Team:Technion Israel/Project/Cofactor

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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 NADPH 2.

Figure 1:Androgen metabolism in the human prostate 1

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 shown that in the presence of NADPH not only is the reduction reaction preferred, but the oxidation reaction is inhibited as well 1.

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 pathway 3,4.

We chose two ways to increase the flux to the pentose phosphate pathway and generate more NADPH:

  1. 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-type 3.
    We received the E.coli MG1655 knockout courtesy of Prof. Toby Fuhrer, Institute of Molecular Systems Biology, Switzerland.
  2. 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:

Figure 2: Main metabolic pathways in E.coli 2. The knockout E.coli MG1655 strain we received has deletions in pgi and UdhaA. Additionally, we over-expressed the zwf gene with BBa_1674005.

zwf expression

The goal of this component of the project was to examine each system separately and determine the implications of the E. coli MG1655 knockout and zwf over-expression in E. coli BL21.

Then, we tried to combine both approaches by cloning the zwf gene into the E. coli MG1655 knockout strain.

To check the expression of the zwf gene under a strong promoter in a good biological expression system, we inserted the gene into the plasmid pSB1C3 containing a pT7 promoter to create BBa_K1674005, and transformed the ligation product into E.coli BL21. A schematic representation of the plasmid can be seen in Figure 3 below.

Figure 3: BBa_K1674005- pSB1C3 plasmid backbone with pT7 + RBS + zwf

Since E.coli MG1655 lacks the T7 polymerase needed to express genes under the pT7 promoter, we sought another inducible promoter. Prof. Roee Amit’s lab gave us a plasmid, A133-rhlR, which includes the rhlR promoter and a rhlR regulator operon. The promoter is almost completely not leaky and induced by butanoyl-homoserine lactone (C4HSL). In the RhlR system, in the presence of the inducer, butanoyl-homoserine lactone (C4HSL), the RhlR protein activates transcription while in the absence of the inducer the RhlR acts as a transcriptional repressor. This makes the promoter almost completely non-leaky. 5

We cloned the zwf gene into this plasmid and then transformed into E.coli MG1655 wild-type and into the MG1655 knockout strain mentioned above. The resulting plasmid structure can be seen in Figure 4 below.

Figure 4: A133-RhlR with zwf

All parts were verified by sequencing.

Experimental verification

Expression

In order to verify the expression of the zwf gene, we performed SDS-page. The result can be seen in Figure 5 below.

Figure 5: SDS-page results for verification of glucose-6-phosphate dehydrogenase overexpression

In order to check intracellular and extracellular NADPH, we checked fluorescence at 340 nm excitation and 460 nm emission. We chose fluorescence as it more specific and sensitive method than absorbance 7. However, it is important to note that NADH and NADPH fluoresce at similar wavelengths 6. See our Proof of Concept protocols for specific information about the experiments.

Click here to see our results.



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.
6. George H. Patterson, Susan M. Knobel, Per Arkhammar, Ole Thastrup, and David W. Piston.: Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet β cells. PNAS 2000 97 (10) 5203-5207.
7. PH (BioTek Instruments). Determination of NADH Concentrations with the SynergyTM 2 Multi-Detection Microplate Reader using Fluorescence or Absorbance. 2007. Available at: http://www.biotek.com/resources/docs/NADH_App_Note.pdf. Accessed September 18, 2015.

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