Separation and purification of N-acetylglucosamine from the fermentation broth of corynebacterium glutamicum by electrodeionisation method

https://www.ajinomoto.com/aboutus/amino-acids/how-amino-acids-are-made This website is maintained by a major company in Japan that produces amino acids via microbial fermentation processes. https://www.sciencedirect.com/science/article/pii/S0734975017301052 This is a review article that focuses on the specificity of the processes used in fermentation to make amino acids via different microbial strains. https://link.springer.com/content/pdf/10.1007%2F978-4-431-56520-8.pdf This is a book on microbial amino acid fermentations. It is comprehensive and covers the history, examples of processes, recent advances and future prospects for the field. https://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=Stickland-Oxidative This page of the MetaCyc database links to reactions that comprise the oxidative branch of the amino acid fermentation metabolism known as the Stickland reactions. https://www.nature.com/articles/ja2017142.pdf?origin=ppub This review article in the Journal of Antibiotics provides a very clear and concise overview of microbial amino acid production. It focuses on the microorganisms, the scale and the economic impacts of these processes. https://www.frontiersin.org/articles/10.3389/fmicb.2018.00683/full While most commercially relevant amino acids are in the L-form, there is increasing evidence of the importance of D-amino acids in the physiology of microorganisms. https://www.nature.com/articles/s41587-019-0345-2 Detecting specific proteinogenic amino acids electrically is described in this report. The method can provide a new, fast way to sequence proteins. https://www.pnas.org/content/115/20/5093 Amino acids have traditionally been derived on scale via microbes that overproduce specific amino acids that they excrete into fermentation broths. This paper describes the potential to use biomass and specific catalysts to produce a number of α-amino acids. If scalable and economically feasible, these methods could compete with the current microbial fermentation processes used in industry. https://www.nature.com/articles/s41598-018-21926-5 This study investigated the high level production of L-valine by Corynebacterium glutamicum. https://www.ncbi.nlm.nih.gov/genome/browse#!/prokaryotes/469/ Corynebacterium glutamicum is the single most used and useful bacterium for producing amino acids. Its genome sequence was first publicly reported in 2003, and currently, dozens of genomes of C. glutamicum strains have been sequenced. Links to the genomes are provided here. https://biocyc.org/CORYNE/organism-summary Metabolic pathways for Corynebacterium glutamicum are provided here. https://link.springer.com/chapter/10.1007/978-3-030-39267-3_10 This review article discusses metabolic engineering of Corynebacterium glutamicum for enhanced amino acid production. The methods covered in this report range from classical mutagenesis, genetic engineering of single genes and systems biology approaches. https://www.biotechnologynotes.com/amino-acids/industrial-production-of-amino-acids-by-microorganism-and-fermentation/13820 This site provides a good basic overview of the range of amino acid products, the microorganisms used and parameters such as yield. https://www.prnewswire.com/news-releases/global-feed-amino-acids-market-overview-2021-with-profiles-of-56-companies--industry-guide-containing-contact-details-for-219-companies-301303039.html This site describes the industrial demand for amino acids and the major global players in their production. https://www.sciencedirect.com/science/article/pii/S2214030120300122 This report describes a selection and bio-sensing system to enhance the production of amino acids by Lactococcus lactis.

Protocatechuate acid (PCA) is a phenolic acid naturally synthesized by various organisms. Protocatechuic acid is synthesized by plants for physiological, metabolic functions, and self-defense, but extraction from plants is less efficient compared to the microbial culture process. The microbial synthesis of protocatechuic acid is sustainable and, due to its high yield, can save energy consumption when producing the same amount. To enhance PCA production using Corynebacterium glutamicum, a statistical optimization of the production medium was performed using full factorial design, the steepest ascent method, and the response surface method. The optimized production medium enabled a PCA production of over 5 g/L in a 72 h batch culture. However, PCA cytotoxicity affected the strain growth and PCA production rate, with an inhibitory concentration of approximately 5 g/L in the fermentation broth. Finally, continuous fermentation was operated for 150 h in the steady-state mode, maintaining the concentration of PCA below 5 g/L. The optimization method established in this study successfully increased PCA production levels, and the findings presented herein are anticipated to contribute to the industrialization of PCA production using C. glutamicum.

LAPS-based monitoring of metabolic responses of bacterial cultures in a paper fermentation broth

Phytate utilization by genetically engineered lysine-producing Corynebacterium glutamicum

BackgroundThe demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important.ResultsIn this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the l-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive laboratory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h−1 by the parental strain to 0.17 h−1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L−1 h−1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of l-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L−1, a yield of 0.23 g g−1 and a volumetric productivity of 0.35 g L−1 h−1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream processing results.ConclusionFlux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies.

Fructose-1,6-bisphosphatase (FBPase) and fructokinase (ScrK) have important roles in regenerating glucose-6-phosphate in the pentose phosphate pathway (PPP), and thus increasing L-lysine production. This article focuses on the development of L-lysine high-producing strains by heterologous expression of FBPase gene fbp and ScrK gene scrK in C. glutamicum lysC (fbr) with molasses as the sole carbon source. Heterologous expression of fbp and scrK lead to a decrease of residual sugar in fermentation broth, and heterologous expression of scrK prevents the fructose efflux. Heterologous expression of fbp and scrK not only increases significantly the activity of corresponding enzymes but also improves cell growth during growth on molasses. FBPase activities are increased tenfold by heterologous expression of fbp, whereas the FBPase activity is only increase fourfold during co-expression of scrK and fbp. Compared with glucose, the DCW of heterologous expression strains are higher on molasses except co-expression of fbp and scrK strain. In addition, heterologous expression of fbp and scrK can strongly increase the L-lysine production with molasses as the sole carbon source. The highest increase (88.4%) was observed for C. glutamicum lysC (fbr) pDXW-8-fbp-scrK, but the increase was also significant for C. glutamicum lysC (fbr) pDXW-8-fbp (47.2%) and C. glutamicum lysC (fbr) pDXW-8-scrK (36.8%). By-products, such as glycerol and dihydroxyacetone, are decreased by heterologous expression of fbp and scrK, whereas trehalose is only slightly increased. The strategy for enhancing L-lysine production by regeneration of glucose-6-phosphate in PPP may provide a reference to enhance the production of other amino acids during growth on molasses or starch.

Recent advances in biotechnology have brought about revolutionary changes across diverse industries, with microbial fermentation technology playing a important role in the production of high-value compounds. Among these, Corynebacterium glutamicum has emerged as a key strain for efficiently producing biochemicals like amino acids. While fermentation processes using C. glutamicum are widely adopted in industries due to their high productivity and cost-effectiveness, the cytotoxicity of protocatechuic acid (PCA), a metabolic product generated during fermentation, has been a significant hindrance to productivity. PCA, known for its potent electrochemical antioxidant properties and wide application in industries such as food, pharmaceuticals, and cosmetics, poses challenges when accumulated at high concentrations during fermentation, inhibiting cell growth and reducing productivity. Therefore, this study aimed to explore the feasibility of applying adsorption processes to enhance PCA productivity in C. glutamicum. Building on previous research, we investigated an approach to effectively remove PCA from fermentation broth using specific ion exchange resins to reduce cytotoxicity and increase overall PCA production. In this study, we improved C. glutamicum strains and optimized production media to enhance PCA production capacity. Subsequently, we analyzed the impact of adsorption processes using ion exchange resins on alleviating cytotoxicity and improving productivity. Through experimentation, we optimized PCA adsorption efficiency and conditions using ion exchange resins, resulting in the production of 3.97 g/L of PCA in fermentation. This research is expected to contribute to the improvement of high-value biochemical production processes based on microbial fermentation.

Applying a genetic algorithm for the optimization of trace element composition in the medium for L-isoleucine production from glucose and DL-α-hydroxybutyric acid with Corynebacterium glutamicum resulted in a reduction of the byproduct L-valine. High L-isoleucine broth concentrations of 20 g/l within 72 h at an L-isoleucine/DL-α-hydroxy butyric acid yield of 70% (w/w) and an L-isoleucine/L-valine ratio of 100 were achieved, if closed-loop control of glucose and of DL-α-hydroxybutyric acid was applied. For the isolation of L-isoleucine from fermentation broth a specific downstream processing was developed and optimized up to semitechnical scale (ultrafiltration, reverse osmosis, first crystallization, active-carbon adsorption, electrodialysis, second crystallization). The economic model of this downstream processing, which was identified by coupling the mass balance and energy balance with the semi-empirical models of the unit operations, was used to quantify the isolation costs as a function of L-isoleucine concentration and L-isoleucine/L-valine ratio in the fermentation broth. A cost reduction for downstream processing from DM 55 to DM 25 (kg L-isoleucine)-1 and an improvement of the L-isoleucine yield in downstream processing from 48% to 80% was achieved using this economic model as the objective function to be minimized by the fermentation process (scenario: production of 70 tonnes L-isoleucine/year).

Coproduction of cell-bound and secreted value-added compounds: Simultaneous production of carotenoids and amino acids by Corynebacterium glutamicum

Aspartate kinase of Brevibacterium flavum QL-5 is inhibited by L-lysine plus L-threonine. Ele-ctrodialysis fermentation was applied to L-lysine production to reduce this inhibition. After 72 hr, the amount of L-lysine produced by electrodialysis fermentation was 57.2 g, which was 1.2 times greater than that by non-electrodialysis fermenta-tion (46.9 g), and similar to that by diffusion dialysis fermentation (57.0g). In electrodialysis fermentation, the L-lysine concentration in dialyz-ing solution was only 0.2%, while that in the fermentation broth was about 2%. It was sug-gested that production of L-lysine was limited by transport of L-lysine across the hollow fiber wall. L-Lysine monohydrochloride was added to the cul-ture broth at various culture stage, but the produc-tion of L-lysine was not influenced by the L-lysine added. Therefore, it was considered that the inhibition by L-lysine plus L-threonine in L-lysine production by Brevibacterium flavum QL-5 was not alleviated even when the L-lysine concentration in the fermentation broth was maintained at low by electrodialysis fermentation or diffusion dialysis fermentation.

The marine carotenoid astaxanthin is one of the strongest natural antioxidants and therefore is used in a broad range of applications such as cosmetics or nutraceuticals. To meet the growing market demand, the natural carotenoid producer Corynebacterium glutamicum has been engineered to produce astaxanthin by heterologous expression of genes from the marine bacterium Fulvimarina pelagi. To exploit this promising source of fermentative and natural astaxanthin, an efficient extraction process using ethanol was established in this study. Appropriate parameters for ethanol extraction were identified by screening ethanol concentration (62.5–97.5% v/v), temperature (30–70 °C) and biomass-to-solvent ratio (3.8–19.0 mgCDW/mLsolvent). The results demonstrated that the optimal extraction conditions were: 90% ethanol, 60 °C, and a biomass-to-solvent ratio of 5.6 mgCDW/mLsolvent. In total, 94% of the cellular astaxanthin was recovered and the oleoresin obtained contained 9.4 mg/g astaxanthin. With respect to other carotenoids, further purification of the oleoresin by column chromatography resulted in pure astaxanthin (100%, HPLC). In addition, a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay showed similar activities compared to esterified astaxanthin from microalgae and a nine-fold higher antioxidative activity than synthetic astaxanthin.

Actinobacteria are important for industrial production of antibiotics, fine chemicals and food and a source of new compounds for drug discovery. Their central metabolism is regulated by a conserved protein GarA that is unique to the Actinobacteria and has been studied in Mycobacterium tuberculosis and Corynebacterium glutamicum. GarA regulates the TCA cycle and glutamate metabolism by direct binding to enzymes to modulate their activity on glutamate and alpha-ketoglutarate. Given the importance of the TCA cycle in the synthesis of acyl-CoA precursors for antibiotic biosynthesis, and increasing evidence for the role of nitrogen regulators in control of secondary metabolism, we hypothesized that engineering GarA could be used to enhance production of valuable metabolites. His6-tagged GarA was introduced into Saccharopolyspora erythraea, an overproducer of the polyketide antibiotic erythromycin. Phosphorylation of GarA was detected at the N-terminal ETTS motif, suggesting that it is regulated by protein kinases like in M. tuberculosis. GarA expression was observed at all growth stages, and a truncated form lacking the phosphorylation site accumulated during late fermentation. Engineered S. erythraea expressing phosphoablative GarA produced twofold more erythromycin, both in standard fermentation broth and in minimal medium. To investigate the mechanism for the increased titre, the engineered strain was characterized for transcription of erythromycin biosynthetic genes, as well as its ability to metabolize glutamate and its intracellular and extracellular aa content. The observed alterations in aa metabolism are consistent with the role of GarA as a TCA cycle regulator that may influence precursor supply for polyketide biosynthesis.

To clarify the mechanism of the γ-L-glutamylpeptide formation in the L-glutamic acid fermentation with Corynebacterium glutamicum, γ-glutamylpeptide synthetic activity of the intact cells and the cell extracts of the bacteria was studied. γ-L-Glu-L-Glu and other various γ-glutamylpeptides were formed by these crude preparations under high substrate amino acid concentrations without direct or indirect biological energy supplying systems. It was re-vealed that these enzyme preparations possessed strong hydrolytic activity to γ-glutamyl-peptides, and that notable amounts of these peptides could be formed by the reverse reaction of the hydrolysis. These reactions were found to be catalyzed by an enzyme. To give the evidence, the equilibrium constants of the hydrolysis of γ-L-glutamylpeptides, the substrate specificity and the distribution of the enzyme among various strains of the bacteria were studied. The partial purification of the enzyme gave sufficient proof to the idea. The mecha-nism was thought to contribute to the γ-L-GIu-L-Glu formation in the fermentation.

Production of Ultraviolet A protectant C50 carotenoid decaprenoxanthin by metabolically engineered Corynebacterium glutamicum.

Lignocellulosic biomass is the most abundant raw material on earth. Its efficient use for novel bio-based materials is essential for an emerging bioeconomy. Possible building blocks for such materials are the key TCA-cycle intermediates α-ketoglutarate and succinate. These organic acids have a wide range of potential applications, particularly in use as monomers for established or novel biopolymers. Recently, Corynebacteriumglutamicum was successfully engineered and evolved towards an improved utilization of d-xylose via the Weimberg pathway, yielding the strain WMB2evo . The Weimberg pathway enables a carbon-efficient C5-to-C5 conversion of d-xylose to α-ketoglutarate and a shortcut route to succinate as co-product in a one-pot process. C.glutamicumWMB2evo was grown under dynamic microaerobic conditions on d-xylose, leading to the formation of comparably high amounts of succinate and only small amounts of α-ketoglutarate. Subsequent carbon isotope labeling experiments verified the targeted production route for both products in C.glutamicumWMB2evo . Fed-batch process development was initiated and the effect of oxygen supply and feeding strategy for a growth-decoupled co-production of α-ketoglutarate and succinate were studied in detail. The finally established fed-batch production process resulted in the formation of 78.4mmolL-1 (11.45gL-1 ) α-ketoglutarate and 96.2mmolL-1 (11.36gL-1 ) succinate. The developed one-pot process represents a promising approach for the combined supply of bio-based α-ketoglutarate and succinate. Future work will focus on tailor-made down-stream processing of both organic acids from the fermentation broth to enable their application as building blocks in chemical syntheses. Alternatively, direct conversion of one or both acids via whole-cell or cell-free enzymatic approaches can be envisioned; thus, extending the network of value chains starting from cheap and renewable d-xylose.