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Recombinant Escherichia coli strains with inducible Campylobacter jejuni single domain hemoglobin CHb expression exhibited improved cell growth in bioreactor culture.

Xu L, Xiong W, Yang JK, Li J, Tao XW - PLoS ONE (2015)

Bottom Line: Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells.To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays.Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

View Article: PubMed Central - PubMed

Affiliation: College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China.

ABSTRACT
Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells. Bacterial hemoglobins are oxygen-binding proteins that transfer oxygen from the environment to metabolic processes and allow bacteria to grow even under microaerophilic conditions. To improve the oxygen utilization efficiency of cells and overcome the oxygen shortage in bioreactors, the gene coding for the Campylobacter jejuni single domain hemoglobin (CHb) gene was artificially synthesized and functionally expressed under the control of inducible expression promoters PT7 and Pvgh in Escherichia coli. The effects of the recombinants PT7-CHb and Pvgh-CHb on cell growth were evaluated in aerobic shake flasks, anaerobic capped bottles and a 5-L bioreactor, and a pronounced improvement in cell biomass was observed for CHb-expressing cells. To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays. Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

No MeSH data available.


Related in: MedlinePlus

CO differential spectrum assays of the CHb in four recombinant cultures in the bioreactor.A: The recombinant PT7-CHb; B: The recombinant Pvgh-CHb.
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pone.0116503.g007: CO differential spectrum assays of the CHb in four recombinant cultures in the bioreactor.A: The recombinant PT7-CHb; B: The recombinant Pvgh-CHb.

Mentions: As the expression of CHb at the transcriptional level may not coincide with protein activity, CO differential spectrum assays were conducted to determine the CHb hemoglobin of each recombinant. The spectrogram scan from 400 nm to 600 nm exhibited an intense absorption peak at approximately 419 nm, with two secondary peaks at approximately 520–560 nm (Fig. 7). To compare the hemoglobin activity of the recombinants, we compared the intensity of the 419-nm peaks among recombinants. The highest hemoglobin activity was observed in recombinant PT7-CHb, with an absorbance value OD = 1.42. The fact that the PT7-CHb strain exhibits the highest hemoglobin activity explains why the cell density and wet cell weight were still higher than those of the control in the stationary phase (Figs. 3, 4, 5), although cell autolysis and growth inhibition occurred in the lag phase and the stationary phase (Figs. 4, 5). We determined that the over-expression of CHb by the PT7-CHb recombinant may represent a heavy burden for cell growth, as commonly occurs in PT7-regulated recombinant expression hosts [18].


Recombinant Escherichia coli strains with inducible Campylobacter jejuni single domain hemoglobin CHb expression exhibited improved cell growth in bioreactor culture.

Xu L, Xiong W, Yang JK, Li J, Tao XW - PLoS ONE (2015)

CO differential spectrum assays of the CHb in four recombinant cultures in the bioreactor.A: The recombinant PT7-CHb; B: The recombinant Pvgh-CHb.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352031&req=5

pone.0116503.g007: CO differential spectrum assays of the CHb in four recombinant cultures in the bioreactor.A: The recombinant PT7-CHb; B: The recombinant Pvgh-CHb.
Mentions: As the expression of CHb at the transcriptional level may not coincide with protein activity, CO differential spectrum assays were conducted to determine the CHb hemoglobin of each recombinant. The spectrogram scan from 400 nm to 600 nm exhibited an intense absorption peak at approximately 419 nm, with two secondary peaks at approximately 520–560 nm (Fig. 7). To compare the hemoglobin activity of the recombinants, we compared the intensity of the 419-nm peaks among recombinants. The highest hemoglobin activity was observed in recombinant PT7-CHb, with an absorbance value OD = 1.42. The fact that the PT7-CHb strain exhibits the highest hemoglobin activity explains why the cell density and wet cell weight were still higher than those of the control in the stationary phase (Figs. 3, 4, 5), although cell autolysis and growth inhibition occurred in the lag phase and the stationary phase (Figs. 4, 5). We determined that the over-expression of CHb by the PT7-CHb recombinant may represent a heavy burden for cell growth, as commonly occurs in PT7-regulated recombinant expression hosts [18].

Bottom Line: Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells.To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays.Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

View Article: PubMed Central - PubMed

Affiliation: College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China.

ABSTRACT
Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells. Bacterial hemoglobins are oxygen-binding proteins that transfer oxygen from the environment to metabolic processes and allow bacteria to grow even under microaerophilic conditions. To improve the oxygen utilization efficiency of cells and overcome the oxygen shortage in bioreactors, the gene coding for the Campylobacter jejuni single domain hemoglobin (CHb) gene was artificially synthesized and functionally expressed under the control of inducible expression promoters PT7 and Pvgh in Escherichia coli. The effects of the recombinants PT7-CHb and Pvgh-CHb on cell growth were evaluated in aerobic shake flasks, anaerobic capped bottles and a 5-L bioreactor, and a pronounced improvement in cell biomass was observed for CHb-expressing cells. To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays. Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.

No MeSH data available.


Related in: MedlinePlus