Upcycling CO2 into succinic acid via electrochemical and engineered Escherichia coli.

Converting CO2 into value-added chemicals still remains a grand challenge. Succinic acid has long been considered as one of the top building block chemicals. This study reported efficiently upcycling CO2 into succinic acid by combining between electrochemical and engineered Escherichia coli. In this process, the Cu-organic framework catalyst was synthesized for electrocatalytic CO2-to-ethanol conversion with high Faradaic efficiency (FE, 84.7 %) and relative purity (RP, 95 wt%). Subsequently, an engineered E. coli with efficiently assimilating CO2-derived ethanol to produce succinic acid was constructed by combining computational design and metabolic engineering, and the succinic acid titer reached 53.8 mM with the yield of 0.41 mol/mol, which is 82 % of the theoretical yield. This study effort to link the two processes of efficient ethanol synthesis by electrocatalytic CO2 and succinic acid production from CO2-derived ethanol, paving a way for the production of succinic acid and other value-added chemicals by converting CO2 into ethanol.

Multi-objectives optimization on microwave-assisted-biological-based biogas upgrading and bio-succinic acid production.

This study represents the first investigation of bio-succinic acid (bio-SA) production with methane enrichment using carbon-dioxide-fixating bacteria in the co-culture of ragi tapai and macroalgae, Chaetomorpha. Microwave irradiation has also been introduced to enhance the biochemical processes as it could provide rapid and selective heating of substrates. In this research, microwave irradiation was applied on ragi tapai as a pre-treatment process. Factors such as microwave irradiation dose on ragi tapai, Chaetomorpha ratio in the co-culture, and pH value were studied. Optimal conditions were identified using Design-Expert software, resulting in optimal experimental biomethane and bio-SA production of 85.7 % and 0.65 g/L, respectively, at a microwave dose of 1.45 W/g, Chaetomorpha ratio of 0.9 and pH value of 7.8. The study provides valuable insights into microwave control for promoting simultaneous methane enrichment and bio-SA production, potentially reducing costs associated with CO2 capture and storage and biogas upgrading.

Chemical composition analysis and transcriptomics reveal the R2R3-MYB genes and phenol oxidases regulating the melanin formation in black radish.

Melanins are dark-brown to black-colored biomacromolecules which have been thoroughly studied in animals and microorganisms. However, the biochemical and molecular basis of plant melanins are poorly understood. We first characterized melanin from the black radish (Raphanus sativus var. niger) 'HLB' through spectroscopic techniques. p-Coumaric acid was identified as the main precursor of radish melanin. Moreover, a joint analysis of transcriptome and coexpression network was performed for the two radish accessions with black and white cortexes, 'HLB' and '55'. A set of R2R3-type RsMYBs and enzyme-coding genes exhibited a coexpression pattern, and were strongly correlated with melanin formation in radish. Transient overexpression of two phenol oxidases RsLAC7 (laccase 7) or RsPOD22-1 (peroxidase 22-1) resulted in a deeper brown color around the infiltration sites and a significant increase in the total phenol content. Furthermore, co-injection of the transcriptional activator RsMYB48/RsMYB97 with RsLAC7 and/or RsPOD22-1, markedly increased the yield of black extracts. Spectroscopic analyses revealed that these extracts are similar to the melanin found in 'HLB'. Our findings advance the understanding of structural information and the transcriptional regulatory mechanism underlying melanin formation in radish.

Production of 1,4-Butanediol from Succinic Acid Using Escherichia Coli Whole-Cell Catalysis.

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 48 hours, 1,4-BDO titers reached 201 mg/L and 1555 mg/L in shake flask and 5 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.

Recent advances in bio-based production of top platform chemical, succinic acid: an alternative to conventional chemistry.

Succinic acid (SA) is one of the top platform chemicals with huge applications in diverse sectors. The presence of two carboxylic acid groups on the terminal carbon atoms makes SA a highly functional molecule that can be derivatized into a wide range of products. The biological route for SA production is a cleaner, greener, and promising technological option with huge potential to sequester the potent greenhouse gas, carbon dioxide. The recycling of renewable carbon of biomass (an indirect form of CO2), along with fixing CO2 in the form of SA, offers a carbon-negative SA manufacturing route to reduce atmospheric CO2 load. These attractive attributes compel a paradigm shift from fossil-based to microbial SA manufacturing, as evidenced by several commercial-scale bio-SA production in the last decade. The current review article scrutinizes the existing knowledge and covers SA production by the most efficient SA producers, including several bacteria and yeast strains. The review starts with the biochemistry of the major pathways accumulating SA as an end product. It discusses the SA production from a variety of pure and crude renewable sources by native as well as engineered strains with details of pathway/metabolic, evolutionary, and process engineering approaches for enhancing TYP (titer, yield, and productivity) metrics. The review is then extended to recent progress on separation technologies to recover SA from fermentation broth. Thereafter, SA derivatization opportunities via chemo-catalysis are discussed for various high-value products, which are only a few steps away. The last two sections are devoted to the current scenario of industrial production of bio-SA and associated challenges, along with the author's perspective.

Response mechanisms of different Saccharomyces cerevisiae strains to succinic acid.

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.

Enhanced direct gaseous CO2 fixation into higher bio-succinic acid production and selectivity.

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.

Targeted and untargeted metabolomic profiles in wild rabbit does (Oryctolagus cuniculus) of different breeding states (pregnant and lactating).

Ecological nutrition aims to unravel the extensive web of nutritional links that drives animals in their interactions with their ecological environments. Nutrition plays a key role in the success of European wild rabbit (Oryctolagus cuniculus) and could be affected by the breeding status of the animals and reflected in the metabolome of this species. As nutritional needs are considerably increased during pregnancy and lactation, the main objective of this work was to determine how the breeding status (pregnant and lactating) of European wild rabbit does affects nutritional requirements and their metabolome (using targeted and untargeted metabolomics), aiming to find a useful biomarker of breeding status and for monitoring nutritional requirements. To address this gap, 60 wild European rabbits were studied. Animals were divided according to their breeding status and only pregnant (n = 18) and lactating (n = 11) rabbit does were used (n = 29 in total). The body weight and length of each animal were analyzed. The relative and absolute chemical composition of the gastric content and whole blood sample were taken, and targeted and untargeted metabolomics were analyzed. As a main result, there were no differences in biometric measurements, gastric content, and targeted metabolomics, except for live weight and nonesterified fatty acids (NEFA), as pregnant animals showed higher live weight (+12%; p = 0.0234) and lower NEFA acid levels (-46%; p = 0.0262) than lactating females. Regarding untargeted metabolomics, a good differentiation of the metabolome of the two breeding groups was confirmed, and it was proven that pregnant animals showed higher plasmatic levels of succinic anhydride (3.48 more times; p = 0.0236), succinic acid (succinate) (3.1 more times; p = 0.0068) and propionic acid (3.98 more times; p = 0.0121) than lactating animals. However, lactating animals showed higher levels of N-[(3a,5b,7b)-7-hydroxy-24-oxo-3-(sulfoxide) cholan-24-yl]-Glycine (cholestadien) (2.4 more times; p < 0.0420), 4-maleyl-acetoacetate (MAA) (3.2 more times; p < 0.0364) and irilone (2.2 more times; p = 0.0451) than pregnant animals, any of these metabolites could be used as a potential biomarker. From these results, it can be concluded that the most notable changes were observed in the metabolome of individuals, with most of the changes observed being due to energy and protein mobilisation.

Effect of adaptive laboratory evolution of engineered Escherichia coli in acetate on the biosynthesis of succinic acid from glucose in two-stage cultivation.

Escherichia coli MLB (MG1655 ΔpflB ΔldhA), which can hardly grow on glucose with little succinate accumulation under anaerobic conditions. Two-stage fermentation is a fermentation in which the first stage is used for cell growth and the second stage is used for product production. The ability of glucose consumption and succinate production of MLB under anaerobic conditions can be improved significantly by using acetate as the solo carbon source under aerobic condition during the two-stage fermentation. Then, the adaptive laboratory evolution (ALE) of growing on acetate was applied here. We assumed that the activities of succinate production related enzymes might be further improved in this study. E. coli MLB46-05 evolved from MLB and it had an improved growth phenotype on acetate. Interestingly, in MLB46-05, the yield and tolerance of succinic acid in the anaerobic condition of two-stage fermentation were improved significantly. According to transcriptome analysis, upregulation of the glyoxylate cycle and the activity of stress regulatory factors are the possible reasons for the elevated yield. And the increased tolerance to acetate made it more tolerant to high concentrations of glucose and succinate. Finally, strain MLB46-05 produced 111 g/L of succinic acid with a product yield of 0.74 g/g glucose. SYNOPSIS.

Advances in succinic acid production: the enhancement of CO2 fixation for the carbon sequestration benefits.

Succinic acid (SA), one of the 12 top platform chemicals produced from biomass, is a precursor of various high value-added derivatives. Specially, 1 mol CO2 is assimilated in 1 mol SA biosynthetic route under anaerobic conditions, which helps to achieve carbon reduction goals. In this review, methods for enhanced CO2 fixation in SA production and utilization of waste biomass for SA production are reviewed. Bioelectrochemical and bioreactor coupling systems constructed with off-gas reutilization to capture CO2 more efficiently were highlighted. In addition, the techno-economic analysis and carbon sequestration benefits for the synthesis of bio-based SA from CO2 and waste biomass are analyzed. Finally, a droplet microfluidics-based high-throughput screening technique applied to the future bioproduction of SA is proposed as a promising approach.

Transcriptome analysis of Kluyveromyces marxianus under succinic acid stress and development of robust strains.

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.

Mutation breeding of high-stress resistant strains for succinic acid production from corn straw.

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.

Succinic Acid Ameliorates Concanavalin A-Induced Hepatitis by Altering the Inflammatory Microenvironment and Expression of BCL-2 Family Proteins.

Autoimmune hepatitis (AIH) is a severe immune-mediated inflammatory liver disease that currently lacks feasible drug treatment methods. Our study aimed to evaluate the protective effect of succinic acid against AIH and provide a reliable method for the clinical treatment of AIH. We performed an in vivo study of the effects of succinic acid on concanavalin A (ConA)-induced liver injury in mice. We examined liver transaminase levels, performed hematoxylin and eosin (HE) staining, and observed apoptotic phenotypes in mice. We performed flow cytometry to detect changes in the number of neutrophils and monocytes, and used liposomes to eliminate the liver Kupffer cells and evaluate their role. We performed bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blotting to detect mitochondrial apoptosis-induced changes in proteins from the B-cell lymphoma 2(Bcl-2) family. Succinic acid ameliorated ConA-induced AIH in a concentration-dependent manner, as reflected in the survival curve. HE and TUNEL staining and terminal deoxynucleotidyl transferase dUTP nick end labeling revealed decreased alanine transaminase and aspartate aminotransferase levels, and reduced liver inflammation and apoptosis. RT-qPCR and enzyme-linked immunosorbent assay revealed that succinic acid significantly reduced liver pro-inflammatory cytokine levels. Flow cytometry revealed significantly decreased levels of liver neutrophils. Moreover, the protective effect of succinic acid disappeared after the Kupffer cells were eliminated, confirming their important role in the effect. Bioinformatics analysis, RT-qPCR, and western blotting showed that succinic acid-induced changes in proteins from the Bcl-2 family involved mitochondrial apoptosis, indicating the molecular mechanism underlying the protective effect of succinic acid. Succinic acid ameliorated ConA-induced liver injury by regulating immune balance, inhibiting pro-inflammatory factors, and promoting anti-apoptotic proteins in the liver. This study provides novel insights into the biological functions and therapeutic potential of succinic acid in the treatment of autoimmune liver injury.

[Possibilities of «therapeutic retargeting¼ of 3-hydroxypyridine and succinic acid derivatives due to their dopaminergic action]. / Vozmozhnosti «terapevticheskogo perenatselivaniya¼ proizvodnykh 3-oksipiridina i yantarnoi kisloty, obuslovlennye ikh dofaminergicheskim deistviem.

The review is devoted to a comparative analysis of the clinical efficacy of the original domestic derivatives of 3-hydroxypyridine and succinic acid (emoxipine, reamberin and mexidol) in comparison with the results of an experimental study of their dopaminergic action. The position that the dopaminomimetic activity of emoxipin, reamberin and mexidol largely determines their anti-ischemic, antihypoxic, insulin-potentiating neuroprotective, nootropic and antidepressant potential has been substantiated. A comparative analysis of the safety profile of emoxipine, reamberin and mexidol was carried out, taking into account potential and real side-effects caused by iatrogenic deviations from the eudopaminergic state. It has been shown that mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate), which is simultaneously a derivative of 3-hydroxypyridine and succinic acid, has the best balance of efficacy and safety. A generalized assessment of the available data on the successful use of off-label derivatives of 3-hydroxypyridine and succinic acid indicates the advisability of a significant expansion of indications for their clinical use. The authors resume that the «therapeutic retargeting¼ of emoxipin, reamberin and mexidol (i.e. their use for qualitatively new indications) will contribute to progress in the treatment of socially significant and most common diseases.

Synthesis of xylan and lignin based hydrogels and their use as growth media for rice (Oryza sativa).

Different hydrogels were synthesised using lignin and xylan (extracted from rice straw) as the raw material, PVA as matrix template, citric acid, and succinic acid as crosslinkers and were structurally characterised by FT-IR and SEM analysis. Citric acid cross-linked hydrogels caused loose bonding between polymers and resulted in formation of large pores whereas succinic acid cross-linked hydrogels were observed to have less porosity and tight bonding network. The swelling ratio and reswelling capacity of hydrogels revealed that lignin hydrogels outperformed all other hydrogels. All the synthesised hydrogels were tested for their use as soil culture media ingredient for the germination of rice seedlings. All the hydrogels performed well in stress conditions as compared to normal conditions. Xylan/lignin hydrogel and lignin hydrogels performed best under stress conditions. These hydrogels could be used effectively as an ingredient of soil culture media owing to their tendency to absorb and retain water properly.

Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membrane.

Mucins are often stained with the basic dye Alcian blue, but mucins with a low acidic glycan content cannot be stained with it. Succinylation-Alcian blue staining is a method that temporarily modifies glycans with succinic acid to visualize mucins with low acidic glycan content. This method can be used to stain mucins on polyvinylidene difluoride (PVDF) membranes separated via supported molecular matrix electrophoresis (SMME) and mucins blotted onto PVDF membranes from gel electrophoreses. The succinyl groups of the modified glycans can be easily and completely removed by releasing O-glycan from the stained mucin bands. Therefore, the glycans can be analyzed using the same methods as those used for mucins with a high acidic glycan content.

A comparative study of Lachancea thermotolerans fermentative performance under standardized wine production conditions.

The study explores diverse strains of Lachancea thermotolerans in single-inoculum wine fermentation conditions using synthetic grape must. It aims to analyze the role of the species without external influences like other microorganisms or natural grape must variability. Commercial strains and selected vineyard isolates, untested together previously, are assessed. The research evaluates volatile and non-volatile chemical compounds in final wine, revealing significant strain-based variations. L. thermotolerans notably produces lactic acid and consumes malic acid, exhibiting moderate ethanol levels. The volatile profile displays strain-specific impacts, affecting higher alcohol and ester concentrations compared to S. cerevisiae. These effects vary based on the specific compounds. Using a uniform synthetic must enables direct strain comparisons, eliminating grape-related, environmental, or timing variables in the experiment, facilitating clearer insights into the behavior of L. thermotolerans in wine fermentation. The study compares for the first time all available commercial strains of L. thermotolerans.

Antagonistic properties of Lactiplantibacillus plantarum MYSVB1 against Alternaria alternata: a putative probiotic strain isolated from the banyan tree fruit.

Alternaria alternata, a notorious phytopathogenic fungus, has been documented to infect several plant species, leading to the loss of agricultural commodities and resulting in significant economic losses. Lactic acid bacteria (LAB) hold immense promise as biocontrol candidates. However, the potential of LABs derived from fruits remains largely unexplored. In this study, several LABs were isolated from tropical fruit and assessed for their probiotic and antifungal properties. A total of fifty-five LABs were successfully isolated from seven distinct fruits. Among these, seven isolates showed inhibition to growth of A. alternata. Two strains, isolated from fruits: Ficus benghalensis, and Tinospora cordifolia exhibited promising antifungal properties against A. alternata. Molecular identification confirmed their identities as Lactiplantibacillus plantarum MYSVB1 and MYSVA7, respectively. Both strains showed adaptability to a wide temperature range (10-45°C), and salt concentrations (up to 7%), with optimal growth around 37 °C and high survival rates under simulated gastrointestinal conditions. Among these two strains, Lpb. plantarum MYSVB1 demonstrated significant inhibition (p < 0.01) of the growth of A. alternata. The inhibitory effects of cell-free supernatant (CFS) were strong, with 5% crude CFS sufficient to reduce fungal growth by >70% and complete inhibition by 10% CFS. Moreover, the CFS was inhibitory for both mycelial growth and conidial germination. CFS retained its activity even after long cold storage. The chromatographic analysis identified organic acids in CFS, with succinic acid as the predominant constituent, with lactic acid, and malic acid in descending order. LAB strains isolated from tropical fruits showed promising probiotic and antifungal properties, making them potential candidates for various applications in food and agriculture.

Succinic acid - A run-through of the latest perspectives of production from renewable biomass.

Succinic acid (SA) production is continuously rising, as its applications in diverse end-product generation are getting broader and more expansive. SA is an eco-friendly bulk product that acts as a valuable intermediate in different processes and might substitute other petrochemical-based products due to the inner capacity of microbes to biosynthesize it. Moreover, large amounts of SA can be obtained through biotechnological ways starting from renewable resources, imprinting at the same time the concept of a circular economy. In this context, the target of the present review paper is to bring an overview of SA market demands, production, biotechnological approaches, new strategies of production, and last but not least, the possible limitations and the latest perspectives in terms of natural biosynthesis of SA.

Succinic acid production from softwood with genome-edited Corynebacterium glutamicum using the CRISPR-Cpf1 system.

Corynebacterium glutamicum is a useful microbe that can be used for producing succinic acid under anaerobic conditions. In this study, we generated a knock-out mutant of the lactate dehydrogenase 1 gene (ΔldhA-6) and co-expressed the succinic acid transporter (Psod:SucE- ΔldhA) using the CRISPR-Cpf1 genome editing system. The highly efficient HPAC (hydrogen peroxide and acetic acid) pretreatment method was employed for the enzymatic hydrolysis of softwood (Pinus densiflora) and subsequently utilized for production of succinic acid. Upon evaluating a 1%-5% hydrolysate concentration range, optimal succinic acid production with the ΔldhA mutant was achieved at a 4% hydrolysate concentration. This resulted in 14.82 g L-1 succinic acid production over 6 h. No production of acetic acid and lactic acid was detected during the fermentation. The co-expression transformant, [Psod:SucE-ΔldhA] produced 17.70 g L-1 succinic acid in 6 h. In the fed-batch system, 39.67 g L-1 succinic acid was produced over 48 h. During the fermentation, the strain consumed 100% and 73% of glucose and xylose, respectively. The yield of succinic acid from the sugars consumed was approximately 0.77 g succinic acid/g sugars. These results indicate that the production of succinic acid from softwood holds potential applications in alternative biochemical processes.