Team:Lund/Applied Design
Product design
Our ambition since the start of the project was to create an elegant approach that integrates the Vitreoscilla hemoglobin (VHb) technology into pre-existing expression systems. We believe that good design constitutes standardization of the internal components, a systematic integration process and flexible customization for the intended final application. This philosophy has been fundamental to us throughout the entire design process.
We believe that many biotechnical processes would benefit in terms of productivity by supplementing with VHb to improve the oxygen utilization. As we explained earlier in design overview, we realized that the main obstacle we would face in realizing this idea is that each expression scenario is unique in terms of oxygen requirements. Different host organisms require different amount of oxygen for their proliferation and production of the recombinant protein. In certain cases, such as high cell density cultivations [1], the oxygen demand is high while vice-versa during fermentation [2] . VHb has been studied in both of these conditions and many more. The results have been significant enough to consider applying the technology at a larger scale [3][4].
However, there exist no practical methodology of integrating the VHb technology into pre-existing systems. On top of this, each expression system has its unique oxygen requirement as mentioned above.
Assembly
All of the constructs come pre-assembled with the VHb gene and a slot for the target protein gene of which the productivity should be improved. By using the collection of Anderson promoters from the iGEM registry, a library of plasmids containing the VHb gene expressed at different levels has been achieved. The user only needs to insert the gene encoding their protein of interest as well as its promoter. See Design for further details of the constructs.
Screening
The different construct are cloned in the organisms of interest using the preferred transformation method of the company and them expressed in small scale (e.g. shake flasks) to verify the success of the transformation through the isolation and quantification of the protein of interest . Later, once all the construct had been successfully cloned and expressed in the organism, they are screened in a bioreactor under the standard conditions of the process to find the optimal promoter strength for the intended application.
Selection
The results of the fermentations are analyzed and the best candidates are chosen based on the product yield and its specific productivity considering that the conditions of the bioreactor resemble the ones used in large scale.
Upscaling
The candidate is later produced in a larger reactor to simulate industrial settings. The importance of this is step is to test the system in a bigger volume due to oxygen pockets being more present in up-scaled settings.
Industry
Finally, the VHb technology is integrated into the companies production pipeline. Based on what we learned from consulting with experts, we do not expect VHb to pose any issues during purification of the final product.
Comparison with other potential solutions
The use of large-scale bioprocesses and recombinant DNA technology has made possible the production of proteins in quantities that are rarely obtained from natural sources [3]. On a large scale, the economical feasibility of an expression system relies on an optimization of the specific productivity and/or the cell productivity which are limited by, among other factors, the oxygen supply and its transfer to the growing cell population [3][4]. Oxygen availability is a common limiting factor in this type of processes due to the poor solubility of oxygen in aqueous solutions and the high oxygen consumption rate of the high cell-density population [1].
The oxygen transfer rate in aqueous solution is influenced by different physical and chemical parameters like the solubility, the turbulence of the fluid, the aeration rate and the oxygen concentration of the gas sparged in the bioreactor among others [5]. Current solutions to increase oxygen transfer are limited to physical approaches due to the highly detrimental economical impact on the process associated with a change of the sparging gas to pure oxygen. In consequence, the most used approaches to increase the oxygen transfer rate in a large- scale bioreactor are increasing the stirring and aeration rates. These methods to control the dissolved oxygen level are popular due to the low capital cost required for their implementation. Nevertheless, the fact that recombinant organisms, like yeasts, are sensitive to mechanical stress constrain the implementation of these solutions to limited ranges of the stirring speed and the aeration rate [5].
Our solution, on the other hand, challenges pre-existing ones by introducing a molecular approach that could potentially remediate the issue of limited growth due to oxygen deprivation without causing any physical damage to the organism and the product (e.g. a recombinant protein). The integration of our system incorporates the plug-and-play concept of synthetic biology. The provided constructs come pre-assembled and ready to use by simply inserting the sequence of the desired protein to up-scale.
Our method provides a way to increase the intrinsic ability of the microorganisms to utilize the available oxygen. The Vitreoscilla hemoglobin platform technology has previously worked in bacteria, yeast and higher eukaryotes such as plants and mammalian cells to improve cell productivity, giving it a wide range of applications.
Benefits
Water-logging, where water saturates the roots of the plant, is a common issue in agriculture with about 6 to 10 million hectares of land affected in India alone [6]. In 2011, the average crop yield of rice, cereals and wheat in India was 2636 kg/hectare according to the Department of Agriculture & Cooperation [7]. To put this in perspective, 6 to 10 millions of hectares equals approximately 16 000 to 26 000 million kg of food affected in negative ways by water-logging every year. VHb co-expression can be a viable solution to alleviate this issue, while also providing the additional benefit of increasing the biomass content of plants [8][9].
A frequent issue in the case of bioremediation is the lack of oxygen in the environment where it is taking place. VHb has been used in various bioremediation scenarios for exactly this reason [10][11][12]. Many of the previously mentioned contaminants can lead to serious disorders in the domestic microbial flora [13] and human population [14].
Ethanol makes up three fourths of biofuel use [15]. Until now it has been produced using cornstarch, sugar canes and sugar beets, however a cost-effective production of bioethanol will necessitate the development of alternative fermentation feedstocks and increased productivity [16].
Results and future vision
When designing our project, we chose green flourescent protein as a proof-of-concept protein. The measured protein concentration prooved to increase significantly with the VHb expression level. See our Results section for further specifics.
We envision our VHb system as future platform technology that other iGEM teams, researchers or companies build upon by co-expressing their own proteins, cultivating their bioremediating bacteria or growing their plants. The VHb technology is not a product per se, it is rather a method, a way of designing your expression system when the goal is to increase the productivity of the desired end product.
Our project promotes the introduction of molecular approaches in the pursuit of higher product yields for the biomanufacturing scene. It also introduces an additional design parameter by allowing the user to tweak the oxygen utilization on a cellular level. VHb has been tested and shown to be successful in various host organisms such as bacteria [12], yeast [7] and higher eukaryotes such as plants [8] and mammalian cells [17].
Multi-organism compatability
Plug-and-play ready
Additional design parameter
References
[1] Lee SY (1996) High cell-density culture of Escherichia coli. Trends Biotechnol 14(3):98–105.
[2] Losen, M. , Frölich, B. , Pohl, M. and Büchs, J. (2004), Effect of Oxygen Limitation and Medium Composition on Escherichia coli Fermentation in Shake‐Flask Cultures. Biotechnol Progress, 20: 1062-1068.
[3] Zhang, L., Li, Y., Wang, Z., Xia, Y., Chen, W., and Tang, K. (2007) Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. Biotechnology Advances 25, 123-136.
[4] Stark, B., Dikshit, K., and Pagilla, K. (2012) THE BIOCHEMISTRY OF VITREOSCILLA HEMOGLOBIN. Computational and Structural Biotechnology Journal 3, e201210002.
[5] Kapat, A. , Jung, J. and Park, Y. (2001), Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition. Journal of Applied Microbiology, 90: 216-222.
[6] Bowonder, B., Ramana, K., and Rajagopal, R. (1986) Waterlogging in irrigation projects. Sadhana 9, 177-190.
[7] Department of Agriculture & Cooperation. (2014) Yield per Hectare of Major Crops. Available at: https://data.gov.in/catalog/yield-hectare-major-crops
[8] Holmberg, N., Lilius, G., Bailey, J., and Bülow, L. (1997) Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production. Nature Biotechnology 15, 244-247.
[9] Cao, M., Huang, J., Wei, Z., Yao, Q., Wan, C., and Lu, J. (2004) Engineering Higher Yield and Herbicide Resistance in Rice by -Mediated Multiple Gene Transformation. Crop Science 44, 2206.
[10] Zhang, Z., Li, W., Li, H., Zhang, J., Zhang, Y., Cao, Y., Ma, J., and Li, Z. (2015) Construction and Characterization of Vitreoscilla Hemoglobin (VHb) with Enhanced Peroxidase Activity for Efficient Degradation of Textile Dye. Journal of Microbiology and Biotechnology 25, 1433-1441.
[11] Kahraman, H., and Geckil, H. (2005) Degradation of Benzene, Toluene and Xylene by Pseudomonas aeruginosa Engineered with the Vitreoscilla Hemoglobin Gene. Engineering in Life Sciences 5, 363-368.
[12] Kim, Y., Webster, D., and Stark, B. (2005) Improvement of bioremediation by Pseudomonas and Burkholderia by mutants of the Vitreoscilla hemoglobin gene (vgb) integrated into their chromosomes. Journal of Industrial Microbiology & Biotechnology 32, 148-154.
[13] Simeonov, L., and Sargsyan, V. (2008) Soil chemical pollution, risk assessment, remediation and security. Springer, Dordrecht.
[14] Science Communication Unit, University of the West of England, Bristol (2013). Science for Environment Policy In-depth Report: Soil Contamination: Impacts on Human Health. Report produced for the European Commission DG Environment, September 2013. Available at: http://ec.europa.eu/science-environment-policy
[15] Arnaldos M, Kunkel S.A, Wang J, Pagilla K.R, Stark B.C, (2012) Vitreoscilla hemoglobin enhances ethanol production by Escherichia coli in a variety of growth media. Biomass Bioenergy 37:1–8
[16] Stark, B.C., Pagilla, K.R. & Dikshit, K.L.. Appl Microbiol Biotechnol (2015) 99: 1627.
[17] Pendse, G., and Bailey, J. (1994) Effect of vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant chinese hamster ovary cells. Biotechnology and Bioengineering 44, 1367-1370.
