Team:Lund/Design/Applications

Design

Applications

As explained in the theory section, VHb can be used to increase the yield of proteins under oxygen-limited conditions. In this section, we present a review of some applications were the technology has proven itself useful. The practical benefits of VHb co-expression have been harnessed in order to increase the production of proteins [1][2] and growth of rice [3], maize [4] and the tobacco plant [5]. VHb can also be used in bioremediation of toxic compounds such as textile dyes [6] and aromatic compounds [7].

Enzymes

β-galactosidase is an enzyme commonly used for removal of lactose from milk, which is required to make it drinkable for more than half of the world’s population [8]. Wu et al. [1] managed to increase the production of this enzyme by 9.9% under non-limiting aeration, proving the benefit of VHb co-expression. The host organism Pichia pastoris demonstrated an improvement of 28.2% in oxygen uptake rate when grown in shake flasks. Another industrial enzyme, ɑ-amylase, was also co-expressed with VHb [2]. An 18% increase of the enzyme concentration was noted when cultivated in a bioreactor under non-limiting aeration. This strengthens the argument that VHb can emit similar benefits when scaling up the process.

Plants

Expression of VHb has also been studied in plants and shown to induce physiological changes. Wang et al. [9] managed to successfully integrate the VHb gene into Arabidopsis thaliana, which resulted in multiple positive effects: substantially taller plants, faster generation time and increased resistance to flooding compared to the wild-type.

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 [10]. In 2011, the average crop yield of rice, cereals and wheat in India was 2636 kg/hectare according to the Department of Agriculture & Cooperation [11]. To put this in perspective, 6 to 10 millions of hectares equals approximately 16 000 to 26 000 million kg of food affected 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 [5][12].

Bioremediation

Contamination of soil and groundwater stems from pharmaceutical residues [13], petroleum [14], aromatic hydrocarbons [15], organic dyes [16], pesticides [17] and heavy metals [18] Many of these contaminants can lead to serious disorders in the domestic microbial flora [19] and human population [20]. Bioremediation is an environmentally friendly solution to this issue and is used to treat heavily contaminated sites [21]. A frequent issue during 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.

Kahraman and Geckil [7] proposed a potential solution to the problem by expressing VHb in yeast capable of using benzene, toluene and xylene as carbon sources. Hypoxic conditions were emulated by limiting the oxygen supply and a significant increase in degradation of the contaminants was observed.

To prevent the release of genetically engineered bioremediating organisms into the environment, Urgun-Demirtas [22] used a membrane bioreactor commonly used in waste-water treatment. They studied the degradation of 2-chlorobenzoic acid by Burkholderia cepacia modified to express VHb. The transformed cells reached higher cell densities compared to their native counterparts, 3.2–5.4 g/L and 2.8–4.7 g/L respectively. Survivability in hypoxic oxygen concentrations also improved.

Biofuels

CO2 emission is expected to increase by approximately 20% before 2035 according to the Intergovernmental panel on Climate Change [23]. Biofuels have become a feasible alternative to fossil-based fuels and more car owners are committing to the change. The consumption of biofuels, mainly ethanol, is predicted to rise from 1.3 barrels/day in 2011 to 4.1 barrels/day by 2035 [24]. In turn, the demand for more efficient biofuel production will increase.

Ethanol makes up three fourths of biofuel use [24]. 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 [25]. Sanny et al. [26] and Arnaldos et al. [27] demonstrate that the expression of small amounts of VHb, despite of being counterintuitive for a fermentation process, resulted in a significant increase in ethanol production.

The delivery of small amounts of oxygen to the bacteria have generated an increase in ethanol production in E. coli using feedstocks as diverse as glucose (30% increase) [26], xylose (119%) [26], corn stover hydrolysate (59%) [26], potato processing waste water (5-18%) [28], cheese whey (21-419%) [29] and whey powder (17-362%) [29] among others. VHb have also been shown to increase ethanol production in yeast [30]. Also investigators in Hong Kong have shown that its expression in Aurantiochytrium sp. enhanced the production of fatty acids that could be used as precursors of the synthesis of biodiesel [31].

References

  1. [1] Wu, J., and Fu, W. (2012) Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia pastoris. Journal of Bioscience and Bioengineering 113, 332-337.
  2. [2] Suthar, D., and Chattoo, B. (2006) Expression of Vitreoscilla hemoglobin enhances growth and levels of α-amylase in Schwanniomyces occidentalis. Applied Microbiology and Biotechnology 72, 94-102.
  3. [3] 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.
  4. [4] Du, H., Shen, X., Huang, Y., Huang, M., and Zhang, Z. (2016) Overexpression of Vitreoscilla hemoglobin increases waterlogging tolerance in Arabidopsis and maize. BMC Plant Biology 16.
  5. [5] 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.
  6. [6] 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.
  7. [7] 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.
  8. [8] Heyman, M. (2006) Lactose Intolerance in Infants, Children, and Adolescents. PEDIATRICS 118, 1279-1286.
  9. [9] Wang, Z., Xiao, Y., Chen, W., Tang, K., and Zhang, L. (2009) Functional expression of Vitreoscilla hemoglobin (VHb) in Arabidopsis relieves submergence, nitrosative, photo-oxidative stress and enhances antioxidants metabolism. Plant Science 176, 66-77.
  10. [10] Bowonder, B., Ramana, K., and Rajagopal, R. (1986) Waterlogging in irrigation projects. Sadhana 9, 177-190.
  11. [11] Department of Agriculture & Cooperation. (2014) Yield per Hectare of Major Crops. Available at: https://data.gov.in/catalog/yield-hectare-major-crops
  12. [12] Cao, M., Huang, J., Wei, Z., Yao, Q., Wan, C., and Lu, J. (2004) Engineering Higher Yield and Herbicide Resistance in Rice by Agrobacterium-Mediated Multiple Gene Transformation. Crop Science 44, 2206.
  13. [13] Wojcieszyńska, D., Domaradzka, D., Hupert-Kocurek, K., and Guzik, U. (2014) Bacterial degradation of naproxen – Undisclosed pollutant in the environment. Journal of Environmental Management 145, 157-161.
  14. [14] Smułek, W., Zdarta, A., Guzik, U., Dudzińska-Bajorek, B., and Kaczorek, E. (2015) Rahnella sp. strain EK12: Cell surface properties and diesel oil biodegradation after long-term contact with natural surfactants and diesel oil. Microbiological Research 176, 38-47.
  15. [15] Rodgers-Vieira, E., Zhang, Z., Adrion, A., Gold, A., and Aitken, M. (2015) Identification of Anthraquinone-Degrading Bacteria in Soil Contaminated with Polycyclic Aromatic Hydrocarbons. Applied and Environmental Microbiology 81, 3775-3781.
  16. [16] Mohamed, A., El-Sayed, R., Osman, T., Toprak, M., Muhammed, M., and Uheida, A. (2016) Composite nanofibers for highly efficient photocatalytic degradation of organic dyes from contaminated water. Environmental Research 145, 18-25.
  17. [17] Moreno-Medina, D.A., Sánches-Salinas, E., Ortiz-Hernández, L. (2014) Removal of Methyl Parathion and Coumaphos Pesticides by a Bacterial Consortium Immobilized in Luffa Cylindrica. Revista Internacional de Contaminación Ambiental 30, 51-63
  18. [18] Wasilkowski, D., Mrozik, A., Piotrowska-Seget, Z., Krzyżak, J., Pogrzeba, M., and Płaza, G. (2014) Changes in Enzyme Activities and Microbial Community Structure in Heavy Metal-Contaminated Soil under in situ Aided Phytostabilization. CLEAN - Soil, Air, Water 42, 1618-1625.
  19. [19] Simeonov, L., and Sargsyan, V. (2008) Soil chemical pollution, risk assessment, remediation and security. Springer, Dordrecht.
  20. [20] 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
  21. [21] Dzionek, A., Wojcieszyńska, D., and Guzik, U. (2016) Natural carriers in bioremediation: A review. Electronic Journal of Biotechnology 23, 28-36.
  22. [22] Urgun-Demirtas, M., Stark, B., and Pagilla, K. (2006) Comparison of 2-chlorobenzoic acid biodegradation in a membrane bioreactor by B. cepacia and B. cepacia bearing the bacterial hemoglobin gene. Water Research 40, 3123-3130.
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