Date Published: April 3, 2017
Publisher: Public Library of Science
Author(s): Yang Sun, Wenwen Guo, Fen Wang, Chunjun Zhan, Yankun Yang, Xiuxia Liu, Zhonghu Bai, Shihui Yang.
Corynebacterium glutamicum (C. glutamicum) is a favorable host cell for the production of recombinant proteins, such as important enzymes and pharmaceutical proteins, due to its excellent potential advantages. Herein, we sought to systematically explore the influence of recombinant protein expression on the transcription and metabolism of C. glutamicum. Two C. glutamicum strains, the wild-type strain and an engineered strain expressing enhanced green fluorescent protein (EGFP), were cultured in parallel in 5-L bioreactors to study the change in metabolism in the process of EGFP expression. The results revealed that EGFP expression had great effects on the growth and metabolism of C. glutamicum and contributed to metabolism-like anaerobic conditions as follows: glycolysis was enhanced, the TCA cycle was shunted, and Glu, Val, Met, lactate and acetate were accumulated to produce sufficient ATP for EGFP production and transfer. Many differentially expressed genes related to ribosomal protein, transcriptional regulators, and energy metabolism were found to be expressed in the presence of EGFP, laying the foundation for identifying genomic loci to change the flow of the host cell metabolism to improve the ability of expressing foreign proteins in C. glutamicum.
Since 1957, when the Gram-positive bacterium Corynebacterium glutamicum (C. glutamicum) was isolated due to its ability to excrete large amounts of L-glutamate , C. glutamicum has been thoroughly investigated and used as a generally regarded-as-safe organism in the fermentation industry. Within the last decades, C. glutamicum was proven to be an excellent production platform for amino acids and organic acids, and a promising alternative bacterial host for the expression of recombinant proteins due to its unique characteristics. First, C. glutamicum is non-pathogenic and does not produce endotoxins. Furthermore C. glutamicum could secrete properly folded proteins into the culture medium and lacks detectable extracellular hydrolysis activity, activities that increase the subsequent purification efficiency . With these advantages, C. glutamicum has been widely used to express recombinant proteins such as staphylococcal nuclease , transglutaminase , human epidermal growth factor , antibody fragment , green fluorescent protein (GFP)  and α-amylase , et al.
We cultivated the wild-type strain (C. glutamicum BZH001) and engineering strains (C. glutamicum EGFP) in a bioreactor to study the effect of EGFP expression on metabolism and identify the targets to improve the ability to express foreign proteins in C. glutamicum. Transcriptome analysis of the response of EGFP expression was carried out for 20 h, and DEG analysis was carried out through KEGG and GO enrichment. MVDA was also used to screen critical genes that play important roles in regulating EGFP expression, and then we detected the content of the intracellular and extracellular metabolites, including organic acids and amino acids, and the activities of the key enzymes in metabolic pathways. Furthermore, we detected the expression of key genes and ATP to study the changes in the material and energy metabolism in the fermentation process. Two effects of EGFP expression were found: 1. metabolism changes in EGFP expression; 2. the effect of EGFP expression on protein synthesis, secretion and expression regulation (Fig 6, S5 Table).