Team:Lund/Description
Project description
Background
Many valuable products such as pharmaceuticals, food additives and enzymes are commonly produced by using microorganisms. Typically, the production takes place in bioreactors where the organisms are grown in large volumes under favorable conditions in order to maximize product yields. A high cell density if often desired and maintaining a sufficient supply of oxygen can then be difficult. The amount of oxygen available to the cells is limited to what can be dissolved in the cultivation medium, and as cell density increases, pockets of very low oxygen concentration may occur. This may cause cells to switch to the less energy-efficient mode of anaerobic metabolism, causing slower growth and increased production of undesired byproducts such as acids. Common strategies to mitigate the problem include increased stirring speed and increased air flow. However, while being easy to implement and in practice widely used, these methods also increase the shear force exerted on the culture, causing damage to or death of the cells.
A completely different, and potentially complementary, route is to approach the issue by leveraging synthetic biology and genetically modifying the organism in question. A novel solution that has been successfully used for this purpose is the co-expression of Vitreoscilla hemoglobin (VHb) in order to increase the oxygen uptake. Instead of attempting to increase the amount of dissolved oxygen in the medium, the organism is modified to more efficiently utilize the dissolved oxygen already present. The approach is simple in the sense that it only requires the initial effort of introducing the gene into the host organism, while the cultivation process can remain essentially unchanged. It has been used to increase yields in various settings such as in polymer, enzyme and biofuel production [1]. However, while this approach is very promising, it is not yet fully understood in which scenarios and under what conditions the utilization of VHb is beneficial, or how it performs in large scale settings.
Our project
The co-expression of VHb to increase recombinant protein yield has been proven successful in various applications. However, studies have indicated that the success is largely dependent on the choice of associated expression system [2][3][4]. This suggests that the level at which VHb is expressed may play an important role in determining whether the effect on recombinant protein yield and productivity is positive or not. Based on this, our project has been dedicated to studying the effect of different levels of VHb co-expression on the production of recombinant target proteins, using Escherichia coli as our host and the set of constitutive Anderson promoters from the iGEM registry as our means of achieving the different VHb expression levels.
To read more about our project design, see the design page.
References
- [1] Stark, B., Pagilla, K., and Dikshit, K. (2015) Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation. Applied Microbiology and Biotechnology 99, 1627-1636.
- [2] Yu, H., Yin, J., Li, H., Yang, S., and Shen, Z. (2000) Construction and selection of the novel recombinant Escherichia coli strain for poly(β-hydroxybutyrate) production. Journal of Bioscience and Bioengineering 89, 307-311.
- [3] Sanny, T., Arnaldos, M., Kunkel, S., Pagilla, K., and Stark, B. (2010) Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose. Applied Microbiology and Biotechnology 88, 1103-1112.
- [4] Tsai, P., Hatzimanikatis, V., and Bailey, J. (1996) Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism. Biotechnology and Bioengineering 49, 139-150.
