Team:KEYSTONE/Engineering
LCC-ICCG
The leaf-branch compost cutinase (LCC) is an enzyme capable of Polyethylene terephthalate (PET) degradation that outperforms PET hydrolase and BTA while having a high thermostability. Further site mutation allowed for further improvement in the activity and thermostability of LCC. The LCC-ICCG used in our experiment, which refered to F243I/D238C/S283C/Y127G, is created by Tournier et al. in 2020.This mutation was among the 25 out of 209 that has 75% or more increased activity compared with the original wild-type LCC. The ICCG mutatation was finally developed as a result of a combination of mutations based on the original variant I among the 25. The thermal stability of LCC-ICCG is maintained without the divalent-metal-binding site with the presence of the disulfide bridge. According to experiments, the LCC-ICCG is the most effective at degrading plastic in bioreactors under pH8 and 72°C condition. Furthermore, the leaf-branch compost cutinase is a very effective enzyme as the terephthalic acid (TPA) and ethylene glycol (EG) formed as a result of PET degradation can be used to make new PET plastic. The resulting plastic has similar properties to the PET plastic from factories [1].
Figure 1: (a) The LCC-ICCG gene was coded onto the standard expression vector pET28a(+) with a 6xHis-tag on both side of N-terminal and C-terminal for more effective purification on Ni-NTA column. (b) The plasmid was transformed into E.coli BL21 (DE3) for LCC production and PET degradation to produce terephthalic acid (TPA) and ethylene glycol (EG) which can be used to produce new PET.
LCC Production & Purification
Characterization of LCC-ICCG. (a) DNA gel electrophoresis of pET28a(+) plasmid coded with LCC-ICCG and transformed in E.coli BL21. (b) Protein gel electrophoresis of LCC expression with 300µM IPTG and purification with 20mM Tris-HCL, 300mM NaCl buffer, showing a molecular mass of 28kDa as expected.
Out of all conditions tested, we were able to produce an optimal yield of 27.3mg/L of LCC with 300µM IPTG induction and purification with 20mM Tris-HCL, 300mM NaCl buffer. By increasing the concentration of NaCl in the buffer from 100mM to 300mM, we were able to increase the yield from 4.22mg/L to 27.3mg/L, as this concentration of NaCl better stabilizes the structure of LCC [1]. However, this yield has been affected due to the considerable amount of LCC proteins that has been remaining in the cell lysis precipitant as shown in figure 2.b, which could have been caused by the following reasons: a) the ultrasonic lysis wasn’t entirely complete/effective, and some bacteria remained intact; b) inclusion bodies formation.
PET Degradation
Since the ICCG variant of the LCC enzyme has an increased thermostability, it is able to achieve optimal degradation at a temperature of 72˚C [1], which allows PET to reach glass transition state and become amorphous, enabling more effective degradation [5] (see more from Dr. Liu’s interview section in Integrated Human_Practices).
We repeated the degradation experiment by Tournier et al. with the LCC proteins that we produced, using 1 ml of a 0.69µM solution of purified protein in 20 mM Tris-HCl, pH 8, 300 mM NaCl, and combined with 100 mg PET powder with 49ml of 100 mM potassium phosphate buffer pH 8 in a 100 ml flask for degradation in a 72˚C environment under 170 rpm agitation. In addition, we tested the effectiveness of degradation with an extended range of LCC concentrations in the 1ml solution, from 0.25µM,-- 0.5µM, 0.69µM, 5µM, 10µM, 15µM to 20µM, as well as a controlled sample of 100mg PET powder in 1ml of 20 mM Tris-HCl, pH 8, 300 mM NaCl and 49ml of potassium phosphate buffer pH 8.
Figure 3: Gas chromatography test for ethylene glycol in LCC degradation supernatant with different LCC concentrations after 24h. *Note: the concentration refers to the concentration of LCC in the 1ml solution that is added to 49ml buffer.
After 24h of degradation, we sent the supernatant samples to gas chromatography testing to test the concentration of ethylene glycol, a product of PET degradation, to examine the effectiveness of LCC in degrading PET. Although a wide range of samples with different concentrations were tested as stated above, a number of them have been mysteriously lost in the process of transportation for GC testing, therefore we were only able to produce the results as shown above.
The standard sample used in the GC test was 20mg/ml EG. In our sample of 100mg PET in 50ml buffer, the maximum concentration of EG that can be obtained is 0.54mg/ml, which would produce a peak with an area of 0.27% of the area of the standard peak. Therefore, although difficult to obtain a precise calculation due to the low concentration, it can be seen that the amount of PET degraded by 5µM and 10µM of LCC was fairly significant, and a distinct difference with the no LCC controlled sample can be seen.
The results might have been affected by the fact that the temperature used for degradation experiment was 70˚C instead of 72˚C due to equipment limitations.
Future steps
Due to time and COVID regulation constraints, we weren’t able to produce all the results we aimed for. We still wish to advance our LCC experiments on the following aspects:
- Enhance and repeat the degradation experiment and gas chromatography in 72˚C temperature with the complete range of protein concentrations we used, including 0.69µM which was used in the original literature.
- Conduct degradation experiments on larger scales for longer periods of time using post-consumer plastic bottles to better simulate the conditions in our hardware
- Simulate the conditions in our hardware by cultivating the E.coli and conduct IPTG induction in 37˚C and directly raise the temperature to 72˚C to start degradation, since this is the mechanism designed to be employed in our bin (for the above two points, please refer to https://2020.igem.org/Team:KEYSTONE/Hardware for context)
- Conduct enzyme activity assays in the process of degradation with a frequency of around once every three hours to produce a mathematical model of the activity of LCC over time, which would suggest the appropriate frequency of renewing the enzymes in our hardware
- Collect samples from the remainder of our degradation and test the feasibility of using the polymerization system comprised of CoA ligase and acyltransferase created by BUCT-China to form new materials from the MHET which is a byproduct of degradation. This system is able to do so by causing esterification of the hydroxyl and carboxyl that make up MHET. (these two above have been initially suggested by BUCT-China, see https://2020.igem.org/Team:KEYSTONE/Partnership for more information)
- An alternative way to address the MHET produced is by linking a MHETase to LCC which hydrolyzes MHET into terephthalic acid and ethylene glycol, potentially by the method used by Knott et al. to link MHETase to PETase, as discussed in our Contribution (https://2020.igem.org/Team:KEYSTONE/Contribution). This would most likely increase the efficiency of PET degradation because it would have been affected by the presence of MHET, as suggested by BUCT-China.
- We’d also like to characterize and compare the degradation efficiency of LCC-ICCG with PETase, an enzyme that had been commonly used in the past for the biodegradation of PET plastic.
- Using Thermophilic Bacteria：LCC is the abbreviation for Leaf Compost Cutinase, which belongs to the α/β-hydrolase superfamily, discovered because they could hydrolyze (break down in a chemical reaction with water) the ester bonds of the plant polymer cutin. However, they are also able to break down other polymers such as PET, which was used in our project. The LCC is found in thermophilic bacteria, which is why the enzyme worked optimally at a temperature of 72 degrees. Therefore, we would like to try and express the LC Cutinase in a thermophilic bacteria host, and add a secretion tag to secrete the protein at a temperature of 72 degrees. In this way, we could preserve our bacteria in the enzyme degradation stage since the new thermophilic bacteria would be able to withstand this temperature. This means that our hardware could be simplified into one reaction container, where the bacteria could be reused for the next rounds of expression and degradation [5].
- Determining Linalool Concentration: As a result of the pandemic, we only had limited time in the lab, and we did not have time to test the concentration of linalool. In determining how much linalool enzyme are present in our broth, we could compare its concentration of fragrance to the concentration of the enzymes, to see a relationship between these two factors, to determine the best concentration of enzyme that releases the fragrance.
Linalool
The linalool production of our experiment is mainly achieved by a plasmid with linalool synthase and the MVA pathway. The MVA pathway was originally constructed to produce limonene. Simple sugars are made into Acetyl-CoA through glycolysis, and it is the first part of the MVA pathway. Through varies reactions, geranyl pyrophosphate (GPP) was finally synthesized from dimethylallyl pyrophosphate by the GPP synthase. GPP can be used to produce most monoterpenes, sesquiterpenes, and diterpenes [2]. The original article successfully produced limonene:
Figure 4: MVA pathway and linalool synthesis pathway for production of limonene [2].
GreatBay_China 2018 applied the same pathway for geraniol production Escherichia coli and Saccharomyces cerevisiae. They were successful in geraniol product with the addition of other plasmids [3].
In our project, we applied the pathway to a new fragrance synthase. The chemical compound of our choice was linalool, which is a monoterpene. The linalool synthase used in our experiments is the linalool synthase from Streptromyces clavuligerus that is called bLIS. It is a very effective linalool synthase. The synthesis of linalool can also be increased by the optimization of the fusion tags and the ribosome binding sites of the protein [4].
Designing Genetic circuits
Figure 5: designing plasmid
We designed four plasmids. The first plasmid is p15A-MVA-ptac-GPPS-bLIS (figure 5A). This plasmid contains MVA and bLIS, which allows the bacteria to produce linalool. pR6K-ptac-GPPS-GES is a plasmid used by Greatbay China 2018 to produce geraniol. They successfully produced geraniol using this plasmid. Therefore, in our plasmid design, we improved this by replacing GES with bLIS, which is responsible for producing linalool. Thus, the second plasmid we designed is pR6K-ptac-GPPS-bLIS (figure 5B). pR6K is a vector with medium copy number, ptac promoter, and GPPS-bLIS segment. However, due to the lack of MVA pathway genes in this plasmid, the plasmid needs to corporate with other plasmids that contain MVA pathway genes. Therefore, we constructed plasmid p15A-MVA (figure 5D). By transforming this plasmid with pR6K-ptac-GPPS-bLIS, the synthesis of linalool can be achieved. We also designed plasmid pSB1K3-ptac-GPPS-bLIS (figure 5C). pSB1K3 is a high copy number vector. Thus, cooperating with p15A-MVA linalool synthesis can be achieved.
polymerase chain reaction
After designing the plasmid, we used polymerase chain reaction to amplify our gene segment. We successfully amplified the segments in p15A-MVA-ptac-GPPS-bLIS (figure 6A) plasmid, since the size of its three segments, bLIS-1, MVA-vector, MVA-GPPS, indicated by figure 6A equal to their theoretical size. We also successfully amplified the segments in p15A-MVA plasmid, since the size of its segments, MVA-1 and MVA-2, equal to their theoretical size (figure 6A). We purified the successfully amplified segments. (figure 6B) . However, we failed to amplify segments of pSB1K3-ptac-GPPS-bLIS.We also failed to amplify pR6K-ptac-GPPS-bLIS, because we used the wrong primer for the reaction.
Due to the important role of plasmid pR6K-ptac-GPPS-bLIS, we repeated the polymerase chain reaction of its segment pR6K using the correct primer. The result we obtained this time is positive (figure 6C), because the size of pR6K indicated in the figure fits its theoretical size. We purified pR6K after successfully amplifying it.
Figure 6: the gel DNA gel electrophoresis results. (A) amplification result of gene segments of p15A-MVA-ptac-GPPS-bLIS (bLIS-1 (1015 bp), MVA-vector (6372 bp), MVA-GPPS (6970 bp)), pR6K-ptac-GPPS-bLIS (bLIS-2 (1016 bp), pR6K (5958 bp)) , pSB1K3-ptac-GPPS-bLIS (bLIS-3 (1016 bp), Lac1-Ptac-GPPS (2371 bp), PSBIC3 (2111 bp)) , p15A-MVA (MVA-1 (5177 bp), MVA-2 (6922 bp)). (B) result of gene segment purification. bLIS-1 (1015 bp), MVA-vector (6372 bp), MVA-GPPS (6970 bp), bLIS-2 (1016 bp), bLIS-3 (1016 bp), Lac1-Ptac-GPPS (2371 bp), (MVA-1 (5177 bp), MVA-2 (6922 bp). (C) amplification result of pR6K (5958 bp). (D) purification result of pR6K (5958 bp).
Gibson assembly
After purifying the segments of plasmid pR6K-ptac-GPPS-bLIS and p15A-MVA, we used Gibson assembly to construct them into plasmids. we transformed both plasmids into E.coli DH5α and conducted a colony polymerase chain reaction of E.coli transformed with pR6K-ptac-GPPS-bLIS, which allows us to determine the success of Gibson assembly (figure 7A). Therefore, we successfully constructed and transformed pR6K-ptac-GPPS-bLIS to E.coli DH5α. After transforming p15A-MVA plasmid to E.coli DH5α, we conducted a genetic sequencing for the bacteria. According to the result of genetic sequencing, we successfully constructed p15A-MVA. However, we failed in constructing MVA-ptac-GPPS-bLIS, because the bacteria transformed with it did not grow in the solid medium with chloramphenicol antibiotic. This may because the size of the plasmid is too huge, which is 14358 bp. As a result, the E.coli may not be able to receive this plasmid, or the plasmid may not be able to function well due to its size.
Figure 7: results of Gibson assembly. (A) gel electrophoresis result of pR6K-ptac-GPPS-bLIS (14358 bp). (B) gene sequencing result of p15A-MVA.
Linalool synthase
Linalool synthase, expressed by bLIS, can convert GPPS to linalool. To further confirm the functioning of pR6K-ptac-GPPS-bLIS, we transformed it into E.coli DH5α, and induced it. result of the protein electrophoresis results indicates that linalool synthase expression was achieved (figure 8). Therefore, plasmid pR6K-ptac-GPPS-bLIS did function well in E.coli DH5α.
Figure 8: protein electrophoresis results of linalool synthase (65.6 kDa)
Linalool synthesis
After confirming the function of pR6K-ptac-GPPS-bLIS, We cotransformed P15A-MVA and pR6K-ptac-GPPS-bLIS plasmid to E.coli DH5α. E.coli DH5αwe induced them with IPTG and two of them with extra glucose. after the inducing process, the bacteria solution was treated with n-hexane to extract purified linalool(figure 9). We can clearly smell the strong fragrance of linalool in those samples, and samples with glucose have a stronger fragrance than samples that are not treated with glucose. This indicates that glucose may promote the synthesis of linalool in E.coli DH5α. We do not need to knock out the gene in E.coli producing stinky smell because the aroma overcomes the smell of E.coli. Therefore, our E.coli won’t affect the local environment and appearance.
Besides, we also compared the smell of our sample to standard linalool sample, and standard geraniol, which is the product of pR6K-ptac-GPPS-GES (the plasmid before our improvement) Based on our observation the aroma of geraniol contains sweetness; whereas, the aroma of standard linalool sample has a slight peppery smell. The two smells are quite distinguishable. The small of our samples also have a slightly peppery aroma, which is close to the linalool standard sample. Thus, we have successfully produced linalool.
In addition, through visual observation, we also confirmed that glucose can promote the synthesis of linalool in E.coli. The transparent liquids in the test tubes are purified linalool (figure 9). The volume of the liquid in samples treated with glucose is larger than samples that aren’t treated by glucose. Therefore, E.coli treated with glucose may be able to produce more linalool in the same condition thanE.coli that is not. Our next step may be finding the optimal condition for linalool synthesis in E.coli. However, due to the Covid-19 situation, we do not have enough time in the lab, so we can only do those experiments in the future.
Figure 9: (A)E.coli DH5α with P15A-MVA and pR6K-ptac-GPPS-bLIS was propagated in four separated conical flasks. After the bacteria solutions reach the suitable OD (0.6-0.8), we added 2.5 ml glucose to two of the mediums during and added 25mM of IPTG to By adding n-hexane (4.5ml) to the induced bacteria solution, we can extract pure linalool (transparent layer)
We have sent our samples for Gas Chromatography and Mass Spectrometry to verify and quantify our production of linalool, however, due to an incidental technical problem with the testing facilities, we are still waiting for the data. Hopefully, we will be able to present it in our presentation video.
Future steps
Citations
[1] Tournier, V., Topham, C.M., Gilles, A. et al (2020). An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, pp.216–219. https://doi.org/10.1038/s41586-020-2149-4.
[2] Alonso-Gutierrez, J., Chan, R., Batth, T. S. et al (2013). Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, volume 19, pp.33-41. https://doi.org/10.1016/j.ymben.2013.05.004.
[3] Greatbay_China (2018). Demonstration. Retrieved Oct 22 2020, from https://2018.igem.org/Team:GreatBay_China/Demonstrate.
[4] Xun, W., Jing, W., Jiaming, C. et al (2019). Efficient biosynthesis of R-(-)-linalool through adjusting expression strategy and increasing GPP supply in Escherichia coli. https://doi.org/10.21203/rs.2.18761/v1.
[5] Luo, L. (2020). Personal Interview.
