Team:UCAS-China/Proof Of Concept

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• proof of concept
• Due to the epidemic
• Protein production
• Protein purification
• Cell-free system
• High temperature adaptation
• Thermal stability of coenzymes
• Improved thermal stability of Cdh by directed evolution
• Improved thermal stability of CkTcS by PROSS
• Reference

To prove the correctness of the concept of our project, there are two main components: to verify the effectiveness of the cell-free caffeine degradation system; and to verify that the system is able to adapt to the high temperature environment of coffee.

Due to the epidemic

Due to the epidemic, our laboratories were not open for the entire time, which prevented us from completing all of our experiments as planned. We will use a combination of experiments and literature references to complete the proof of concept.

Protein production

During the production of the proteins, we performed detailed experimental documentation, although limited photos were taken. To see the complete experimental record, please refer to the notebook section.

When we first started trying to produce protein, the strain we chose was BC102 from Biomed. however, because this strain was not suitable for protein production, the od600 value in culture falied to reach 0.5, leading to setbacks in our subsequent experiments. The following figures shows the record of our initial experiments.

Fig1. Experiment steps of transformation

After the attempt, we were advised to change the strain by consulting our teachers and professional researchers. Subsequently, we transformed and expanded the culture using BL21-receptive E. coli and took colony PCR to verify whether the plasmid had been successfully transformed.

However, PCR again suffered a setback: the target bands did not appear several times. After some adjustments to the parameters and extracting plasmids for PCR as a control, we finally determined that the problem was that the primers provided by Genetics were not appropriate. It took some time to reorder the primers, but fortunately, we eventually obtained new primers and verified that the transformation was successful.

Below are the experiment steps of PCR and pictures of the gel.

Fig2. Experiment steps of recover PCR products and test for purity and concentration

Fig3. PCR result of Cdh plasmids and colonies

Fig4. PCR result of CkTcS plasmids and colonies

After confirming the successful transformation, we then proceeded to the amplification culture of the engineered bacteria with induced protein expression. Subsequently, we collected a small amount of the bacterium by centrifugation, crushed it and performed SDS-PAGE gel electrophoresis, and observed bands that matched the molecular weight of our target protein. This proved that our protein expression was successful.

Fig5. SDS-PAGE result of cell crushing solution

However, unfortunately, due to epidemic prevention measures and limited laboratory opening time, we did not have time to complete the large-scale culture of the engineered bacteria and the subsequent protein purification process. However, we have performed cryofreezing of the culture medium and will continue to complete this part of the experimental work in the future.

Protein purification

Despite not being able to perform the experiment as planned, we searched the literature to ensure that our design for protein purification worked. We found a detailed protocol to guide our entire experimental procedure.

What's more, fortunately, in the article about the 3D structure of CkTcS, the enzyme used in our second reaction, researchers just produced it in E.coil and purified it using SUMO tags. This not only directly provided a successful precedent for a portion of our protein purification work, but also demonstrated the feasibility of using SUMO to express complex eukaryotic proteins in a prokaryotic expression system.

Based on the information, we identified the basic steps for purifying the protein, see the Project-Protein purification section. The detailed procedure can also be found in the protocol. From the "methods" section of the above-mentioned literature on the expression and purification of CkTcS with the help of SUMO, we also learned that if the final protein obtained by following the steps in the protocol is not sufficiently pure, the purity can be further improved by ion exchange chromatography or gel filtration chromatography.

Cell-free system

Although we have not had sufficient time to complete the construction of the cell-free system due to the epidemic, there is sufficient evidence that that the two enzymes we selected can catalyze the reaction normally in a cell-free system. CkTcS is able to catalyze the production of theacrine at pH=7.0 and in the presence of MgCl2 (Yue-Hong Zhang et al., 2020)^[1] Cdh is able to catalyze the conversion of caffeine to 1,3,7-trimethyluric acid at pH=7.8 and in the presence of CoQ0 (K. M. Madyastha et al. 1999)^[2]So the cell-free system we build can ensure the efficiency of the reaction.

High temperature adaptation

Coffee lovers are well aware of the impact of temperature on sensory properties of coffee. In order to expand the usage scenarios of our coffee cup and maintain the most of coffee flavor and user experience, we have demonstrated that the cell-free system can function in high temperature environments.

Thermal stability of coenzymes

We have determined the thermal stability of SAM with the aid of HPLC analysis. Here are the data and images we obtained.

This shows that SAM can be regarded as loss-free for 1 hour at room temperature, with a loss of approximately 4.7% for 5 hours. At 70°C the loss of SAM for 1 hour is approximately 32%.

Consider our design of decaffeinated coffee cups.

(1) During transport, transporting SAM at room temperature will cause greater loss and cold chain transport should be used for long distances. However, short term storage at room temperature when the user brings the tea bags at home or outside will not affect the function of the cartridge.

(2) During use, the actual use of the cup by the user for enzymatic reactions is at most ten minutes, even at high temperatures, the heat loss of SAM is still limited, i.e. the poor heat resistance of SAM will not have an impact on the actual function of the cup.

Improved thermal stability of Cdh by directed evolution

Directed evolution is a synthetic process that harnesses the power of selection to evolve target protein to have desired properties from large, stochastically permuted pools or combinatorial libraries. This is a simple idea that imitate the process of natural selection. Directed evolution has the advantage of its simplicity of operation. There is no need to figure out all the details of proteins, like structure.

There has already been general directed evolution strategy based on error-prone PCR. In the first step we choose Error-prone PCR, which can make the gene mutate more easily when doing PCR, by rising the concentration of Mn(ii). After expression, we select the protein with better thermostability, amplify its gene and use it in the more round until we get a proper mutant.

There have been a series studies which demonstrate the feasibility of the directed evolution of protein based on error-prone PCR, which can usually successful in improve enzymes’ thermostability, high or low pH tolerance and catalytic activity.

Lipase catalyze can serve as hydrolysis of fats and oils, which have been widely used in industrial fields. But it’s low thermostability limits its application in many fields. Guan, L. et al. (2020) used error-prone PCR to screen for enhanced thermostability and successfully got 2 mutants out of over 3000 mutants, which with Tm 2.5 and 6℃ respectively higher than that of the wild type.

Endoglucanases is a useful tool for hydrolyzing cellulose homopolymers. In 2017, Yang, et al. used Error-prone PCR together with DNA shuffling to select transformants with higher thermostability. Among 1370 transformants, they chose one clone finally and found that it’s thermostability was increased 1.53-fold (21.4 min vs. 14.0 min), which is beneficial for producing fermentable sugars.

The glycoside hydrolase, alpha-L-rhamnosidase, is used to remove the bitter taste of citrus juices, but most of them are easy to deactivate at high temperature. Li, et al. developed a mutant by directed evolution and site-directed mutagenesis and improved it’s thermostability.

Xing H, et al. did their work in 2020 that create a variant with a 420-fold increase in half-life at 70℃ by Error-prone PCR and integrating the beneficial residues in many mutants.

Qiu J, et al. (2020) also used two rounds error-prone PCR to enhance the activity and thermal stability of a phthalate-degrading hydrolase and selected a mutants showed over 50% improvement in thermostability.

Through a large number of literature research, we found that directed evolution of protein based on error-prone PCR has been widely used to improve the activity and thermostability of enzymes used in various industrial production, and this method can often achieve a significant improvement in the thermostability and catalytic activity of enzymes. But to achieve such effect, a lot of experiments and screening are necessary. Consider screening efficiency, finding a suitable high-throughput screening method is essential for the error-prone PCR-based directed evolution of enzymes. The most significant changes which can be easily measured before and after the reaction is the absorbance of the reaction substrate coQ0. The obvious difference between the absorbance of the oxidation state and the reduced state coQ0 fortunately provides us with the possibility of high-throughput screening, furthermore, a chance to conduct the directed evolution of cdh enzyme based on error-prone PCR. However, due to the limited time and the epidemic control in the laboratory, we did not complete this part of the experiment. We hope that in future plans, we can have the opportunity to verify the feasibility of our plan, and provide a better answer for the goal of improving the thermostability of cdh enzyme and ensuring the product taste.

Improved thermal stability of CkTcS by PROSS

We hope to increase the reaction temperature of the system to at least 70 Celsius without affecting its catalytic efficiency, but the optimal reaction temperature of wild-type CkTcS is only about 30 Celsius, so we need to make great improvements to the thermal stability of CkTcS. Under these circumstances normal methods including "directed evolution" may cost us large amounts of time to obtain a thermally stable and catalytically active protein. Worsely, we may not get the desired results.

Fortunately, Adi Goldenzweig et al (2016) has developed an algorithm called PROSS( Protein Repair One Stop Shop) for improving protein stability. They had successfully increased hAChE’s resistance to heat inactivation of up to 20 Celsius with nearly 2,000-fold higher bacterial expression and intact activity. Other designed enzymes also show high stability and/or soluble expression. You can find introduction to the principle of the algorithm in our Model part.

Our PI has used PROSS to design some proteins successfully. Using the PROSS platform, we obtained 9 results in a short time, some of them contain few mutations (<5%), and some produced a large number of mutations (>20%). Yue-Hong Zhang et al (2020) have pointed out important amino acids for the enzyme's executive function ( Fig6.). We removed the mutations on these sites, and back-translate the amino acid sequence to DNA sequence. We then designed plasmid vectors and handed them over to a gene company for synthesis.

Fig6. Important amino acids for the protein function

Reference

Guan, L., Gao, Y., Li, J., Wang, K., Zhang, Z., Yan, S., . . . Lu, S. (2020). Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR. Front Bioeng Biotechnol,8, 1034. doi:10.3389/fbioe.2020.01034

Ulrich, H. D. (2009). SUMO Protocols. Preface. Methods Mol Biol, 497, v-vi. doi:10.1007/978-1-59745-566-4

Xing, H., Zou, G., Liu, C., Chai, S., Yan, X., Li, X., . . . Zhou, Z. (2021). Improving the thermostability of a GH11 xylanase by directed evolution and rational design guided by B-factor analysis. Enzyme Microb Technol,143, 109720. doi:10.1016/j.enzmictec.2020.109720

Yang, M.-J., Lee, H. W., & Kim, H. (2017). Enhancement of thermostability of Bacillus subtilis endoglucanase by error-prone PCR and DNA shuffling. Applied Biological Chemistry, 60(1), 73-78. doi:10.1007/s13765-017-0254-3

Zhang, Y. H., Li, Y. F., Wang, Y., Tan, L., Cao, Z. Q., Xie, C., . . . He, R. R. (2020). Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea. Nat Commun, 11(1), 1473. doi:10.1038/s41467-020-15324-7