Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements)

Abstract

Background: In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain.

Results: From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA).

Conclusions: Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C. glutamicum, emphasizing the importance of developing IS element free host strains.

Fig. 1

FACS sorting of low fluorescent cells in the cultivation of wild type C. glutamicum harboring pCES-H36-GFP. a The histogram of wild type C. glutamicum harboring pCES-NMCS (negative control). b, c, d The histogram of wild type C. glutamicum harboring pCES-H36-GFP (b), the 1st round sorted cells (c) and the 2nd round sorted cells (d). The bars indicate the sorting region

Fig. 2

Analysis of the isolated cells by FACS screening. a Confirmation of plasmids by agarose gel electrophoresis. Lane M represents DNA size markers (kb). Lanes 1 represents plasmid from the original cell cultivation harboring pCES-H36-GFP. Lanes 2 and 3 represent plasmids from the 1st round sorted cells and the 2nd round sorted cells. The dashed and solid arrows indicate the correct pCES-H36-GFP and IS element-inserted plasmid, respectively. b Location of IS element insertion in egfp. Gray bar indicates the open read frame of egfp. Arrows indicate the IS element insertion site. Upper and lower region represent ISCg1 and ISCg2, respectively. Double or triple triangles means double or triple clones

Fig. 3

Determination of cell population change during co-culture of C. glutamicum wild type harboring pCES-H36-porBss-Amy and pCES-H36-porBss-IS-Amy. a Growth curves during the cultivation. Open circles and closed circles represent C. glutamicum harboring pCES-H36-porBss-Amy and C. glutamicum harboring pCES-H36-porBss-IS-Amy, respectively. Triangles, squares and diamonds represent co-culture of C. glutamicum (pCES-H36-porBss-IS-Amy) and C. glutamicum (pCES-H36-porBss-Amy) with the ratio of 1:10³, 1:10⁴, and 1:10⁵, respectively. b α-Amylase activity of the co-culture. 1, 2, 3, and 4 represent enzyme activity in the cultivation of C. glutamicum (pCES-H36-porBss-Amy), co-culture with the ratio of 1:10³, 1:10⁴, and 1:10⁵, respectively

Fig. 4

Comparison of GFP production in IS element-deleted mutants. a Analysis of fluorescence intensity in the cell by FACS. C. glutamicum WJ004 harboring pCES-H36-GFP and C. glutamicum WJ008 harboring pCES-H36-GFP are represented by squares and circles, respectively. Open triangles represent wild type C. glutamicum harboring pCES-H36-GFP. b Analysis of GFP production by Western blotting. Lanes 1, 2, and 3 represent the protein sample from wild type C. glutamicum, WJ004, and WJ008 harboring pCES-H36-GFP at 18 h. Lanes T and S represent total protein fraction and soluble protein fraction. Protein sample was prepared from the same cell concentration which was normalized to OD₆₀₀ of 4

Fig. 5

Production of a secondary metabolite. a Production of P(3HB) in the wild type C. glutamicum, WJ004 and WJ008 harboring pCES-H36-PhaCAB. All samples were prepared after 24 h of cultivation. b Production of GABA in the wild type C. glutamicum, WJ004 and WJ008 harboring pHGmut. All samples were prepared after 72 h of cultivation

Fig. 6

Transformation efficiency of the wild type C. glutamicum, WJ004 and WJ008. All error bars represent standard deviations of six-times repeated experiments