Part:BBa_K2770003
AceA Generator
Usage and Biology
Glycolic acid is a simple α-hydroxy acid. Through the functional hydroxy- and acid-groups the molecule is highly soluble in water. This property makes glycolic acid polymers attractive for many applications in industry, e.g. in the textile, leather, oil, and gas industry[1]. The polymer of glycolic acid exhibits excellent gas barrier properties, which is an optimal base for e.g. packaging materials[2]. To enable the production of glycolic acid, we modified the glyoxylate cycle, which branches off the TCA cycle.
When acetyl-CoA yielding C2-units, e.g. acetate or ethanol, act as a sole carbon source for organisms such as E. coli and S. cerevisiae, the glyoxylate cycle functions as an anaplerotic pathway replenishing succinate. The isocitrate lyase converts isocitrate into succinate and glyoxylate and thereby enables the bypassing of both decarboxylation steps of the TCA cycle. Another key enzyme is the malate synthase, which condenses glyoxylate with two acetyl-CoA molecules to isocitrate. The intermediate glyoxylate can also be reduced by a glyoxylate reductase to glycolic acid. [3]
The isocitrate lyase is called aceA, which catalyzes the conversion of isocitrate into glyoxylate and succinate. It is natively found in the E. coli strain K12, but not expressed under normal conditions. Because of this, a T7 lac promotor (BBa_K921000) and a B0034-based ribosomal binding site (BBa_K2380024) were inserted upstream of the coding sequence via the BioBrick assembly to express YcdW. Together with the enzyme YcdW (BBa_K2770002), a codon optimized version of the gene aceA was used to produce glycolic acid. The product could then be used for polymerization with further monomers, e.g. Lactic acid and Caprolactone.
Figure 1: Tetrameric structure of the isocitrate lyase (AceA) with a molecular mass of 47.5 kDa per subunit.
To learn more about AceA and its part in our project, visit our
[http://2018.igem.org/Team:TU_Darmstadt/Project/Glycolic_acid/E_coli wiki].
Mechanism
The isocitrate lyase catalyzes the conversion of isocitrate into glyoxylate and succinate. It uses Mg2+ as a cofactor. [4].
Figure 2: Reaction mechanism of AceA.
Methods
Cloning
The sequence aceA was modified with a His-tag, ordered from Integrated DNA Technologies (IDT), and inserted into the vector pSB1C3. For this purpose, the BioBrick assembly (BBa) was used. A T7 lac promotor (BBa_K921000) and a B0034-based ribosomal binding site (BBa_K2380024) were inserted upstream of the coding sequence via the BBa as well. E. coli TOP10 were transformed with generated plasmids and positive colonies were identified via colony PCR and DNA sequencing.
Figure 3: pSB1C3 plasmid including expression cassette of aceA.
SDS-PAGE and Western Blot
To verify that AceA was produced, a SDS-PAGE was performed, followed by a western blot. The resulting bands were compared to the molecular weight of AceA.
Purification
After production of AceA in E. coli BL21, by induction of the T7 lac promotor with IPTG, an ÄKTA chromatography system (GE Healthcare, Illinois, USA) was used to purify the desired His-tagged enzyme.
Activity assay
The purified enzymes were spectrophotometrically assayed for their activity using a plate reader. For the assay of the isocitrat lyase (AceA), the enzyme activity was tested via a phenylhydrazine-dependent reaction. As soon as AceA converts isocitrate into glyoxylate, the product reacts with phenylhydrazin to glyoxylate-phenylhydrazone, which possesses an absorption maximum at 324 nm. The change in absorption at 324 nm was measured over time. A calibration curve was created to calculate the glyoxylate-phenylhydrazone conversion rate. Therefore, the absorption of different concentrations of glyoxylate and phenylhyrazine was used.
Figure 3: Reaction mechanism of phenylhydrazine-dependent assay for the verification of AceA enzyme activity.
Results
The aceA gene was cloned in E. coli TOP 10. Furthermore, the plasmid was successfully transformed into BL21 cell lines to enable protein production under the control of an IPTG inducible T7-lac promotor (BBA_K921000). An attached His-tag was used to purify the encoded enzymes via an ÄKTA system. The subsequent SDS-PAGE showed bands of the expected protein size. We also proved the successful production and purification of the enzymes via western blot (see fig.4). The expected size was 47.5 kDa for AceA, respective bands could be detected on the western blot.
Figure 4: western blot analysis of purified proteins AceA and YcdW. Thermo Scientific PageRuler Prestained Protein Ladder was used. The nitrocellulose membrane was incubated with an anti-His antibody. For protein detection a mouse anti-rabbit antibody conjugated with a horseradish-peroxidase was used.
As seen in figure 4 on the left side, AceA shows a signal at the expected size. This indicates its successful purification and production in E. coli. Hence, we started the protein characterization via in vitro enzyme assays.
Assay
We verified the enzyme activity of AceA via a phenylhydrazine-dependent assay with the collected protein fractions from the ÄKTA purification. Isocitrate was included as a substrate. By adding phenylhydrazine to the reaction, glyoxylate produced during the assay (reaction time of 30 minutes) will further react to glyoxylate-phenylhydrazone, which absorbs light at a wavelength of 324 nm (see [http://2018.igem.org/Team:TU_Darmstadt/Project/Glycolic_acid/E_coli#Enzyme_assays methods]). Hence, we were able to indirectly measure glyoxylate production over time. We also included a negative control without enzyme, which is expected to stay on a constant absorption level, because no isocitrate is converted into glyoxylate. We used different enzyme concentrations (0.03 µg/µL; 0.15 µg/µL; 0.3 µg/µL; 0.6 µg/µL) and temperatures (21 °C/25 °C/37 °C) to screen for the best conditions.
Referring to Robertson et al. [5], we first carried out assays with different enzyme concentrations at 25 °C. Figure 5 shows the calibration curve for the AceA assay, which is used to calculate the substrate turnover per time. To create the calibration curve, the absorption of different glyoxylate-phenylhydrazone concentrations was measured and compared to the resulting assay absorptions (Figure 6). For the AceA enzyme assay it was expected that a higher concentration would result in a faster substrate conversion.
Figure 5: Calibration curve for AceA enzyme assay with different glyoxylate concentrations (0 mM, 0.125 mM, 0.1875 mM, 0.25 mM, 0.375 mM, 0.5 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM, 3 mM and 4 mM).
Figure 6: AceA enzyme assay at 25 °C and 324 nm. The graph shows the average absorption in correlation to the time (min). Reactions with different enzyme concentration were measured, as well as a negative control sample without enzyme.
All used samples reach an absorption (324 nm) of approximately 3.7, but with different substrate conversion rates. Higher absorption values than 3.7 are not detectable by the plate reader. Hence, it is not possible to determine the exact yield. The negative control stays on a constant absorption level, which indicates that the increase in absorption of the enzyme containing samples is based on enzymatic activity.
As expected, figure 6 shows that a higher enzyme concentration leads to a faster turnover of glyoxylate. The highest concentration of 0.6 µg/µL results in a substrate conversion rate of 0.912 µM/s. Thus, we decided to carry out further analysis regarding the optimal temperature with an enzyme concentration of 0.6 µg/µL.
To study the enzyme activity at different temperatures, we next performed the assay at 21 °C and 37 °C. The substrate conversion per time (µM/s) is shown in table 1. To achieve this, the substrate conversion values were recorded in the linear range of the particular curve in a time frame of 3.5 minutes. It should be noted that the determination does not start at the exact same time for all samples, but the time frame of 3.5 minutes was retained. Three replicates were measured for every assay.
Table 1: Substrate conversion of AceA per time (µM/s) in correlation to different temperatures (21 °C, 25 °C and 37 °C).
| Temperature [°C] | Substrate conversion
per second [µM/s] |
|---|---|
| 21 | 0.82 |
| 25 | 0.912 |
| 37 | 0.914 |
Table 1 shows that the enzyme performs best at a temperature of 25 °C and 37 °C with a substrate conversion of 0.912 µM/s and 0.914 µM/s. At a temperature of 21 °C the enzyme shows its lowest conversion rate of 0.82 µM/s. Referring to Robertson et al. [5] the 25 °C assay was used for further analysis.
A Student's t-test was performed to check the significance of our data. For this test, five data points from the 25 °C assay were chosen (0, 5, 10, 20 and 30 minutes) and plotted in figure 7 for a better visualization.
Figure 7: Enzyme assay absorption for 0, 5, 10, 20 and 30 minutes with standard error. n=3
The absorptions, as well as the particular standard errors, are also shown in table 2. Significance is indicated by *.
Table 2: Calculated significance of AceA data sets for 0, 5, 10, 20 and 30 minutes, 25 °C. The table also shows the absorption as well as the particular standard error. A= Absorption, *= Significance (p-value < 0.05), n= 3
| AceA [µg/µL] | A (t=0 min) | A (t=5 min) | A (t=10 min) | A (t=20 min) | A (t=30 min) |
|---|---|---|---|---|---|
| 0.03 | 1.046±0.016* | 1.665±0.018 | 1.32±0.058 | 1.645±0.258 | 1.937±0.249 |
| 0.15 | 1.071±0.009* | 1.609±0.1* | 2.456±0.222* | 3.639±0.114* | 3.732±0,003* |
| 0.3 | 1.089±0.003 | 1.793±0.04* | 2.84±0.091* | 3.19±0.017* | 3.732±0.012* |
| 0.6 | 1.175±0.023 | 2.698±0.225* | 3.795±0.083* | 3.909±0.027* | 3.759±0.02* |
| neg. | 1.105±0.005 | 1.13+0.007 | 1.154+0.007 | 1.191±0.006 | 1.209±0.006 |
As shown in table 2, most of the data points show a p-value < 0.05, which indicates that the data is significant. Non-significant p-values appear in the data set of the lowest enzyme concentration.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 230
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ Dischert et al., United States Patent Application Publication, Jul.12,2012 [1].
- ↑ Engineering Escherichia coli for glycolic acid production from D-xylose through the Dahms pathway and glyoxylate bypass. [2].
- ↑ Stryer, Lubert(2002): Biochemistry, 5th Edition, New York: W H Freeman.
- ↑ UniProtKB – P0A9G6 (ACEA_ECOLI), 10/14/2018 [3].
- ↑ 5.0 5.1 Eugene F. Roberston and Henry C. Reeves, Purification and characterization of isocitrate lyase from Escherichia coli, Current Microbiology Vol.14 (1987), pp. 347-350 [4].
| None |