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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

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
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Fig3: 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

Mentions: Cells containing IS element-inserted plasmid cannot produce the target protein, and in many reports, it is well recognized that cells producing recombinant proteins have slower cell growth (very poor growth in some cases) compared to that of non-producing cells due to the metabolic burden or other problems including the toxicity of the proteins to the host cells, etc. [23]. Particularly, in large-scale production which generally needs long operation time, the fast growth of non-producing cells can be a very serious issue due to a significant decrease in protein productivity by the overgrowth of non-producing cells. To confirm this deleterious effect of IS element insertion on cell growth, two expression systems were constructed: (1) α-Amylase gene expression under the constitutive PH36 promoter (pCES-H36-porBss-Amy) and (2) IS element-inserted version (pCES-H36-porBss-IS-Amy) in which ISCg1 was inserted in the middle of signal sequence. C. glutamicum harboring each plasmid were cultivated in flasks, and the growth of each strain was compared. Additionally, IS element-inserted C. glutamicum cells were mixed with C. glutamicum producing α-amylase cells at different ratios (1:103, 1:104, and 1:105), and their growth rates were also compared. As shown in Fig. 3, C. glutamicum harboring the pCES-H36-porBss-Amy grew slowly (specific growth rate, μ = 0.268 ± 0.052 h−1) after a long lag phase and reached stationary phase around 36 h. In contrast, the IS element-inserted version (C. glutamicum harboring pCES-H36-porBss-IS-Amy) grew much faster (μ = 0.552 ± 0.012 h−1) after a short lag phase and reached stationary phase much earlier (around 12 h). In the co-culture with the IS element-inserted cells and α-amylase-producing cells, the overall growth rates were also increased as the ratio of the IS element-inserted cells increased from 1:105 to 1:103 (μ = 0.305 ± 0.021, μ = 0.405 ± 0.041, and μ = 0.498 ± 0.039, respectively). In the co-culture experiments, the change in population during cultivation was analyzed by PCR with individual clones. At the end of each cultivation (at 36 h), cells were spread onto an agar plate, and 30 colonies from each plate were randomly selected, and the plasmids were confirmed by PCR experiment. For both ratios of 1:103 and 1:104, all the colonies were IS element-inserted clones, and for the ratio of 105, IS element-inserted clones were also the major population (86 %) (data not shown). We also checked α-amylase activity in all cultivations at 36 h time-point, and we found that the cultivation of C. glutamicum harboring pCES-H36-porBss-Amy (without mixing with IS element-inserted clones) showed much higher activity (6.14 ± 0.44 U/mL) than those of other mixed cultures (Fig. 3b). In the cultivation of cells containing pCES-H36-porBss-Amy only, the possible insertion of IS element into plasmid was also analyzed by PCR but we could not find any insertion in the plasmid during the cultivation (data not shown). These results clearly show that the insertion of the IS element causes a significant decrease in protein productivity by the overgrowth of non-producing cells, and it is postulated that the removal of the IS element in the chromosome could be an important strategy for enhancing the production of target molecules in C. glutamicum.Fig. 3


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)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: 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
Mentions: Cells containing IS element-inserted plasmid cannot produce the target protein, and in many reports, it is well recognized that cells producing recombinant proteins have slower cell growth (very poor growth in some cases) compared to that of non-producing cells due to the metabolic burden or other problems including the toxicity of the proteins to the host cells, etc. [23]. Particularly, in large-scale production which generally needs long operation time, the fast growth of non-producing cells can be a very serious issue due to a significant decrease in protein productivity by the overgrowth of non-producing cells. To confirm this deleterious effect of IS element insertion on cell growth, two expression systems were constructed: (1) α-Amylase gene expression under the constitutive PH36 promoter (pCES-H36-porBss-Amy) and (2) IS element-inserted version (pCES-H36-porBss-IS-Amy) in which ISCg1 was inserted in the middle of signal sequence. C. glutamicum harboring each plasmid were cultivated in flasks, and the growth of each strain was compared. Additionally, IS element-inserted C. glutamicum cells were mixed with C. glutamicum producing α-amylase cells at different ratios (1:103, 1:104, and 1:105), and their growth rates were also compared. As shown in Fig. 3, C. glutamicum harboring the pCES-H36-porBss-Amy grew slowly (specific growth rate, μ = 0.268 ± 0.052 h−1) after a long lag phase and reached stationary phase around 36 h. In contrast, the IS element-inserted version (C. glutamicum harboring pCES-H36-porBss-IS-Amy) grew much faster (μ = 0.552 ± 0.012 h−1) after a short lag phase and reached stationary phase much earlier (around 12 h). In the co-culture with the IS element-inserted cells and α-amylase-producing cells, the overall growth rates were also increased as the ratio of the IS element-inserted cells increased from 1:105 to 1:103 (μ = 0.305 ± 0.021, μ = 0.405 ± 0.041, and μ = 0.498 ± 0.039, respectively). In the co-culture experiments, the change in population during cultivation was analyzed by PCR with individual clones. At the end of each cultivation (at 36 h), cells were spread onto an agar plate, and 30 colonies from each plate were randomly selected, and the plasmids were confirmed by PCR experiment. For both ratios of 1:103 and 1:104, all the colonies were IS element-inserted clones, and for the ratio of 105, IS element-inserted clones were also the major population (86 %) (data not shown). We also checked α-amylase activity in all cultivations at 36 h time-point, and we found that the cultivation of C. glutamicum harboring pCES-H36-porBss-Amy (without mixing with IS element-inserted clones) showed much higher activity (6.14 ± 0.44 U/mL) than those of other mixed cultures (Fig. 3b). In the cultivation of cells containing pCES-H36-porBss-Amy only, the possible insertion of IS element into plasmid was also analyzed by PCR but we could not find any insertion in the plasmid during the cultivation (data not shown). These results clearly show that the insertion of the IS element causes a significant decrease in protein productivity by the overgrowth of non-producing cells, and it is postulated that the removal of the IS element in the chromosome could be an important strategy for enhancing the production of target molecules in C. glutamicum.Fig. 3

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