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Microorganisms are used to produce many valuable products such as pharmaceuticals, food additives and enzymes. However, the production process can be expensive and achieving a high yield is sometimes difficult, with the oxygen supply being a common bottleneck. One approach in combating this problem is the co-expression of Vitreoscilla hemoglobin (VHb). The co-expression is often linked with increased cell densities and higher protein yields. However, few studies have to our knowledge investigated to what degree the protein should be expressed in order to provide beneficial effects. In addition, we have not found any standardized approach in implementing VHb into already existing systems.

Vitreoscilla hemoglobin is a protein found in the aerobic bacterium Vitreoscilla. It has been proposed that its main role is to deliver oxygen to terminal respiratory oxidases and thereby increase respiration when the oxygen availability is low, and it may also function as a terminal oxidase itself¹. Several studies have shown that expression of the gene coding for VHb, vgb, can improve cell growth^2,3,4 as well as increase the production of, among other things, enzymes^5,6, antifungals⁷ and ethanol⁸. </p>

We hypothesize that expressing only a small amount of vgb will lead to an insufficient oxygen utilization and consequently non-optimal cell productivity. On the other hand, overexpressing vgb will result in a depletion of cell resources as the relative amount of energy gained from the increased metabolism will be overtaken by the amount of protein expressed, The optimal conditions should therefore be approachable by an expression level which is somewhere in-between. </p>

To investigate the hypothesis, our aim is to co-express VHb under promoters of varying strength along with a target protein in Escherichia coli. This will be accomplished by using the library of Anderson promoters from the iGEM registry, which are characterized by their RNA polymerase binding strengths. Consequently, we want to measure various properties such as protein yields, cell densities and metabolite concentrations in order to study the effect of increased VHb expression. As such, we wish to first investigate the effect of increased VHb levels and then test our system in a real upscale setting. </p>

In our contribution to iGEM, our ambition is to provide a library of vgb expression systems with varying promoter strengths. With this available, the amount of increased oxygen uptake can be tailored specifically for the intended application. This gives a new degree of freedom in the experimental design when considering the use of oxygen carriers in synthetic biological systems. The parts could for example be used to increase protein yields under upscaling or increase cell survivability in low oxygen environments.</p>

[1] Stark, B. C., Dikshit, K. L. and Pagilla, K. R. (2012). The Biochemistry of Vitreoscilla hemoglobin. Computational and Structural Biotechnology Journal 3, e201210002. http://doi.org/10.5936/csbj.201210002
[2] Khosla, C., and Bailey, J. (1988) Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli. Nature 331, 633-635.
[3] Wu, JM. and Fu, WC. (2012) Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia pastoris. J Biosci Bioeng 113(3), 332–337.

[4] Pablos, T. E., Mora, E. M., Le Borgne, S., Ramírez, O. T., Gosset, G. and Lara, A. R. (2011), Vitreoscilla hemoglobin expression in engineered Escherichia coli: Improved performance in high cell‐density batch cultivations. Biotechnology Journal, 6: 993-1002. doi:10.1002/biot.201000405

[5] Khosravi, M., Webster D. A. and Stark, B.C. (1990). Presence of the bacterial hemoglobin gene improves α-amylase production of a recombinant Escherichia coli strain. Plasmid 24(3), 190-194.

[6] Wu, JM., Wang, SY., Fu, WC. (2012). Lower Temperature Cultures Enlarge the Effects of Vitreoscilla Hemoglobin Expression on Recombinant Pichia pastoris. Int. J. Mol. Sci. 13(10), 13212-13226; doi:10.3390/ijms131013212

[7] Wang S, Liu F, Hou Z, Zong G, Zhu X, Ling P. Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) World J Microb Biot. 2014;30:1369–1376. doi: 10.1007/s11274-013-1561-4

[8] Sanny, T., Arnaldos, M., Kunkel, S.A. et al (2010). Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose. Appl Microbiol Biotechnol 88, 1103-1112. https://doi.org/10.1007/s00253-010-2817-7

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Contact: igemlund@gmail.com

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Welcome to iGEM Lund 2018!