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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

Transformation efficiency of the wild type C. glutamicum, WJ004 and WJ008. All error bars represent standard deviations of six-times repeated experiments
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Fig6: Transformation efficiency of the wild type C. glutamicum, WJ004 and WJ008. All error bars represent standard deviations of six-times repeated experiments

Mentions: An earlier study [8] showed that the deletion of prophages in the chromosome of C. glutamicum (ATCC 13032) resulted in an increase in the transformation efficiency due to the deletion of the restriction modification (RM) system present in the prophage gene. Takahashi N et al. [27] also reported that the movement of the restriction modification system in Escherichia coli was linked with an IS element. In this regard, the deletion of IS elements may also have a positive effect on the transformation efficiency; thus, the effect of the IS element deletion was examined on the transformation efficiency as another characteristic of the IS element-deleted strains. The two IS element deleted mutants (WJ004 and WJ008) and wild-type C. glutamicum strain were transformed with the same plasmid (pCES-H36-GFP), and their transformation efficiencies were compared by checking the colony forming units (CFUs) on selective agar plates. As shown in Fig. 6, the WJ004 and WJ008 strains had about a four- and six-fold higher transformation efficiency (12.2 ± 0.94 × 105 cfu/μg and 19.8 ± 1.2 × 105 cfu/μg) than that of the wild type strain (3.86 ± 0.18 × 105 cfu/μg), respectively.Fig. 6


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)

Transformation efficiency of the wild type C. glutamicum, WJ004 and WJ008. All error bars represent standard deviations of six-times repeated experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Transformation efficiency of the wild type C. glutamicum, WJ004 and WJ008. All error bars represent standard deviations of six-times repeated experiments
Mentions: An earlier study [8] showed that the deletion of prophages in the chromosome of C. glutamicum (ATCC 13032) resulted in an increase in the transformation efficiency due to the deletion of the restriction modification (RM) system present in the prophage gene. Takahashi N et al. [27] also reported that the movement of the restriction modification system in Escherichia coli was linked with an IS element. In this regard, the deletion of IS elements may also have a positive effect on the transformation efficiency; thus, the effect of the IS element deletion was examined on the transformation efficiency as another characteristic of the IS element-deleted strains. The two IS element deleted mutants (WJ004 and WJ008) and wild-type C. glutamicum strain were transformed with the same plasmid (pCES-H36-GFP), and their transformation efficiencies were compared by checking the colony forming units (CFUs) on selective agar plates. As shown in Fig. 6, the WJ004 and WJ008 strains had about a four- and six-fold higher transformation efficiency (12.2 ± 0.94 × 105 cfu/μg and 19.8 ± 1.2 × 105 cfu/μg) than that of the wild type strain (3.86 ± 0.18 × 105 cfu/μg), respectively.Fig. 6

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