Team:British Columbia/6CNA
6-CNA Detoxification
6-chloronicotinic acid (6-CNA) is an intermediate in the imidacloprid degradation that is both toxic to bees [1], and a persistent environmental contaminant [2]. It is suggested that neonicotinoids such as imidacloprid, after modification under environmental conditions can spontaneously convert to 6-CNA. This may explain the accumulation of 6-CNA in the environment given that there is no other potential source other than imidacloprid [3].
Figure 1: Photodegradation of imidacloprid into 6-CNA under environmental conditions. Additionally, hydroxylation by P450s followed by a spontaneous degradation of the intermediate provides an alternative route for 6-CNA production.
To fully protect bees from the effects of imidacloprid, our probeeotic must be capable of fully detoxifying this pesticide. That also includes the degradation of 6-CNA.In 2012 Madhura Shettigar et al. [4] isolated a bacterium, Bradyrhizobiaceae SG-6C, able to degrade 6-CNA. SG-6C was found to contain a chlorohydrolase able to dechlorinate 6-chloronicotinic acid to 6-hydroxynicotinic acid (6-HNA). This enzyme, known as Cch2, was cloned into E. coli giving the bacterium the ability to convert 6-CNA to 6-HNA a non-toxic product.
Figure 2: Schematic image of dechlorination of 6-CNA by Cch2 into 6-HNA.
We selected Cch2 to form the basis of our 6-CNA detoxification system.
After synthesizing a codon-optimized cch2, we used standard assembly to created a composite part composed of cch2 driven by the tac promoter (BBa_K1813037) and followed by a double terminator (BBa_B0014).
The tac promoter contains a lac promoter sequence that can be bound by LacI, the lac repressor protein, allowing inducible expression by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Our cch2 cassette was assembled behind a lacI cassette (BBa_K1813019) to give us the ability to control the expression of cch2.
All cch2 constructs are contained within standard pSB1C3 vectors.
Figure 3: Schematic of Cch2 composite part. This part was constituted by a reversed LacI, pTAC promoter, RBS, cch2 and a double terminator. It was employed for all experiments that followed, including a 6-CNA degradation rate experiment.
Once we had assembled our cch2 composite part our first goal was to test for the expression cch2 and the activity of the gene product. Our cch2 construct was transformed into E. coli and soluble production of Cch2 was assessed by SDS-PAGE. We found that Cch2 was strongly produced and soluble when grown at 25°C.
Once we had produced soluble Cch2 we wanted to ensure that our enzyme was able to de-chlorinate 6-CNA. E. coli cell lysate containing Cch2 was incubated with 6-CNA. The reaction mixture was extracted and run on GC/MS, showing that Cch2 had converted 6-CNA into 6-HNA.
Figure 4: Cch2 is optimally expressed at 25°C. This optimal expression temperature was used for a resting cell assay which was used to analyze degradation by Cch2 of 6-CNA. Samples were analyzed by gas-chromatography and mass spectrometry. The resting cell assay was done for 48 hours. There was complete degradation by Cch2. The no insert control had no degradation of 6-CNA.
Shettigar et al. [3] reported that intact E. coli cells, expressing cch2, are able to de-chlorinate 6-CNA when added to a resting cell assay. We set out to replicate these results. Using a resting cell assay with our cch2 constructs we followed the consumption of 6-CNA and the formation of 6-HNA by GC/MS.
Figure 5: A two hour rate experiment of Cch2 was done to obtain a rate of degradation of the enzyme at 30°C. The samples were analyzed using gas chromatography and mass spectrometry. Cch2 completely degraded 6-HNA by 45 minutes, thereafter 6-HNA began to appear on the spectrum and peaked at 60 minutes. The no insert control had no degradation over the one hour time course.
We found that we had complete consumption of 6-CNA within 60 min, with a transformation rate of 43.3 + 11.2 µM/min. Our transformation rate was approximately 8-fold higher than that reported by Shettigar et al. [3]who showed 70% consumption over 6 hours under similar conditions.
Also, we observed that large amounts of 6-HNA appeared in the culture media while 6-CNA is consumed. As Cch2 is produced as cytosolic enzyme within our E. coli, this suggests that the transport of 6-CNA into the cell and 6-HNA out of the cell is a dynamic and rapid process. It is unlikely that these polar molecules are passively diffusing through the membrane. Therefore, it is likely that a general transporter in E. coli facilitates the transport of these molecules.
In the bee gut, our probeeotic must be able to quickly remove 6-CNA to protect the bees from the toxic effect of 6-CNA. Thus, identification of the 6-CNA transport process in the cell might lead to future improvements to our specialty chassis.
To further improve our 6-CNA degradation system we sought to create a metabolic pathway that would lead to the complete mineralization of 6-CNA. This would accomplish two goals. First, prevent the buildup of metabolites that could be harmful to our bees, and second, to give our probeeotic a competitive advantage by providing the means to utilize the carbon and nitrogen contained within 6-CNA.
We chose to use the nicotinate degradation pathway (nic pathway) found in Pseudomonas putida KT2440. First described by Tang et al. in 2012 [4], this pathway is well suited for our system. Pseudomonas is a Proteobacteria suggesting that the enzymes in the pathway are likely functional in both E. coli and our two bee specific chassis, all of which are also proteobacteria.
Figure 6: The biochemical pathway for degradation of 6-CNA by Cch2 and the Nic cluster. This produces fumaric acid which can be utilized in the TCA cycle for central metabolism.
The nic genes were codon-optimized, synthesized, and individually assembled using standard assembly to created composite parts driven by the tac promoter (BBa_K1813037) and followed by a double terminator (BBa_B0014). As with the cch2 cassette, the nic expression cassettes were assembled downstream of a lacI cassette (BBa_K1813019) to give us inducible control of expression.
All nic constructs are contained within standard pSB1C3 vectors.
Figure 7: This is the assembly of constructs used for Sole Carbon Source experiment. Each individual part is composed of a ptac, rbs, the gene and a double terminator. Additionally, a single reversed lacI is present at the beginning of the assembly.
We tested our individual nic constructs for soluble production in E.coli using SDS-PAGE. Three of our five nic genes were strongly expressed. The remaining two (nicC and nicX) were unable to be easily detected using SDS-PAGE.
Figure 8:
Though we were unable to see clear expression of two of our nic genes, it is possible that they are being expressed at low levels. We moved forward, testing if our two large composite parts (see Figure 9) enable E. coli to grow on 6-CNA or 6-HNA as a sole carbon or nitrogen source. However, we were not able to finish the growth experiments because of poor growth of our engineered strains.
| Sample: |
| Glucose in M9 minimal media |
| E1, E2, E3 |
| No Insert Control |
| E4, E5, E6 |
| cch2 |
| E7, E8, E9 |
| cch2 and nic cluster |
| E10, E11, E12 |
| nic cluster |
Figure 9: A sole carbon and nitrogen growth experiment was done with a plate reader. Of the tested constructs, cch2, nic cluster, cch2, nic cluster and a no insert control. Each had 6-CNA, 6-HNA or glucose added to the cultures. Only the glucose cultures had increases changes in their optical densities, however they began to plateau thereafter.
We think that one problem with our current system was insufficient repression of our composite parts by LacI. This resulted in significant “leaky” expression of our genes, presumably imposing a major metabolic burden on our cells, which led to growth defects. These growth defects have made it very difficult to asses if our system is performing as designed because we were not able to generate enough cell material.
Conclusion and Achievements:
The 2015 UBC iGEM team created a system that produces catalytically active Cch2, a chlorohydrolase able to convert the toxic metabolite 6-CNA into the harmless intermediate 6-HNA.
We have used this system to show that E.coli expressing cch2 was able to rapidly detoxify media containing high amounts 6-CNA. This system applied inside the bee’s intestinal tract by using an engineered probiotic bacterium may render bees resistant to the toxic effects of the degradation product of imidacloprid.
Future Directions:
Moving forward, we have two major objectives to improve our system:
- Modification of our nicC and nicX constructs to show that soluble overexpression is possible. Our first strategy would be to increase the strength of the ribosome binding site, using the Salis lab ribosome binding site calculator and optimization algorithms.
- Reduce the metabolic burden of our composite parts: First, by increasing the transcriptional repression of our constructs by providing stronger expression of lacI. Another strategy would be to switch to a weaker and more easily tunable promoter system such as the arabinose inducible Pbad promoter system.
References:
[1] Nauen, R., Ebbinghaus-Kintscher, U. and Schmuck, R. (2001) Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae) Pest. Manag. Sci. 57 (7) DOI: 10.1002/ps.331
[2] Rouchaud J, Gustin F, Wauters A (1996) Imidacloprid insecticide soil metabolism in sugar beet field crops. Bull Environ Contam Toxicol 56: 29–36. doi: 10.1007/s001289900005
[3] Shettigar M, Pearce S, Pandey R, Khan F, Dorrian SJ, et al. (2012) Cloning of a Novel 6-Chloronicotinic Acid Chlorohydrolase from the Newly Isolated 6-Chloronicotinic Acid Mineralizing Bradyrhizobiaceae Strain SG-6C. PLoS ONE 7(11): e51162. doi: 10.1371/journal.pone.0051162
[4] Tang, H., Yao, Y., Wang, L., Yu, H., Ren, Y. et al. (2012) Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation. Scientific Reports 2, Article number: 377 doi:10.1038/srep00377
UBC iGEM 2015