to sugars) for the production of the platform chemical succinic acid (SA) via a reductive, carbon dioxide fixing and redox-neutral pathway in a production host robust for organic acid production. Expression cassettes for three enzymes converting oxaloacetate to SA in the cytosol ("SA module") were integrated into the genome of UBR2 CBS-DHA, an optimized CEN.PK derivative. Together with the additional expression of the heterologous dicarboxylic acid transporter DCT-02 from Aspergillus niger, a maximum SA titer of 10.7 g/L and a yield of 0.22 ± 0.01 g/g glycerol was achieved in shake flask (batch) cultures. Characterization of the constructed strain under controlled conditions in a bioreactor supplying additional carbon dioxide revealed that the carbon balance was closed to 96%. Interestingly, the results of the current study indicate that the artificial "SA module" and endogenous pathways contribute to the SA production in a highly synergistic manner.

Oleaginous yeast Yarrowia lipolytica is a prospective host for production of succinic acid. The interruption of tricarboxylic acid cycle through succinate dehydrogenase gene (SDH) deletion was reported to result in strains incapable of glucose utilization and this ability had to be restored by chemical mutation or long adaptive laboratory evolution. In this study, a succinate producing strain of Y. lipolytica was engineered by truncating the promoter of SDH1 gene, which resulted in 77% reduction in SDH activity but did not impair the ability of the strain to grow on glucose. The flux toward succinic acid was further improved by overexpressing the genes in the glyoxylate pathway and the oxidative TCA branch, and expressing phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes. A short adaptation on glucose reduced the lag phase of the strain and increased its tolerance to high glucose concentrations. The resulting strain produced 7.8 ± 0.0 g/L succinic acid with a yield of 0.105 g/g glucose in shake flasks without pH control, while mannitol (11.8 ± 0.8 g/L) was the main by-product. Further investigations showed that mannitol accumulation was caused by low pH stress and buffering the fermentation medium eliminated mannitol formation. In a fed-batch bioreactor in mineral medium at pH 5, at which point according to Ka values of succinic acid, the major fraction of product was in acidic form rather than dissociated form, the strain produced 35.3 ± 1.5 g/L succinic acid with 0.26 ± 0.00 g/g glucose yield.

Immune checkpoint blockade therapy with anti-PD-1 monoclonal antibody (mAb) is a treatment for colorectal cancer (CRC). However, some patients remain unresponsive to PD-1 blockade. The gut microbiota has been linked to immunotherapy resistance through unclear mechanisms. We found that patients with metastatic CRC who fail to respond to immunotherapy had a greater abundance of Fusobacterium nucleatum and increased succinic acid. Fecal microbiota transfer from responders with low F. nucleatum, but not F. nucleatum-high non-responders, conferred sensitivity to anti-PD-1 mAb in mice. Mechanistically, F. nucleatum-derived succinic acid suppressed the cGAS-interferon-β pathway, consequently dampening the antitumor response by limiting CD8+ T cell trafficking to the tumor microenvironment (TME) in vivo. Treatment with the antibiotic metronidazole reduced intestinal F. nucleatum abundance, thereby decreasing serum succinic acid levels and resensitizing tumors to immunotherapy in vivo. These findings indicate that F. nucleatum and succinic acid induce tumor resistance to immunotherapy, offering insights into microbiota-metabolite-immune crosstalk in CRC.

To improve the fermentation efficiency of Propionibacterium acidipropionici, a semi-continuous coupled fermentation process was established to achieve co-production of propionic acid (PA) and succinic acid (SA). First, the optimal proportion of glucose (Glc) and glycerol (Gl) as a mixed carbon source was determined, and the feeding procedure of Gl was optimized to make more energy flow in the direction of product synthesis. Then, ZGD630 anion exchange resin was used for efficient adsorption of PA, thereby eliminating the feedback inhibition effect of PA. Finally, an efficient, coupled fermentation process of P. acidipropionici characterized by membrane separation and chromatography technology was developed. The concentrations of PA and SA reached 62.22 ± 2.32 and 20.45 ± 1.34 g L-1, with corresponding productivity of 0.43 and 0.14 g L-1 h-1, increased by 65.38% and 48.54%, respectively. Membrane separation coupled fermentation of PA and SA could significantly improve the process economics of P. acidipropionici, and has good prospects for industrial application.

Xylose is the most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast that produces industrially important metabolites. The yeast is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economica feasibility of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory.

Bacterial vaginosis (BV), a common condition in women, is associated with increased shedding of HIV in the female genital tract. While the Lactobacillus species that comprise a healthy vaginal microbiota produce lactic acid, the bacteria common in BV produce high concentrations of short chain fatty acids (SCFAs) and succinic acid. Macrophages are abundant in the lower genital tract mucosa and are thought to play an important role in HIV infection. In this study, we investigated whether SCFAs and succinic acid impacted HIV expression in monocyte-derived macrophages. Monocytes differentiated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) were infected with either HIVBal or an HIV-luciferase reporter virus and treated with SCFAs, succinic acid, or lactic acid. Butyric acid suppressed HIV expression while succinic acid significantly increased expression in macrophages differentiated with either GM-CSF or M-CSF. Acetic, propionic, and lactic acids had no effect on HIV expression. Only succinic acid resulted in a significant increase in interleukin-8 production by infected macrophages. Our results suggest that succinic acid present in increased concentrations in the genital tract of women with BV plays a pro-inflammatory role and increases HIV expression. This could be one factor contributing to increased virus shedding seen in women with BV.

Copolymerization of xylitol usually yields cross-linked materials. In this work, microwave-assisted polyesterification of xylitol and succinic acid produced materials with diverse molecular weights and different branching degrees, and more importantly, no cross-linking was observed, as supported by the solubility behavior and spectroscopic data. Reactions were carried out for short times, less than 20 min, which is not common for production of industrial polyesters. Control over the branching degree was achieved by tuning the reaction conditions, such as temperature, time of exposure, and monomer ratio, during microwave irradiation. No solvent or catalyst was employed during the step-growth polymerization.

This study focuses on succinic acid production by Actinobacillus succinogenes in batch fermentation from whey and lactose widely encountered in dairy effluents. The effects of initial whey and lactose concentration, CO2 rate on succinic acid production were investigated. The optimal succinic acid production was obtained with 25 g L-1 of lactose and 35 g L-1 of whey with yields and productivities respectively of 65% and 0.9 g L-1 h-1 for lactose and 62.1%, 0.81 g L-1 h-1 for whey. The maximum yield and productivity of succinic acid was obtained with lactose in comparison with whey. Productivity and yield decreased when the amount of initial lactose was increased. Biomass, acetic acid and formic acid increased when whey was used as a substrate compared to lactose. Succinic acid production by anaerobic fermentation is a green biotechnology alternative to valorize whey and lactose from dairy effluent and to reduce their impact on the environment.

Baltic amber teething necklaces have been popularized as a safe and natural alternative to conventional or pharmacological medicines for the management of teething pain. However, claims made by retailers regarding the efficacy and mechanism of action of these necklaces lack scientific or clinical basis. The claim most closely resembling science is the assertion that succinic acid will leach out of the beads and through the skin of the wearer and carry out anti-inflammatory and analgesic effects. The objective of the current research is to scientifically assess this claim.