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.
Highlights
To achieve efficient aerobic cultivation in a bioreactor, it is critical to maintain an appropriate concentration of dissolved oxygen in the broth
Three types of hemoglobin have been identified in bacteria far, single domain hemoglobins, flavohemoglobins (FHbs) and truncated hemoglobins [5]
As revealed by the first and most thoroughly studied bacterial hemoglobins Vitreoscilla hemoglobin (VHb), bacterial hemoglobin can bind to oxygen, at low concentrations, and carry and deliver that oxygen to the terminal respiratory oxidase to enhance ATP production under hypoxic conditions [19,20,21]
Summary
To achieve efficient aerobic cultivation in a bioreactor, it is critical to maintain an appropriate concentration of dissolved oxygen in the broth. As an iron-containing oxygen-transport metalloprotein, it carries oxygen from inner respiratory cells and delivers the oxygen as an input to the metabolic processes that provide energy and sustain multiple organismal functions This oxygen-binding protein was first found in mammals, recent findings have indicated the almost ubiquitous presence of Hb in mammals, nonvertebrates, plants, and bacteria [4]. Recent studies demonstrated that the heterologous expression of the Vitreoscilla hemoglobin gene (vgh) substantially promoted both bacterial protein synthesis and oxygen diffusion, improving the aerobic metabolism of the host This finding has intrigued the interest of scientists in the field of genetic engineering due to the potential of the heterologous expression of the vgh gene to improve industrial protein production systems by enhancing cell density, protein synthesis and oxidative metabolism, under oxygen-limited conditions [5, 8,9,10,11]
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