Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum

• Published in: Applied microbiology and biotechnology Vol. 102; no. 2; pp. 873 - 883
• Main Authors: Choi, Jae Woong, Yim, Sung Sun, Jeong, Ki Jun
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 2018; Springer; Springer Nature B.V
• Subjects: Amino acids; Applied Genetics and Molecular Biotechnology; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Batch Cell Culture Techniques; Batch culture; Bioconversion; Biomedical and Life Sciences; Biotechnology; Cell culture; Codon, Nonsense; Copy number; Corynebacteria; Corynebacterium glutamicum; Corynebacterium glutamicum - genetics; Cultivation; Culture Media; Dehydrogenase; Dehydrogenases; Directed Molecular Evolution; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - metabolism; Evolution; Fluorescence; Gene Dosage; Genetic aspects; Green Fluorescent Proteins; Growth rate; Laboratories; Life Sciences; Metabolic Engineering; Microbial Genetics and Genomics; Microbiology; Mutation; Natural history; Nonsense mutation; Plasmids; Plasmids - genetics; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Xylose; Xylose - metabolism; Adaptive evolution; Plasmid copy number; ParB; Corynebacterium glutamicum
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-017-8653-2

Beyond its traditional role as an L-amino acid producer, Corynebacterium glutamicum has recently received significant attention regarding its use in the production of various biochemicals and recombinant proteins. However, despite these attributes, limitations in genetic tools are still hampering the engineering of C. glutamicum for use in more potential hosts. Here, we engineered a C. glutamicum via adaptive laboratory evolution to enhance the production of recombinant proteins. During the continuous cultivation, C. glutamicum producing enhanced green fluorescent proteins was screened using high-speed flow cytometer, and in the end, we successfully isolated an evolved strain with a fluorescence intensity 4.5-fold higher than that of the original strain. Extensive analysis of the evolved strain confirmed that the plasmid prepared from the evolved strain contains the nonsense mutation in the parB locus, which mutation contributed to increasing the copy number of plasmid by approximately 10-fold compared to that of the wild type. To validate the usefulness of the high-copy-number plasmid, we examined the secretory production of endoxylanase and the bioconversion of xylose to xylonate using xylonate dehydrogenase. In the fed-batch cultivation, the use of the high-copy-number plasmid led to 1.4-fold increase in the production of endoxylanase (~ 1.54 g/L in culture medium) without cell growth retardation comparing cultivation with cells harboring original plasmid. The expression of xylonate dehydrogenase in the high-copy-number plasmid also improved the bioconversion into xylonic acid by approximately 1.5-fold compared to the original plasmid.