Part:BBa_K2770002
ycdW 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] This enzyme is called YcdW. It is a NADPH-dependent glyoxylate reductase, which 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 AceA (BBa_K2770003), a codon optimized version of the gene ycdW 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: Structure of the NADPH-dependent glyoxylate reductase (YcdW) with a molecular mass of 35.3 kDa.
To learn more about YcdW and its part in our project, please visit our [http://2018.igem.org/Team:TU_Darmstadt/Project/Glycolic_acid/E_coli wiki].
Mechanism
As written above, YcdW is a NADPH dependent glyoxylate reductase, that catalyzes the conversion of glyoxylate into glycolic acid [4].
Figure 2: Reaction mechanism of YcdW
Methods
Cloning
The sequence ycdW 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 ycdW.
SDS-PAGE and Western Blot
To verify that YcdW was produced, a SDS-PAGE was performed, followed by a western blot. The resulting bands were compared to the molecular weight of YcdW.
Purification
After production of YcdW 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 enzyme was spectrophotometrically assayed for its activity using a plate reader. The assay for the glyoxylate reductase YcdW is based on the different absorption maxima of NADPH (absorption maximum at 340 nm) and NADP (maximum at 260 nm). During the reaction the enzyme uses NADPH as a cofactor. NADPH is converted into NADP+ which leads to a decrease of absorption at 340 nm. By measuring the absorption level over time, it is possible to quantify the enzyme activity and infer glycolic acid production.
HPLC analysis
To detect the produced monomer glycolic acid, as well as the precursors, high-performance liquid chromatography (HPLC) was employed. An organic acid separation column as stationary phase and sulfuric acid as mobile phase allowed the separation of isocitrate, glyoxylate and glycolic acid. Signals were recorded by a refractive index detector.
M9 assay
The growth of E. coli BL21 transformed with an YcdW-generating plasmid was studied in M9 minimal media with and without a supplemented carbon source. M9 minimal medium consists of numerous salts to achieve an isotonic environment, but no carbon source. Hence, bacterial growth in M9 minimal media is inhibited. However, by supplementing either glucose, glyoxylate or glycolic acid in M9 media, the potential metabolization of these compounds of interest as a carbon source can be investigated. Therefore, the OD600 of E. coli cultures with the different carbon sources of interest were measured over a period of four days and compared to a carbon source-deficient culture.
Results
The ycdW 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.2). The expected size was 35.34 kDa for YcdW, 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 right side, YcdW 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.
Assays
We verified the enzyme activity of YcdW via a NADPH assay with the collected protein fractions from the ÄKTA purification. YcdW oxidizes NADPH to reduce glyoxylate to glycolic acid. NADPH absorbs light at a wavelength of 340 nm, but NADP+ does not. Hence, the absorbance at 340 nm decreases when YcdW is actively turning glyoxylate into glycolic acid. In this experiment we included a positive control, which included NADP+ with enzyme and a negative control, which included NADPH without enzyme. For both the positive and negative control it was expected, that they stay on a constant absorption level. For the negative control, containing NADPH without the desired enzyme, the absorption should stay constant because no NADPH should be converted into NADP+. For the positive control, containing NADP+, the absorption is expected to stay on a constant absorption level, which should be lower than the absorption for NADPH at a wavelength of 340 nm. Different enzyme concentrations (0.1 µg/µL; 0.2 µg/µL; 0.4 µg/µL) and temperatures (21 °C/25 °C/37 °C) were tested to find optimal assay conditions.
Referring to Nuñez et al., [5] we carried out assays with different enzyme concentrations at 25 °C (Figure 14). Figure 13 shows the calibration curve for the YcdW assay, which is used to calculate the substrate turnover per time. To create the calibration curve, the absorption of different NADPH concentrations were measured and compared to the resulting assay absorptions (Figure 14). For the YcdW assay it was expected that a higher enzyme concentration would result in a faster substrate conversion.
Figure 5: Calibration curve for YcdW enzyme assay with different NADPH concentrations (1 mM, 0.5 mM, 0.25 mM, 0.125 mM and 0 mM).
Figure 6: NADPH dependent enzyme assay of YcdW at a temperature of 25 °C. The graph shows the average absorption at 340 nm in correlation to the time (min). Different enzyme concentration were used, as well as a postive (NADP+) and negative control (NADPH without enzyme).
As expected, figure 6 shows that a higher enzyme concentration leads to a faster turnover of glyoxylate. The highest concentration of 0.4 µg/µL reaches an absorption of 0.29 after approximately 15 minutes, which equals a total substrate conversion rate of 0.345 µM/s. Hence, we decided to carry out further analysis regarding the optimal temperature with an enzyme concentration of 0.4 µg/µL. The positive and negative control both stayed on a constant absorption level, which indicates that the absorption decrease of the enzyme containing samples is based on enzymatic activity. However, figure 14 also shows that the enzyme does not reach the level of the positive control, which is presumably due to residual NADPH in the sample. A possible reason could be a too small amount of glyxoylate when starting the assay.
To study the enzyme activity at different temperatures we performed the assay at 21 °C and 37 °C. The substrate conversion per time (µM/s) is shown in table 1. Therefore, 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 YcdW per time (µM/s) in correlation to different temperature (21 °C, 25 °C and 37 °C)
| Temperature [°C] | Substrate conversion
per second [µM/s] |
|---|---|
| 21 | 0.31 |
| 25 | 0.345 |
| 37 | 0.26 |
Table 1 shows that the enzyme performs best at a temperature of 25 °C with a substrate conversion of 0.345 µM/s, followed by a conversion rate of 0.31 µM/s at 21 °C. At a temperature of 37 °C the enzyme shows its lowest conversion rate of 0.26 µM/s.
After showing that the YcdW enzyme performs best at a temperature of 25 °C, a Student's t-test was performed to check the significance of our data. Five different data sets (0, 5, 10, 20, 30 min) were chosen (Figure 7). For a better visualization the data sets were plotted.
Figure 7: Enzyme assay absorption for 0, 5, 10, 20 and 30 minutes with standard error n=3.
The absorptions and corresponding standard errors are also shown in table 2. Significance is indicated by *.
Table 2: Calculated significance of YcdW data sets for 0, 5, 10, 20 and 30 minutes, 25 °C. The table also shows the NADPH-absorption as well as the particular standard error. A= Absorption, *= Significance (p-value < 0.05), n= 3
| YcdW [µg/µL] | A (t=0 min) | A (t=5 min) | A (t=10 min) | A (t=20 min) | A (t=30 min) |
|---|---|---|---|---|---|
| 0.1 | 0.691±0.0003* | 0.635±0.01* | 0.58±0.008* | 0.451±0.004* | 0.326±0.005* |
| 0.2 | 0.693±0.009 | 0.623±0.018* | 0.563±0.028* | 0.429±0.056* | 0.31±0.08* |
| 0.4 | 0.868±0.043 | 0.57±0.07 | 0.38±0.064* | 0.3±0.06* | 0.293±0.054* |
| pos. | 0.096±0.0008* | 0.098±0.001* | 0.099±0.001* | 0.1±0.001* | 0.101±0.001* |
| neg. | 0.724±0.0017 | 0.719±0.002 | 0.714±0.002 | 0.705±0.003 | 0.696±0.003 |
As shown in table 2, most of the data points show a p-value < 0.05, which indicates that the data is significant.
The results show clearly that the glyoxylate reductase YcdW performs best at 25 °C and the highest concentration of 0.4 µg/µL, which allows a further characterization of the reaction and production of the desired monomer glycolic acid via high-performance liquid chromatography (HPLC).
HPLC Analysis
In order to verify the production of glycolic acid (product of YcdW) high-performance liquid chromatography (HPLC) was performed. Therefore, three in vitro assays were performed as described in the methods section. For the YcdW assay, previous HPLC-analysis showed a low conversion rate of gyloxylate into glycolic acid, which indicated a NADPH-shortage. Hence, NADPH was used in a higher concentration. Additionally an in vitro assay, containing both enzymes was carried out in the YcdW reaction buffer to analyze the simultaneous performance of both enzymes, starting from isocitrate as a substrate. In order to analyze the in vivo production of glycolic acid, ycdW-transformed E. coli cells were disrupted and centrifuged. The supernatant was sterile-filtered and analyzed.
1. In vitro YcdW assay with NADPH surplus (Figure 8)
2. In vitro YcdW with AceA in YcdW reaction buffer (Figure 9)
3. Analysis of disrupted ycdW-transformed E. coli cells (Figure 10)
To compare the resulting retention times from our samples, we used external references, which showed the following retention times:
1. Isocitrat: 10.25 min
2. Glyoxylate: 12.193 min
3. Glycolic acid: 15.749 min
For assay 1 (in vitro YcdW assay) a peak after 15.701 minutes confirms the production of glycolic acid from glyoxylate (12.210 min), (Figure 8).
Figure 8: HPLC analysis of YcdW in vitro enzyme assay with NADPH surplus. Glycolic acid peak can be seen at 15.701 minutes and glyoxylate peak at 12.210 minutes.
While testing the parallel performance of YcdW and AceA, a white precipitate was noticed. Since the HPLC (Figure 9) shows no peaks for neither glyoxylate nor glycolic acid, it is likely that AceA precipitated in the YcdW reaction buffer (potassium phosphate), resulting in a loss of catalytic activity.
Figure 9: HPLC analysis of in vitro assay containing both desired enzymes in the YcdW reaction buffer. No expected retention times can be seen.
The cell lysate (Figure 10) shows, besides others, peaks corresponding to the retention time of glyoxylate (12.066 min) and glycolic acid (15.568).
It can therefore be concluded that the production of glycolic acid in E. coli was successful.
Figure 10: HPLC analysis of supernatant of disrupted ycdW-transformed E. coli cells. Glyoxylate and glycolic acid peaks can be seen at 12.066 minutes and 15.568 minutes.
The results show, that a simultaneous in vitro performance of both enzymes in the YcdW potassium phosphate buffer, starting from isocitrate as a substrate, does not result in the production of glycolic acid (Figure 10). To switch from a successive in vitro synthesis of glycolic acid to a simultaneous one, it is necessary to find an optimized buffer for maximum performance of both enzymes. The in vitro productions of glycolic acid as well as the in vivo production of glycolic acid were successful (Figures 8 and 9).
Growth Assay
To find out in which way the cloned ycdW effect the growth efficiency of our expression strain E. coli BL21, we performed a growth assay. The results showed no significance. However, in other experiments, these cells showed no distinguishable difference in growth compared to non-transformed cells. Hence, we suspect that the overexpression of ycdW has no great influence on growth. To proof this hypothesis, further growth assays must be carried out.
Sequence and features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 353
- 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 – P75913 (YCDW_ECOLI), 10/14/2018 [3].
- ↑ M. Felisa Nuñez, M. Teresa Pellicer, Josefa Badia, Juan Aguilar and Laura Baldoma, Biochemical characterization of the 2-ketoacid reductases encoded by ycdW and yiaE genes in Escherichia coli, Biochemical Journal, 354, 707-715 [4].
| None |