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

Choi JW, Yim SS, Kim MJ, Jeong KJ - Microb. Cell Fact. (2015)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering (BK Plus program), KAIST, 291 Daehakro, Yuseong-gu, Daejeon, 34141, Republic of Korea. jwoongci@gmail.com.

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.

No MeSH data available.


Related in: MedlinePlus

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4696348&req=5

Fig2: 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

Mentions: In each round of FACS screening, plasmids were prepared from the sorted cells, and after digestion with the BamHI restriction enzyme, they were analyzed on agarose gels. In the original population, a single band for the plasmid was clearly observed for which the size coincided well with that of pCES-H36-GFP (~6.6 kb) (lane 1 of Fig. 2a). However, in the samples after the first round of sorting, another distinct band was also observed at approximately 8 kb. The ratio of the densities between the two bands was about 50:50 (lane 2 of Fig. 2a). After the second round of sorting, it was clearly observed that the 8 kb-long plasmid became dominant in the sorted cells (lane 3 of Fig. 2a). After the second round of sorting, 14 clones were randomly selected, and GFP expression in each clone was analyzed by SDS-PAGE. As expected, all 14 clones did not produce GFP (Additional file 1: Figure S1). We performed sequencing experiment for the DNA from 72-bp upstream of H36 promoter to 71-bp downstream of GFP stop codon. The sequences of the GFP coding regions for the 14 clones were determined by sequencing which showed that all 14 clones had insertions of IS elements at various positions in the GFP coding region (Fig. 2b). However, we could not find any insertion of IS element in promoter region. From the sequencing analysis, it was found that two types of IS elements were inserted, and the IS elements were identified to be part of the ISCg1 and ISCg2 family, by searching the C. glutamicum genome database. Among the 14 clones, 11 clones contained insertions from the ISCg2 family, and the rest (3 clones) contained insertions from the ISCg1 family (Fig. 2b).Fig. 2


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

Choi JW, Yim SS, Kim MJ, Jeong KJ - Microb. Cell Fact. (2015)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696348&req=5

Fig2: 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
Mentions: In each round of FACS screening, plasmids were prepared from the sorted cells, and after digestion with the BamHI restriction enzyme, they were analyzed on agarose gels. In the original population, a single band for the plasmid was clearly observed for which the size coincided well with that of pCES-H36-GFP (~6.6 kb) (lane 1 of Fig. 2a). However, in the samples after the first round of sorting, another distinct band was also observed at approximately 8 kb. The ratio of the densities between the two bands was about 50:50 (lane 2 of Fig. 2a). After the second round of sorting, it was clearly observed that the 8 kb-long plasmid became dominant in the sorted cells (lane 3 of Fig. 2a). After the second round of sorting, 14 clones were randomly selected, and GFP expression in each clone was analyzed by SDS-PAGE. As expected, all 14 clones did not produce GFP (Additional file 1: Figure S1). We performed sequencing experiment for the DNA from 72-bp upstream of H36 promoter to 71-bp downstream of GFP stop codon. The sequences of the GFP coding regions for the 14 clones were determined by sequencing which showed that all 14 clones had insertions of IS elements at various positions in the GFP coding region (Fig. 2b). However, we could not find any insertion of IS element in promoter region. From the sequencing analysis, it was found that two types of IS elements were inserted, and the IS elements were identified to be part of the ISCg1 and ISCg2 family, by searching the C. glutamicum genome database. Among the 14 clones, 11 clones contained insertions from the ISCg2 family, and the rest (3 clones) contained insertions from the ISCg1 family (Fig. 2b).Fig. 2

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering (BK Plus program), KAIST, 291 Daehakro, Yuseong-gu, Daejeon, 34141, Republic of Korea. jwoongci@gmail.com.

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.

No MeSH data available.


Related in: MedlinePlus