Proteomics of Corynebacterium glutamicum microbiologystudy

Proteomics, a complete set of proteins, is the study of proteins that includes proteome experimentation and data analysis combination, protein composition analysis, structure, expression, modification status, and the interactions between proteins.

Introduction of Corynebacterium glutamicum

• Corynebacterium glutamicum, a facultative anaerobic bacterium producing L-glutamate, is a non-pathogenic, fast-growing, non-motile, rod-shaped, gram-positive, saprophytic bacterium used to develop novel products.
• Corynebacterium glutamicum, a non-lethal and non-emulsifying bacterium, was first developed in Japan based on its native ability to secrete glutamate under suitable conditions.
• When cultivated under aerobic conditions, it is found in soil or on the skin of fruits and vegetables and reaches high cellular densities.
• It consists of a typical plasma membrane bilayer with an outer lipid layer of a mycolic acid attached by an arabinogalactan–peptidoglycan polymer complex. It forms a thick cell wall glycan core and a crystalline surface S-layer of high molecular mass glycans and arabinomannans. Moreover, it also forms various proteins and lipids.
• It produces primary metabolites, such as amino acids, nucleotides, and vitamins.
• It is used for industrial amino acid production, such as L-glutamate and L-lysine, and for synthesizing metabolites and proteins.
• Other industrially essential amino acids include l-threonine and l-tryptophan via microbial fermentation.
• It can maintain a stable intracellular pH of 7.5 at medium pH values between 6 and 9. For growth, It can also utilize aromatic compounds.
• In C. glutamicum, the first chromosomally encoded small antisense RNA has been identified.
• C. glutamicum is a crucial Gram-positive model organism for the closely related human pathogens C. diphtheriae and Mycobacterium tuberculosis.

Proteomics of Corynebacterium glutamicum

• The study of Corynebacterium glutamicum proteomics includes protein functions and mechanisms analysis, posttranslational protein modifications, protein levels and spatial distributions, protein interactions, expression over time, and turnover.
• The culture supernatant shows a low protease activity in the culture supernatant and releases protease-sensitive proteins.
• To produce therapeutic proteins, the gram-negative bacterial surface component lipopolysaccharide (endotoxin) from C. glutamicum should be removed, which may increase the heterologous protein yield by minimizing the purification steps.
• The proteome of the C. glutamicum experiment consists of the simple steps of sample preparation for MS analysis of the peptides for protein identification by extraction, purification and concentration, denaturation and reduction for protein separation, followed by enzymatic digestion. MS analyses facilitate the fast, sensitive, and reliable identification of proteins. The sub-maps of cytoplasmic proteins, membrane fraction proteins, cell wall-associated proteins, and secreted proteins are available by proteome analysis.
• The conventional method used in proteomics is the 2D PAGE method- 2D gel electrophoresis uses physical parameters, such as molecular mass and isoelectric point, to separate proteins. The C. glutamicum proteins cover a range of masses, ranging from 22 amino acids for the hypothetical protein Cg0491 to 2,996 amino acids for the fatty acid synthase IA (Cg2743).
• The conventional method used in proteomics is the 2D PAGE method- 2D gel electrophoresis uses physical parameters, such as molecular mass and isoelectric point, to separate proteins. According to the charge, Proteins are separated by a polyacrylamide gel strip containing an immobilized pH gradient. Here, proteins move to the isoelectric point- they have no net charge. Then, it is placed over SDS-PAGE, which separates proteins according to their molecular mass. After electrophoresis, the protein spots are detected. These proteins can be determined via protease digest and ESI MS or MALDI-TOF MS, or eventually by micro-sequencing.
• The new secreted proteins from C. glutamicum R were identified by 2D gel electrophoresis that detected 100 protein spots in the pH range 4.5–5.5 and corresponding to molecular masses ranging from 10 to 50 kDa. The two hypothetical proteins encoded by cgR_1176 and cgR_2070 were observed to be expressed, and an active form of a-amylase from Geobacillus stearothermophilus may be secreted by using the expected signal sequences.
• Therefore, C. glutamicum R can release exoproteins from its signal sequences that can be a host for protein creation in biotechnology.
• The proteome of C. glutamicum genome sequences allowed the prediction of integral membrane proteins (IMPs). The protein separation and analysis by classical 2D PAGE is limited. The consecutive combination of anion exchange chromatography and SDS-PAGE (AIEC/ SDS-PAGE) is a technique for C. glutamicum that provides the separation of intact membrane proteins to meet the requirements for separating intact IMPs. According to their charge, the proteins are separated in AIEC technology. According to their sizes, the SDS-PAGE separation resolves proteins.
• LC-MS/MS analysis helps to determine proteins in the secretome of strains producing β-lactoglobulin and wild-type at different growth phases.
• Over the past decades, post-electrophoretic stains have been developed for gel-based proteomics to facilitate the visualization and identification of proteins and specific detection of posttranslational modifications such as phosphorylation, glycosylation, or oxidation. MALDI and ESI are used for C. glutamicum proteomes analysis and studies of post-translational modifications.

Metabolism

Corynebacterium glutamicum, a model organism for the actinobacteria, is a widely studied bacteria used as a microbial cell factory.

The three approaches present in the metabolic processes of C. glutamicum may be rationally modified for the production of various biochemicals: 

(1) amplification of biosynthetic pathway enzymes to increase target products, 

(2) reduction of by-product formation, and 

(3) introduction of essential enzyme feedback controls to optimize target biomaterials.

Under anaerobic conditions, It is used for industrial amino acid production, such as L-glutamate and L-lysine, and for synthesizing metabolites and proteins. It stops its growth and produces L-lactate and succinate as fermentation products. L-lactate is the sole carbon and energy source for Corynebacterium glutamicum. It also forms other industrially essential amino acids after the deletion of genes and the introduction of heterologous genes.

Under aerobic conditions, Carbon fux synthesizes ATP through substrate phosphorylation and electron transport systems by enzymatic reactions. It also synthesizes cellular building blocks for cell replication in which carbon from glucose is released as carbon dioxide (CO₂) in the wild-type C. glutamicum.

Metabolically engineered strategies

• Metabolically engineered C. glutamicum strains produce many biochemicals (biopolymers, organic acids, rare sugars, etc.).
• C. glutamicum has been engineered to produce the diamines putrescine/diaminobutane.
• C. glutamicum has been metabolically engineered to produce l- and d-lactate and succinate by the deletion of genes combined with heterologous genes that produce other fermentation products under anaerobic conditions, such as ethanol, isobutanol, 3-methyl-1-butanol, 2,3-butanediol, l-alanine, and l-valine.
• C. glutamicum has been metabolically engineered to produce commodity chemicals for the materials (plastic) and transportation industries, such as organic acids (e.g., lactate and succinate), poly(3-hydroxybutyrate), isopropanol, or ethanol.
• Under aerobic and anaerobic conditions, C. glutamicum has emerged from being an amino acid producer to a versatile cell factory that allows industrially essential chemicals and fuel production. The development of metabolic engineering technology helps in the metabolic pathway of C. glutamicum, which utilizes amino acids as precursors for high-value-added chemical production, such as bio-based monomers and building blocks for healthcare products and pharmaceuticals.

References

1. Matamouros, S., Gensch, T., Cerff, M., Sachs, C. C., Abdollahzadeh, I., Hendriks, J., Horst, L., Tenhaef, N., Tenhaef, J., Noack, S., Graf, M., Takors, R., Nöh, K., & Bott, M. (2023). Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli. Nature Communications, 14(1). https://doi.org/10.1038/s41467-023-41176-y
2. Wendisch, V. F., & Polen, T. (2012). Transcriptome/Proteome Analysis of Corynebacterium glutamicum. In Microbiology monographs (pp. 173–216). https://doi.org/10.1007/978-3-642-29857-8_6
3. Tsuge, Y., & Yamaguchi, A. (2021). Physiological characteristics of Corynebacterium glutamicum as a cell factory under anaerobic conditions. Applied Microbiology and Biotechnology, 105(16–17), 6173–6181. https://doi.org/10.1007/s00253-021-11474-w
4. Vertès, A. A. (2012). Protein Secretion Systems of Corynebacterium glutamicum. In Microbiology monographs (pp. 351–389). https://doi.org/10.1007/978-3-642-29857-8_13
5. Balasubramanian, S., Køhler, J. B., Jers, C., Jensen, P. R., & Mijakovic, I. (2024). Exploring the secretome of Corynebacterium glutamicum ATCC 13032. Frontiers in Bioengineering and Biotechnology, 12. https://doi.org/10.3389/fbioe.2024.1348184
6. Lee, M. J., & Kim, P. (2018). Recombinant Protein Expression System in Corynebacterium glutamicum and Its Application. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.02523
7. Cui, M., Cheng, C., & Zhang, L. (2022). High-throughput proteomics: a methodological mini-review. Laboratory Investigation, 102(11), 1170–1181. https://doi.org/10.1038/s41374-022-00830-7