Part:BBa_K1674004
zwf - Glucose-6-Phosphate-Dehydrogenase from E.coli
Introduction
The zwf gene encodes to Glucose-6-Phosphate-Dehydrogenase enzyme (G6PD) which catalyzes the oxidation of glucose-6-phosphate to 6-phosphoglucono-δ-lactone [1].|
Figure 1: Glucose-6-Phosphate-Dehydrogenase catalyzes the reaction between glucose-6-phosphate to 6-phosphoglucono-δ-lactone while generates one molecule of NADPH. Drew with https://www.emolecules.com |
G6PD is a key enzyme as it catalyzes the first step to the Pentose-phosphate (PP) and Embden-Meyerhoff (ED) pathways [2]. Consequently, regulation of this enzyme allows controlling of the bacteria metabolism. Respectively, over-expression of G6PD causes over-production of NADPH as the G6PD reaction itself generates one mole of NADPH followed by another reaction that gives one more NADPH - 2 NADPH moles in total, as can be seen in figure 2.
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Figure 2: One of the major NADPH resources in E.coli is pentose phosphate pathway. Glucose-6-Phosphate-Dehydrogenase is the key enzyme to this pathway [4]. |
Many processes within the cell, like reduction and oxidation reactions, need NADPH as a reduction power. As the commercial substitutes of NADPH are expensive and unstable, we searched a way to produce NADPH using synthetic biology.
In order to achieve excess of NADPH we engineered an E.coli
BL21 with the zwf gene under a T7 promoter to get over-expression of G6PD.
The NADPH measurements were done using NADPH fluorescence.
Our experiment results show that zwf over-expression leads to enhancement in extracellular fluorescence which implies an increasing NADPH concentration in comparison to the E.coli wild-type.
The over-expression of Glucose-6-Phosphate-Dehydrogenase
is a great way to ensure sufficient NADPH supply without using external
analogs for NADPH which are expensive and sensitive.
The zwf gene was cloned into pSB1C3 as well as to pSB1C3 with T7 promoter (BBa_K1674005) in order to over-express it in E.coli BL21.
The BioBrick sequence was validated by sequencing.
SDS-PAGE
SDS-PAGE shows the protein Glucose-6-Phosphate-Dehydrogenase at the expected size of 55 kDa.
The protein zwf under T7 promoter giving a strong band compared to a weak band in the E.coli BL21 wild-type.
Figure 3:
The SDS-PAGE gel displays the protein Glucose-6-Phosphate-Dehydrogenase
at the right size of 55kDa. It is over-produced in BL21 with zwf under T7 promoter.
Extracellular NADPH Assay
The protocol for this assay can be seen
here.
We checked NADPH fluorescence at 340 nm excitation and 460 nm emission. We chose to check
fluorescence as opposed to absorbance since it has been found to be more specific and sensitive
method than absorbance. The NADPH measurements were done by reading fluorescence in a 96-well plate.
The fluorescence readings were normalized by the culture’s O.D at 600 nm at a given time.
Glucose-6-phosphate dehydrogenase gene activity
At first, we wanted to see whether the zwf over-expression under the pT7 promoter
(BBa_K1674005)
in E.coli BL21 gave us the expected enhancement in NADPH.
We observed that, as expected, the over-expression of glucose-6-phosphate dehydrogenase
generated more NADPH, as can be seen in Figure 4, since there was a higher
fluorescence/O.D.600nm in E.coli BL21 with zwf compared to E.coli BL21 wild-type
Figure 4:
Normalized Extracellular NADPH fluorescence vs. Time for E. coli BL21 strains
Figure 4 illustrates that from time 0 until 12 hours, the fluorescence levels
were similar in both in E.coli BL21 wild-type and in E.coli BL21 with zwf. After
12 hours a significant increase in the fluorescence/O.D.600nm was observed in the
strain containing the zwf compared to the wild-type.
Consultations with academic staff led us to believe that NADPH is not
excreted from the system. Therefore, we assume that the drastic change
in fluorescence after 12 hours is probably because of natural lysis of the
cells in the medium. If so, the values observed may give an indication of the
amount of intracellular NADPH in the strains. This assumption is supported by the
decrease in O.D.600nm parallel to the increase in fluorescence values at this point in time.
Intracellular NADPH Assay
The protocol for this assay can be seen
here.
In order to further investigate the effect of the over-expression of zwf
with BBa_K1674005, we measured the intracellular NADPH. In order to do so,
we had to disrupt the cells without oxidizing the NADPH molecules. After a
few unsuccessful measurements of the intracellular content using a sonication
method, we decided to perform an experiment using various lysis procedures on
E. coli BL21 with and without the zwf gene.
The results showed a higher intracellular NADPH concentration in the strain
with the zwf gene for every lysis method used (Figure 5). Thus we concluded
that the gene successfully enhances intracellular NADPH. The preferred lysis
methods for NADPH concentrations, according to the results, are methods #3 and #7.
Figure 5:
Intracellular NADPH fluorescence in E. coli BL21 with and without the zwf gene
In the future, we hope to use these results to perform further experiments using the
E. coli MG1655 knockout strain along with a plasmid for the over-expression of G6PD.
NADPH Overproduction as a Function of Glucose Concentrations
We attempted to check the NADPH concentrations when
the bacteria were grown with and without glucose in
the medium. Glucose is a precursor of glucose-6-phosphate,
the substrate of glucose-6-Phosphate dehydrogenase. Therefore,
we wanted to check if the addition of more substrate would lead to more NADPH production.
This experiment’s results were not conclusive, so further experiments are needed.
Conclusion
For more accurate results in the future, we plan to use alternative
methods such as HPLC, allowing the separation of NADPH from NADH,
potentially giving us better insight into the NADPH concentrations in the clones.
combined experiment
After achieving NADPH enhancement with E.Coli BL21 carrying
BBa_K1674005
we progressed into a combined experiment involving
the 3α-hydroxysteroid dehydrogenase (3α-HSD) enzyme.
In this experiment we used the extracellular medium of E.coli BL21 with BBa_K1674005 after 25
hours of growth, enriched with NADPH over-produced by G6PD. Next we added lysate of E.coli
BL21 with BBa_K1674002,
over-expressing the 3α-HSD enzyme, along with the substrate dihydrotestosterone (DHT).
The results show similar behavior, as in the experiments involving the
commercial NADPH, indicating that the NADPH in the supernatant is in adequate concentration of NADPH for the activity of the
over-expressed 3α-HSD enzyme. Still further condition optimization is needed.
The results are shown at figure 6:
Figure 6:
combined experiment results. Extracellular medium of E.coli
BL21 with BBa_K1674005 combined with lysate of E.coli BL21
with BBa_1674002 along with the substrate dihydrotestosterone (DHT).
Design considerations
Experiments and results
references
Sequence and Features
| None |