- Human Practices
The ecology of natural tea garden has been deeply destroyed due to the widely use of pesticides, resulting in numerous examples of excessive glyphosate in tea food.
After brainstorming, we focused on glyphosate of tea and decided to engineer strains that can rapidly detect and effectively degrade glyphosate.
Currently, chromatography and other traditional methods are still widely used in glyphosate detection. These methods are time-consuming, laborious and complicated. We hope to design a simple and portable glyphosate bioassay system based on the knowledge of synthetic biology.
We convert glyphosate into AMPA and glyoxylate through a glyphosate oxidase (GOX) from Echinochloa colona named EcAKR4-11. Then glyoxylate can be transformedinto glycolic acid by the function of a human glyoxylate reductase named GRHPR, with a molecule of NADPH converted to NADP+ simultaneously (Fig. 1)2.
Fig. 1 Mechanism of GOX and GRHPR.
iNap3,4, an NADPH sensor which is derived from a fusion protein of T-Rex and cpYFP, is chosen to detect the amount of NADPH in the system. When NADPH exists, the conformational changes of the fusion protein will make it become fluorescent activity (Fig. 2). Therefore, the amount of glyphosate can be indirectly quantified by determining the amount of NADPH formed in the process from fluorescence intensity. This idea makes it possible to get our detection device portable and easy to use, which also guided us to design our hardware.
Fig. 2 iNap mechanism.
Because NADPH is widely distributed in the cell as a metabolite, GOX, GRHPR and iNap are anchored on the surface of engineered bacteria via being fused with anchor protein5. A comprehensive analysis of the characteristics of different kinds of anchor proteins is played and we ultimately determine the following:
Table. 1 Three kinds of anchor protein.
In present work, we try to test and evaluate all the nine fusion proteins that can be used in our project, and our design is shown on Fig. 3. Some of fusion protein have been proved to work outstandingly compared to the others, such as INPNC-GOX and INPNC-GRHPR, more details could be seen in Results page.
Fig. 3 Gene circuits of detection system.
As many biological creatures can implement a complete glyphosate and methylphosphonate degradation process in their body, we planned to place the working apparatus into our chassis bacteria and get the metabolic pathways and catalytic enzymes to function right. Moreover, the bacteria are expected to proliferate well in soil, able to uptake glyphosate from the ambience and eventually convert the chemical compounds to innocuous small particles.
There are two pathways found to degrade glyphosate (Fig. 4):
1. Glyphosate generates AMPA and glyoxylic acid through the C-N cleavage
2. Glyphosate generates sarcosine and phosphate acid through the C-P cleavage.
Fig. 4 Glyphosate oxidation pathway6.
Under natural conditions, a majority of microorganisms choose to produce AMPA in the former pathway, while AMPA has serious perniciousness to environment and physiological health remaining in soil and tea products concomitantly. Fortunately, the primary product of C-P cleavage, sacrosine, can be easily oxidized to the final product: formaldehyde7, which can recur or transport in/out cells. Thus, the C-P cleavage pathway is expected to degrade glyphosate in our design.
E. coli BL21 (DE3) turned to be an ideal option for our degradative design, for its existing intrinsic dysfunctional C-P cleavage-degradative system and broad potential in genetic engineering and advantaged genetic background. To remodel a functional and efficient degradation workshop in E. coli, three prerequisites should be included:
I. Phosphonate acid transmembrane transport.
II. C-P cleavage of glyphosate.
III. AMPA derivation and degradation
In order to meet the demands, we selected relevant proteins from different bacterial strains with high catalytic activity. The proteins-genes-strains relationships are listed at Table 2. We plan to introduce the correlated genes into E. coli to expand its ability to transport and degrade glyphosate and AMPA.
Table. 2 phn cluster.
*Sinorhizobium meliloti 1021 transports 85% of glyphosate in less than an hour (liquid medium) , Enterobacter cloacae K7 degrades 40% of glyphosate in 5 days, Salmonella enterica can derivatizes AMPA to utilize it.
Fig. 5 phn gene cluster mechanism.
In Sinorhizobium meliloti 10218, we discovered its specialty in glyphosate and AMPA absorption, conducted by the spontaneous mutation in phn genes (phnCDE1E2). Enterobacter cloacae K7, a plant-growth-promoting rhizobacteria, contains the capability of strong glyphosate degradation, in which its gene, phnJ, merely encodes the core part that required in degradation9. phn gene cluster (phnCDEFGHIJKLM) shows great importance in glyphosate uptake and degradation, is expressed in many bacterial strains with distinctive degrading abilities and we identify the excellent ones. Therefore, the less effective homologs in Escherichia coli would be altered by those necessary phn genes which take in charge of the work efficiency in Enterobacter cloacae K7 and Sinorhizobium meliloti 1021 strain (Fig. 5).
Furthermore, we discovered that phnO gene was reported with AMPA-degrade capacity in Salmonella enterica LT210, so we switched the wanted phnO gene with the intrinsic one in E. coli.
RNA interference sequences were designed in order to hinder endogenous genes (phnFJ) from expression. Constant expression of phnF in E. coli represses the whole phn gene cluster, thus a corresponding siRNA sequence targeting phnF was designed to activate the asleep degradation function. Moreover, siRNA for phnJ was specialized to endogenous phnJ, which prevents the disturbance in exogenous phnJ expression.
Fig. 6 Gene circuit of degradation system.
For different applications of our engineered bacteria, we designed different kill switches to ensure bio-safety.
We introduce the inverter into our kill switch systems which consist of a cI repressor and a pR promoter from E. coli λ phage, where cI repressor can inhibit pR promoter. In this case, activation of the upstream promoter of cI repressor will lead to high expression of it, thereby reducing the expression efficiency of downstream pR promoter. By using an inverter, inducible promoters can obtain two-way uses, while reducing the complexity of circuit construction and eliminating the need of precise control of steady state. It is a basis of our kill switches design.
1. Kill switch of engineered bacteria in detection system
Since our engineered bacteria in detection system will work in a certain device, we need to make the engineered bacteria survive only in a special environment where some particular substances exist.
We introduce the inverter to make engineered bacteria survive when certain inducers are present and die when they do not exist. The circuit in the Fig. 7 could make engineered bacteria survive when the concentration of L-arabinose is high enough, but die when is low. L-arabinose is added into the culture medium in the device to ensure that the engineered bacteria could survive and work robustly. Once engineered bacteria escape to the external environment, the expression of mazF will cause the engineered bacteria die due to MazF protein's toxicity.
Fig. 7 Gene circuit of kill switch in detection system.
2. Kill switch of engineered bacteria in degradation system
The working environment of degrading engineered bacteria is simple. Given the bacteria would be added to the soil directly as bacterial fluid, however, it is not easy for us to find the conditions that trigger the kill of the engineered bacteria in the environment, so we look for the conditions that cause kill from the internal of engineered bacteria.
Fig. 8 Gene circuit of kill switch in degradation system.
Formaldehyde will be produced during the process of degrading glyphosate,. When the concentration of glyphosate is too low, the concentration of formaldehyde in the engineered bacteria will also be in a low level. The formaldehyde promoter (pHCHO) will be activated when the concentration of formaldehyde is high, and the activity is positively correlated with the concentration of formaldehyde. Regarding the concentration of formaldehyde as an input signal to turn on the kill switch seems to be a possible option, however, it is contrary to our purpose which the engineered bacteria could degrade glyphosate with formaldehyde generated subsequently while they are alive. So a construction of inverter is also placed between the formaldehyde promoter and mazF (Fig. 8), just like the case in kill switch's design for detection system, which makes it possible to achieve that at high concentrations almost no toxic proteins express while when the concentration of formaldehyde is low as the concentration of glyphosate in the soil is low enough, the engineered bacteria will die.
Fig. 9 Effect of kill switch in degradation system.
3. Brief introduction to the design we developed when brainstorming
We've designed several kill switch systems when brainstorming, some of them were abandoned due to the limited lab time this year. But we still want to share the designs with you and hope they can inspire the iGEMers in the future. And we are also pleased that the design of MazF-MazE system has already inspired our Partner Team CSU-China this year. See more in the page of Partnership.
This is a balanced system constructed by multiple promoters and their inducers or repressors. This balance will be broken through changing the external environment. When ATc is added, the inhibited state of the pLtetO-1 promoter will be stopped and lacI will express, thereby pTrc-2 and pTrc-2 derivative promoters will be blocked and engineered bacteria will be alive. And if IPTG is added, the inhibition of these two promoters will be stopped and the engineered bacteria will die (Fig. 10).
Fig. 10 Gene circuit alternative of kill switch in detection system.
2) MazF-MazE system
This is a timing kill switch system. We use MazF-MazE, a toxin-antitoxin module, to control the death or survival of engineered bacteria by regulating the expression of them. Under normal conditions, our engineered bacteria express mazE and tetR under the control of constitutive promoter. TetR can inhibit the expression of mazF by inhibiting pTet promoter, which ensures that no affect will be made to the survival of our engineered bacteria. However, after the addition of ATc, the inhibition of pTet will be stopped, delta begins to express and activate the pPO promoter. Then delta and mazF under the control of pPO promoter begin to express, which furtherly activate pPO promoter in a positive feedback manner. The expression of MazF protein will increase exponentially, surpass MazE and cause the death of engineered bacteria in the time we want.
Fig. 11 Gene circuit of alternative kill switch in degradation system.
1 Pan, L. et al. Aldo-keto Reductase Metabolizes Glyphosate and Confers Glyphosate Resistance in Echinochloa colona. Plant Physiol 181, 1519-1534, doi:10.1104/pp.19.00979 (2019).
2 Rumsby, G. & Cregeen, D. P. Identification and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1446, 383-388, doi:https://doi.org/10.1016/S0167-4781(99)00105-0 (1999).
3 Tao, R. et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism. Nat Methods 14, 720-728, doi:10.1038/nmeth.4306 (2017).
4 Zhao, Y. et al. SoNar, a Highly Responsive NAD+/NADH Sensor, Allows High-Throughput Metabolic Screening of Anti-tumor Agents. Cell Metabolism 21, 777-789, doi:10.1016/j.cmet.2015.04.009 (2015).
5 van Bloois, E., Winter, R. T., Kolmar, H. & Fraaije, M. W. Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol 29, 79-86, doi:10.1016/j.tibtech.2010.11.003 (2011).
6 Fu, G. M. et al. Pathway and rate-limiting step of glyphosate degradation by Aspergillus oryzae A-F02. Prep Biochem Biotechnol 47, 782-788, doi:10.1080/10826068.2017.1342260 (2017).
7 Hove-Jensen, B., Zechel, D. L. & Jochimsen, B. Utilization of glyphosate as phosphate source: biochemistry and genetics of bacterial carbon-phosphorus lyase. Microbiol Mol Biol Rev 78, 176-197, doi:10.1128/MMBR.00040-13 (2014).
8 Singh, S. et al. Glyphosate uptake, translocation, resistance emergence in crops, analytical monitoring, toxicity and degradation: a review. Environmental Chemistry Letters 18, 663-702, doi:10.1007/s10311-020-00969-z (2020).
9 Kryuchkova, Y. V. et al. Isolation and characterization of a glyphosate-degrading rhizosphere strain, Enterobacter cloacae K7. Microbiological Research 169, 99-105, doi:https://doi.org/10.1016/j.micres.2013.03.002 (2014).
10 Errey, J. C. & Blanchard, J. S. Functional Annotation and Kinetic Characterization of PhnO from Salmonella enterica. Biochemistry 45, 3033-3039, doi:10.1021/bi052297p (2006).
11 Chan, C. T., Lee, J. W., Cameron, D. E., Bashor, C. J. & Collins, J. J. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol 12, 82-86, doi:10.1038/nchembio.1979 (2016).