Team:METU HS Ankara/Basic Part

METU HS IGEM

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Basic Parts

Name Type Description Designer Length
BBa_K2571000 FucO /L-1,2-propanediol oxidoreductase Tugba Inanc & Ceyhun Kayihan 1152bp
BBa_K2571001 Bifunctional gamma-glutamate-cysteine ligase/Glutathione synthetase Tugba Inanc & Ceyhun Kayihan 2268bp

FucO (BBa_K2571000)

FucO is a protein-coding region that codes for L-1,2-propanediol oxidoreductase which is an NADH-linked, homodimer enzyme having the role of acting on furfural. Furfural is a highly toxic substance which is a toxic inhibitor of microbial fermentations causing cell wall and membrane damage, DNA breakdowns, DNA cleavages and reduced enzymatic activities (Zheng, 2013; Liu, Ma & Song, 2009).

In the presence of furfural, NADPH-dependent oxidoreductases go active in order to reduce furfural to its less toxic alcohol derivative - furfuryl alcohol (Zheng, 2013; Wang et al., 2013; Allen et al., 2010). In this pathway, the expression of oxidoreductases that are NADPH-dependent, such as YqhD, are shown to inhibit the growth and fermentation in E. coli by competing with biosynthesis for NADPH (Zheng, 2013).

Because the native conversion of NADH to NADPH in E. coli is insufficient to revitalize the NADPH pool during fermentation, the actions shouldn’t be interfering with NADPH metabolism (Wang et al. 2011). Thus, the overexpression of plasmid-based NADH-dependent propanediol oxidoreductase (FucO) gene reduces furfural to ultimately improve furfural resistance without detrimentally affecting the biosynthesis of NADPH (Wang et al. 2011).


Figure 1: BBa_K2571000: fucO was cloned into pSB1C3.

Figure 2: BBa_K2571000 check with FucO left and VR primers. Expected band length: 625 bp. FucO basic well shows positive results.

We’ve inserted the gene FucO, which is our basic part 1, to pSB1C3 backbone and transformed it to DH5-alpha. After plasmid isolation, we’ve checked the orientation with FucO left and VR primers and expected to see a band of 625 bp.
FucO and VR primers are as below:
FucO left: GTGATAAGGATGCCGGAGAA
VR: ATTACCGCCTTTGAGTGAGC

GSH (BBa_K2571001)

GSH is a protein-coding region that codes for Bifunctional gamma glutamate cysteine ligase/Glutathione synthetase.

Glutathione (GSH) is known to be an important antioxidant that is a sulfur compound; a tripeptide composed of three amino acids (cysteine, glycine and glutamic acid) and a non-protein thiol (Pizzorno, 2014; Lu, 2013). GHS is, furthermore, found in thiol-reduced form which accounts for its strength as an antioxidant.

Reactive oxygen species (ROS) are harmful substances that distort protein based matters by taking electrons and also causes oxidative stress (Lu, 2013) which occur during the fermentation process and is another major setback. The chemical structure of the protein-based substances such as the DNA are altered and become therefore become dysfunctional because of ROS (Lu, 2013; Burton & Jauniaux, 2011).

GSH is generally found in the thiol-reduced form which is crucial for detoxification of ROS and free radicals. which cause oxidative stress. (Lu, 2013; Burton & Jauniaux, 2011).

Antioxidants like GSH play an important role in the detoxification of ROS and reactive oxygen species by directly acting as electron donors, changing the unbalanced electron state of the free radicals and turning them into less harmful substances or affect them indirectly by getting in the way of the expression of free radical generating enzymes (Lü et al., 2014).


Figure 3:BBa_K2571001: GSH was cloned into pSB1C3.

Figure 4: BBa_K2571001 check with GSH specific primers. Expected band length: 225 bp. GSH basic well shows positive results.

We’ve inserted the gene GSH, our basic part 2, to pSB1C3 backbone and transformed it to DH5 alpha. After plasmid isolation, we’ve checked the orientation with GSH specific primers and expected to see a band of 225 bp.
GSH left and right primers are shown as below:
GSH left: TCGGAGGCTAAAACTCAGGA
GSH right: GTGGGCAGTCCAGTCGTAAT

References

  • Allen, S. A., Clark, W., McCaffery, J. M., Cai, Z., Lanctot, A., Slininger, P. J., … Gorsich, S. W. (2010). Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae. Biotechnology for Biofuels, 3, 2. http://doi.org/10.1186/1754-6834-3-2
  • Chou, H.-H., Marx, C. J., & Sauer, U. (2015). Transhydrogenase Promotes the Robustness and Evolvability of E. coli Deficient in NADPH Production. PLoS Genetics, 11(2), e1005007. http://doi.org/10.1371/journal.pgen.1005007
  • Liu, Z.L., Ma M., Song, M.(2009). Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways. Mol Genet Genomics 282, 233-244. doi: 10.1007/s00438-009-0461-7
  • Wang, X., Miller, E. N., Yomano, L. P., Zhang, X., Shanmugam, K. T., & Ingram, L. O. (2011). Increased Furfural Tolerance Due to Overexpression of NADH-Dependent Oxidoreductase FucO in Escherichia coli Strains Engineered for the Production of Ethanol and Lactate. Applied and Environmental Microbiology, 77(15), 5132–5140. http://doi.org/10.1128/AEM.05008-11
  • Zheng, H., Wang, X., Yomano, L.P., Geddes, R.D, Shanmugan, K. T., Ingram, L.O. (2013). Improving Escherichia coli FucO for Furfural Tolerance by Saturation Mutagenesis of Individual Amino Acid Positions. Applied and Environmental Microbiology Vol 79, no 10. 3202–3208. http://aem.asm.org/content/79/10/3202.full.pdf+html
  • Lu, S. C. (2013). Glutathione Synthesis. Biochemical et Biophysica Acta, 1830(5), 3143–3153. http://doi.org/10.1016/j.bbagen.2012.09.008
  • National Center for Biotechnology Information. PubChem Compound Database; CID=124886, https://pubchem.ncbi.nlm.nih.gov/compound/124886 (accessed July 18, 2018). https://pubchem.ncbi.nlm.nih.gov/compound/124886#section=Top
  • Pizzorno, J. (2014). Glutathione! Integrative Medicine: A Clinician’s Journal, 13(1), 8–12. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684116/
  • Burton, G. J., & Jauniaux, E. (2011). Oxidative stress. Best Practice & Research. Clinical Obstetrics & Gynaecology, 25(3), 287–299. http://doi.org/10.1016/j.bpobgyn.2010.10.016
  • Lü, J.-M., Lin, P. H., Yao, Q., & Chen, C. (2010). Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems. Journal of Cellular and Molecular Medicine, 14(4), 840–860. http://doi.org/10.1111/j.1582-4934.2009.00897.x