Part:BBa_K2770010
CHMO Generator
Usage and biology
The CHMO from Acinetobacter calcoaceticus is a flavin-dependent Baeyer-Villiger monooxygenase (BVMO), which catalyzes the reaction of cyclohexanone into Ɛ-caprolactone. The enzyme uses NADPH as a co-substrate and requires molecular oxygen for the reaction. Since methionine and cysteine are amino acids that are easily affected by oxidation, which leads to a partial loss of the enzymes function, a double mutant (C376L/M400I) without the respective amino acids was used[1]. The CHMO coding sequence was cloned into the pSB1C3 vector, containing the BioBrick prefix and suffix, a T7/lac Promotor (K921001), an RBS (B0034), an N-terminal 10x His-tag for purification and a glycine-serine-linker. The coding sequence consists of 1629 bp which are translated to 543 amino acids. CHMO has a molecular mass of 60.8 kDa.
Figure 1: 3D-structure of CHMO.
CHMO was characterized together with the alcohol dehydrogenase (ADH), so the methods and results for this enzyme are the same.
To learn more about CHMO and its part in our project, please visit our [http://2018.igem.org/Team:TU_Darmstadt/Project/Caprolactone Wiki].
Mechanism
CHMO converts cyclohexanon to caprolactone, using NADPH as a cosubstrate.
Figure 2: Reaction mechanism of CHMO.
Methods
Cloning
The sequence for chmo was modified with a His-tag, ordered from Integrated DNA Technologies (IDT) and inserted into the pSB1C3 plasmid. For this purpose, the BioBrick assembly (BBa) was used[2]. 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 the generated plasmid and positive colonies were identified via colony PCR and DNA sequencing.
Figure 3: pSB1C3 with the chmo gene.
SDS-PAGE and Western Blot
To verify that chmo was expressed and the corresponding protein was translated, a SDS-PAGE and western blot were performed. The resulting bands were compared to the expected protein size of CHMO.
Purification
After expression of chmo 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 enzymes.
Enzyme assays
Enzyme activity was measured by following the depletion of NADPH at 340 nm. When CHMO is active NADPH is being converted into NADP+. Thioanisole was used as a substrate. The change in absorption over time was used to calculate the specific enzyme activity utilizing a calibration curve of NADPH[3].
M9 growth assay
The growth of E. coli BL21 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, cyclohexanon or ε-caprolactone 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 for 14 houres at 37 °C using a Tecan Spark.
Growth assay
The aim of the growth assay is to find out in what way the cloned plasmids and the enzyme production affects the growth efficiency of our expression stain E. coli BL21. The tests were performed in a 24 well blade with the Tecan Infinit M200PRO. Everything was tested in triplicates. LB-media was used as a blanc. The OD600 was measured every 10 minutes after 8 minutes of shaking at 37 °C. The measurement took place for 20 hours. The blades were adjusted to a starting OD of 0.1.
Results
Cloning
After ligating chmo into the pSB1C3 vector through the Biobrick system, a cPCR was performed using VF2 and VR primers. The PCR product (1996 bp) was successfully detected after running the sample on an agarose gel. The ligation of chmo (1629 bp) into pSB1C3 was also verified via DNA sequencing.
SDS-PAGE and western blot
The enzymes CHMO and ADH in pSB1C3 could not be detected using SDS-PAGE or western analysis. A possible reason for this is that the translation was somehow hindered, or the enzymes were degraded before a measurement could be done. Therefore, a different construct was used to determine whether there was a production of both enzymes. E. coli BL21 were transformed with the plasmid pRSFDuet (provided to us by working groupe Prof. Dr. Bornscheuer) encoding the genes of N-terminally His-tagged ADH and CHMO under the control of a T7 lac promoter. Positive colonies were identified through white-red selection, cultured, and protein expression was induced by addition of IPTG. Samples were taken after 16 hours and separated via SDS-PAGE. We demonstrated successful protein expression via western blot (Figure 4). For this, we used primary Rabbit anti His-tag antibodies and secondary Mouse anti rabbit antibodies conjugated to a horse radish peroxidase (HRP).
Figure 4: Western blot. Lane 1: Protein prestained Ladder (PageRuler™ Prestained Protein Ladder, 10 to 180 kDa, Thermo Fisher Scientific). Lane 2: Negative control. Lane 3: Overexpressed enzymes ADH and CHMO in pRSFDuet in E. coli BL21.
The western blot shows two bands at approximately 30 and 60 kDa, which are the expected molecular weights of CHMO (62.5 kDa) and ADH (28.5 kDa). There are two possible reasons for the different intensities of the bands. First, the ADH possesses an S-Tag, which has a lower affinity towards the anti His-tag antibodies than His-tags. The other reason could be, that the promoters for ADH and CHMO are not equally strong, because the optimal ratio of the two enzymes is 1:10 (ADH: CHMO) for ε-caprolactone production and the promotors are adjusted to this ratio by design. Therefore, the differences between the two enzyme bands were not surprising to us.
Purification
After a successful western analysis of the enzymes on vector pRSFDuet, they were purified using FPLC (Äkta Purifier). There were 3 distinguished peaks detectable in the chromatogram (Figure 5).
Figure 5: Chromatogram of the ÄKTA purification of ADH and CHMO.
The wash fraction (3) and fractions 13-22 were collected in tubes. ADH was expected to be highly concentrated in fractions 13-16 and CHMO in 17-22. Fraction 3, 13, 14, 17, 19, 20, 21 were analyzed via SDS-PAGE (Figure 6). Also, a sample of the pellet after the cell disruption and a sample of crude protein extract before ÄKTA purification was applied.
Figure 6: SDS-PAGE. Lanes, from left to right: PageRuler Prestained Protein Ladder (10 to 180 kDa), Pellet after disruption, supernatant before ÄKTA purification, fraction 3 (wash fraction), fraction 13 (ADH), fraction 14 (ADH), fraction 17 (CHMO), fraction 19 (CHMO), fraction 20 (CHMO), fraction 21 (CHMO).
The SDS-PAGE shows, that there is a high concentration of CHMO in the chosen samples, but no ADH in the expected fractions. ADH could, however, be collected in the wash fraction. Fraction 3 (wash fraction) was used for later ADH assays and fraction 19 for CHMO assays.
Enzyme assays
Enzyme activity was measured by following the formation or depletion of NADPH at 340 nm. After mixing either 0.2 or 1 µM of the enzyme with the substrate of choice in the reaction buffer, 300 µM NADP+ or NADPH was added, mixed in a well, and the extinction was measured over time (Tecan Infinite M200 PRO). 10 mM cyclohexanol was used as a substrate for ADH activity measurements and 0.5 mM thioanisole or cyclohexanone was used for CHMO. The changes in absorption over time were used to calculate the specific enzyme activity utilizing a calibration curve of NADPH[3].
Figure 7: NADPH dependent enzyme assay showing the degradation and formation of NADPH using enzymes CHMO and ADH over 30 minutes.
The depletion of NADPH using CHMO and its formation by ADH was observed, individually (Figure 7). 200 nM and 1 µM of the respective enzyme were used for the assays. It is shown, that higher enzyme concentrations result in faster formation/degradation of NADPH. An assay with NADPH/NADP+ without added enzyme was used as a negative/positive control. These controls’ absorption didn’t change over time, indicating an activity of our enzymes. The mean specific enzyme activity (n (product in µmol))/(t (in min)*m enzyme in mg)) of CHMO and ADH was determined to be 0.17 and 0.22. The assays successfully proofed the activity of our tested enzymes. Future experiments could determine optimal temperature and pH environments for the highest activity
Growth assay
The aim of the growth assay is to find out in what way the cloned plasmids affect the growth efficiency of our expression strain E. coli BL21. For this reasons, different colonies with different vectors cloned inside where tested. E. coli BL21 untransformed was used as a positive control. The gene adh in E. coli BL21 was tested both induced as not induced. The same was done with chmo in E. coli BL21. E. coli BL21 transformed with a vector with both adh and chmo was tested induced and not induced as well.
Figure 8: Growth assay with E. coli BL21 with different vectors and induced or not induced.
In figure 8 the results of the growth assay are shown. It can be seen, that the E. coli BL21 colony with the induced adh plasmids grows better than the other colonies including the positive control E. coli E. coli BL21 without an added plasmid. It can furthermore be seen that the chmo strain induced and the positive control reach the lowest plateau with an OD600 of 0.5 while the E. coli BL21 with adh induced grows until an OD600 of 0.7. We expected the positive control without an added plasmid and without inducing to grow best since it does not have a metabolic burden added. It was also expected, that the induced E. coli BL21 stain with chmo and adh added has the lowest growth rate due to the large added plasmid and to the two enzymes with have to be expressed additionaly. In the experiment this coloniy shows an average grow with a final OD600 of 0.55. Possible error sources might be that an error accourded when inocunating the LB-media in the well with E. coli BL21. As a conclusion it can still be said that the added plasmid and the protein expression do not seem to affect the growth of E. coli BL21 severely. These means for us that or modified E. coli BL21 is still able to grow and also produce ε-caprolactone
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 822
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
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