Difference between revisions of "Team:Technion Israel/Project/Cofactor"
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<p>The manipulations detailed above are, as stated, shown in Figure 2 below:</p> | <p>The manipulations detailed above are, as stated, shown in Figure 2 below:</p> | ||
| − | <figure><img class="img_ctr" src="https://static.igem.org/mediawiki/2015/e/ | + | <figure><img class="img_ctr" src="https://static.igem.org/mediawiki/2015/e/e4/Technion_Israel_2015_zwf_overview_figure2.png"/><figcaption>Figure 2:Androgen metabolism in the human prostate<sup><a href="#fn1" id="ref1">1</a></sup></figcaption></figure> |
Revision as of 23:07, 17 September 2015
Cofactor
Introduction
Aim
To express the NADPH cofactor in order to create a better environment for the activity of the 3α-HSD enzyme.
The Cofactor
Co-factors such as NADH and NADPH are essential for most enzymatic reactions, 3α-hydroxysteroid dehydrogenase (3α-HSD) among them. 3α-HSD enzyme catalyzes the reaction between 5α-dihydrotestosterone (DHT) and 3α-androstanediol (3α-Diol) using NADH and NADPH as co-factors. Studies show that this enzyme has higher affinity to NADPH2.
While the reaction is reversible, and the oxidation direction isn’t preferable in our system, there is a necessity to ensure that the reaction only occurs in the desired direction in our system: the reduction of 5α-DHT into 3α-Diol. Additionally, studies have showed that in the presence of NADPH not only is the reduction reaction preferred, but the oxidation reaction is inhibited as well1.
Therefore, we searched for a way to overproduce NADPH through synthetic biology and thereby provide the 3α-HSD enzyme with a cofactor, in excess.
Genetic approach
NADPH, a molecule with major reducing power in organisms, is mostly generated in the pentose phosphate pathway3,4.
We chose two ways to increase the flux to the pentose phosphate pathway and generate more NADPH:
- We used E.Coli MG1655 with knockouts of the pgi and UdhA genes. The deletion of pgi prevents the cells from using the glycolysis pathway and forces the metabolism into pentose phosphate pathway. The UdhA gene encodes a soluble transhydrogenase, which is believed to play a role in maintaining the balance between NADH and NADPH levels in the cell. Deletion of the UdhA gene inhibits the transformation of NADPH into NADH, thus enhancing the intracellular levels of NADPH. See Figure 2 for a schematic outline of the relevant metabolic pathways. As a result of these modifications, the E.coli MG1655 Δpgi ΔUdhA strain is able to produce more NADPH than the E.coli MG1655 wild-type3. We received the E.coli MG1655 knockout courtesy of Prof. Toby Fuhrer, Institute of Molecular Systems Biology, Switzerland.
- Glucose-6-phosphate dehydrogenase (G6PD), encoded by the zwf gene, is a key enzyme in bacterial metabolism, as it is involved in both the glycolysis and pentose phosphate pathways. The G6PD enzyme catalyzes the oxidation of glucose-6-phosphate to 6-phosphoglucono-δ-lactone while generating NADPH 4. Over-expressing zwf leads to over-production of the G6PD enzyme, which will yield higher concentrations of NADPH when compared to the wildtype. See Figure 2 for a schematic outline of the relevant metabolic pathways. We cloned zwf from E.coli DH5α origin into a plasmid, and over-expressed it under the control of 2 different promoters. The zwf gene was kindly contributed to us by Prof. Hanna Engleberg-Kulka’s lab, Hebrew University of Jerusalem, Israel.
The manipulations detailed above are, as stated, shown in Figure 2 below:
1. Tea L. R.; Hsueh K. L.; Donna M. P.; Stephane S.; David R. B.; Trevor M. P.: Human type 3 3α-Hydroxysteroid Dehydrogenase (aldo-keto reductase 1C2) and androgen metabolism in prostate cells. Endocrinology. 2003. 144(7). 2922-2932. 2. Susan S. H.; John E. P.; Pedro M. A.; Trevor M. P.; Mitchell L.: Tree-dimensional structure of rat liver 3α-hydroxysteriod/dihydrodiol dehydrogenase: A member of the aldo-keto reductase superfamily. PNAS. 1994.91. 2517-2521. 3. Fabrizio C.; Tracy A. H.; Sylvia H.; Taotao W.; Thomas S.; Uwe S.: Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA. FEMS Microbiology Letters. 2001. 204. 247-252. 4. Sang-Jun L.; Young-Mi J.; Hyun-Dong S.; Yong-Hyun L.: Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E.coli transformant harboring a cloned phbCAB operon. Journal of bioscience and bioengineering. 2002. 93. 543-549. 5. Gerardo M.; Katy J.; Brenda V.; Gloria S.C.: Mechanism of Pseudomonas aeruginosa RhlR transcriptional regulation of the rhlAB promoter. Journal of bacteriology. 2003. 185(2). 5976-5983.