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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

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
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Fig5: 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

Mentions: The production of useful metabolites in C. glutamicum can be achieved by the expression of biosynthesis genes. However, if the IS element is inserted into the biosynthesis genes, those genes cannot be expressed, and the productivity of the target metabolites subsequently decreases. In this regard, the use of an IS element-deleted mutant can be beneficial for the biosynthesis of metabolites. To demonstrate this beneficial concept, the production of (1) poly(3-hydroxybutyrate) [P(3HB)], biodegradable polymers [25], and (2) gamma-aminobutyric acid (GABA), the bioactive component in various foods and pharmaceutical products [26], were examined with the IS element-deleted mutants. For the biosynthesis of P(3HB) in C. glutamicum, C. glutamicum wild type, WJ004 and WJ008 strains were transformed with the plasmid, pCES-H36-PhaCAB, in which the expressions of three genes (phaC, phaA and phaB) from Ralstonia eutropha were under the constitutive PH36 promoter. In these cultivations, all cells showed almost same growth profiles (Additional file 4: Figure S4). After flask cultivation for 24 h, the P(3HB) content in each strain was analyzed by gas chromatography. As shown in Fig. 5a, the use of the WJ008 strain resulted in a higher content (23.4 ± 0.36 wt%) than that in the WJ004 and wild type C. glutamicum strain (19.1 ± 0.44 wt% and 17.1 ± 0.55 wt%, respectively). For the biosynthesis of GABA in C. glutamicum, the plasmid pHGmut, in which the expression of glutamate decarboxylase (gadB) from Escherichia coli is under the constitutive PH36 promoter, was transformed into the WJ004 and WJ008 strains, and were cultured for 72 h. A wild type C. glutamicum grew a little faster than others, but all strains reached almost same optical density after 36 h cultivation (Additional file 4: Figure S4). The GABA content in each strain was analyzed by liquid chromatography. Like in the previous results, higher production of GABA (9.43 ± 0.52 g/L) could be obtained in the WJ008 strain than in the WJ004 and wild type C. glutamicum strain (8.34 ± 0.62 and 8.17 ± 0.66 g/L, respectively) (Fig. 5b).Fig. 5


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)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: 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
Mentions: The production of useful metabolites in C. glutamicum can be achieved by the expression of biosynthesis genes. However, if the IS element is inserted into the biosynthesis genes, those genes cannot be expressed, and the productivity of the target metabolites subsequently decreases. In this regard, the use of an IS element-deleted mutant can be beneficial for the biosynthesis of metabolites. To demonstrate this beneficial concept, the production of (1) poly(3-hydroxybutyrate) [P(3HB)], biodegradable polymers [25], and (2) gamma-aminobutyric acid (GABA), the bioactive component in various foods and pharmaceutical products [26], were examined with the IS element-deleted mutants. For the biosynthesis of P(3HB) in C. glutamicum, C. glutamicum wild type, WJ004 and WJ008 strains were transformed with the plasmid, pCES-H36-PhaCAB, in which the expressions of three genes (phaC, phaA and phaB) from Ralstonia eutropha were under the constitutive PH36 promoter. In these cultivations, all cells showed almost same growth profiles (Additional file 4: Figure S4). After flask cultivation for 24 h, the P(3HB) content in each strain was analyzed by gas chromatography. As shown in Fig. 5a, the use of the WJ008 strain resulted in a higher content (23.4 ± 0.36 wt%) than that in the WJ004 and wild type C. glutamicum strain (19.1 ± 0.44 wt% and 17.1 ± 0.55 wt%, respectively). For the biosynthesis of GABA in C. glutamicum, the plasmid pHGmut, in which the expression of glutamate decarboxylase (gadB) from Escherichia coli is under the constitutive PH36 promoter, was transformed into the WJ004 and WJ008 strains, and were cultured for 72 h. A wild type C. glutamicum grew a little faster than others, but all strains reached almost same optical density after 36 h cultivation (Additional file 4: Figure S4). The GABA content in each strain was analyzed by liquid chromatography. Like in the previous results, higher production of GABA (9.43 ± 0.52 g/L) could be obtained in the WJ008 strain than in the WJ004 and wild type C. glutamicum strain (8.34 ± 0.62 and 8.17 ± 0.66 g/L, respectively) (Fig. 5b).Fig. 5

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