| Corynebacterium glutamicum is commonly used for industrial glutamate production. It has unique metabolic traits, such as fermenting different types of biomass substrates and has high resistance to high sugar concentration Therefore, a comprehensive understanding of the physiological and metabolic characteristics of C. glutamicum is important for developing its potential for industrial production. This study on one hand constructed a GSMM of C. glutamicum through a combined use of automatic reconstruction and manual refinement, on the other hand knocked out amn gene in C. glutamicum and study its effect on strain metabolism. The main results were as follows:(1) The genome-scale metabolic model i JM658 was reconstructed based on the published genome sequence of C. glutamicum S9114 on the Matlab platform, combined with BLAST and Model SEED annotation methods, as well as data from biochemical databases and literature. The model contained 658 genes, 984 metabolites and 1065 reactions, which were distributed in two cellular compartments(extracellular and cytosol). It had a gene coverage of 22%, about 62% higher than the other two models. In addition, 883 reactions were gene-associated, and 182 reactions were non- gene-associated, and were added based on their phenotypes or literature reports.(2) Model i JM658 investigated the metabolism of C.glutamcium from the perspective of cell growth phenotype and glutamate production capacity. ①Model i JM658 was simulated for ability for growth on different carbon nitrogen sources. The result showed that 23 carbon sources and 11 nitrogen sources were predicted to support cell growth; ②Gene and reaction essentiality analysis reviewed that 129 genes and 165 reactions were predicted to be essential for cell growth; ③A map illustrating the central metabolism of glutamate production from various carbon sources in C. glutamicum S9114 was provided; ④Comparison of carbon flux distribution between the cell growth phase and glutamate production phase by FBA method showed three key metabolic nodes for glutamate production: pentose phosphate pathway, pathway from phosphoenolpyruvate to oxaloacetate and a cytosolic reductive pathway from oxaloacetate to malate; ⑤Robustness analysis revealed a relative low oxygen level(1.21 mmol·g DW-1·h-1) would improve growth rate and glutamate production rate; ⑥Single- gene deletion method simulated target genes for enhancing γ-aminobutyrate(mdh) and isoleucine(ldh, pdh and pqo) production rate.(3) To study the effect of adenosine monophosphate nucleosidase gene amn on C. glutamicum metabolism, we constructed amn deficient strain △amn and anaplerosis strain p DXW-8-amn. Analysis of the glutamate fermentation performance and low p H tolerance showed that compared with wild type strain, the biomass of △amn strain increased by 16.2% while the glutamate production reduced by 58.8%;the intracellular ATP level increased by 3.0, 3.7 and 2.2 times those of △amn strain in 10 h, 25 h and 40 h, respectively; the activity of IC L increased by 17.1%, 4.9% and 44.5% in 10 h, 25 h and 40 h, respectively while the ICDH activity decreased by 76.9%, 74.6% and 5.0% and the GDH activity decreased 42.4%, 50.8% and 42.4%; under condition of p H4.0, the survivability of △amn strain droped 64.9%, however, the intracellular ROS and protein carbonylation level showed a 31.5% and 22.5% increase, respectively. |
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