Proof of Concept

Design: Measure the Enzyme Functions

In order to confirm whether β-glucuronidase can hydrolyze the reactant substance to drop a glucuronic acid, we selected an E. coli strain, E.coli BL21 (DE3), which is commonly used to express proteins for experiments and measurements. We chose to engineer the plasmid pet28a(+) that is known to be compatible with this strain for expression of β-glucuronidase protein by inserting the corresponding Gus sequence. To Distinguish from the previous works, we call our sequence the bG sequence. The construction of the plasmid is shown in the schematic drawing on the right.

Figure 1. Construction of plasmid T7-bG-pet28a(+) for expression of the enzyme in E. coli BL21(DE3).

In order to measure the production of glycyrrhetinic acid, HPLC is the most commonly used method in the reference articles. It can quantitatively measure the amount of the reduction of GL (glycyrrhizic acid) and the production of GA (glycyrrhetinic acid), but the apparatus is highly costly for our lab to own. We have consulted a few iGEM teams in our HP and colab activities, and also consulted a few commercial companies about HPLC measurements, and from the responses, we learned this method still costs a lot even for a single measurement run because the special container is not recyclable. At the same time, HPLC measurements may take a long time, even up to 3 weeks, due to limited access and complex sample preparations. Therefore HPLC method is not a proper method in the first stage of experiments for optimization of the enzyme expression.

Principles of β-glucuronidase enzyme activity measurement and its function converting GL (glycyrrhizic acid) to GA (glycyrrhetinic acid)

In our interviews, we learned there is a special test toolkit for β-glucuronidase. The reactant substance prepared in the toolkit contains Phenolphthalein β-D-glucuronide. After being hydrolyzed with the presence of β-glucuronidase, the product releases free Phenolphthalein in the buffer. By adding NaOH into the buffer after reaction, Phenolphthalein will show pink to purple color. The amount of Phenolphthalein released is proportional to the light intensity at OD540 according to the toolkit documentation, and the measurement can directly indicate the level of (the total) enzyme activity of β-glucuronidase. The method can not tell the amount of β-glucuronidase produced in each cell. In the following figure, we show the chemical theory for the production of GA by β-glucuronidase hydrolysis and the principle of enzyme activity measurement with the toolkit.

In summary, for quantitative measurement of enzyme activity, we need:
OD540: the light emission intensity at 540 nm which indicates the amount of Phenolphthalein;
OD600: the light emission intentisy at 600 nm which indicates the concentration of bacteria solution;
and we take OD540/OD600 value, normalizing by the bacteria concentration for enzyme activity value.

Figure 2. (1) β-glucuronidase can hydrolyze glycyrrhizic acid into glycyrrhetinic acid，of which the product can be detected by HPLC; (2) Phenolphthalein β-D-glucuronide can be converted into Phenolphthalein under the hydrolysis of β-glucuronidase. The product Phenolphthalein can be observed as pink to purple color under basic conditions. The product can be quantitatively determined by OD540.

Strategy to optimize for enzyme species

According to our literature searches and Human Practices interviews with Prof. Lv Bo, we selected three different kinds of β-glucuronidase which is likely to have high catalytic effectiveness, namely the β-glucuronidase from E. coli source [3], from Aspergillus oryzae [4] and from Thermotoga maritima [5].

All three enzymes were expressed in E. coli BL21(DE3), and we carried out SDS-PAGE experiments and enzyme activity experiments to evaluate their effectiveness. The results show only the β-glucuronidase enzyme from E. coli source has been successfully expressed, while β-glucuronidase (Aspergillus oryzae) and β-glucuronidase (Thermotoga maritima) expression had to be further explored. Similarly in the enzyme activity measurement, the β-glucuronidase enzyme from E. coli source has the highest enzyme activity. Therefore, we carried out further experiments based on this enzyme.

The failure to express β-glucuronidase (Aspergillus oryzae) and β-glucuronidase (Thermotoga maritima) might be the result of the following reasons.
1. The protein structure of the two enzymes are different from that of the E. coli source and therefore the translation and folding of this protein might not be compatible with the specific plasmid and E. coli strain we currently use.
2. The expression condition might need to be further optimized for these two enzyme such as the timing for induction, the working concentration of IPTG and/or the strain we are using.
Because we had only limited time in an iGEM season, we first focused on the experiments with the successfully expressed species, and if we had a chance in the future, we would carry out more experiments to explore the expression conditions at various induction conditions such as adding IPTG at OD600=0.6~0.8, or changing the IPTG concentration from 0.5mM to 2mM, or varying the incubation temperature from 16 degree Celsius to 37 degree Celsius, or using different expression strains like E. coli Rosetta or E. coli Plyss.

Strategy to optimize E. coli strain for more effective expression

There are a few more E. coli strains frequently and routinely used for protein expression and production in the factories. Under certain circumstances, they could be advantageous compared to the conventional BL21(DE3). We then tested the enzyme activities of β-glucuronidase expressed in different E. coli expression strains and found in our case the enzyme activities do not show a significant difference. For subsequent experiments, we decided to keep using E. coli BL21(DE3) strain with the enzyme from E. coli source.

Strategy to optimize expression strength using sfGFP measurement with different promoters

The appropriate enzyme expression strength may contribute to the catalytic efficiency in the unit volume bacteria culture. With this consideration in mind, we plan to control the catalytic power of the bacteria culture by optimizing the expression of β-glucuronidase in E. coli. In the expression systems of E. coli, T7 promoter is a most commonly used promoter originating from the T7 phage. Researchers have obtained a series of modified T7 promoters with different expression strength through engineered mutations (Ref [1], [2]). We selected to test C4, H9 and G6 promoters on a reporter gene sfGFP. To our surprise, the fluorescence intensities we measured do not show a significant difference between the mutated promoter and the original T7.

Strategy to optimize expression strength using enzyme activity measurement with different promoters

Considering that the intensity of sfGFP does not necessarily represent the expression of β-glucuronidase enzyme, We used different promoters to connect the β-glucuronidase enzyme from E. coli source to detect the enzyme activity under these conditions. β-glucuronidase enzymes from Aspergillus oryzae and from Thermotoga maritima were tested again. The results indicated the promoters indeed do not bear a significant difference. Therefore, the T7 promoter can still be used as the first choice for promoters. Promoters with different strengths will be obtained through mutation or directed evolution in the future.

Of course, we believe that the induction temperature and induction method may also affect the final catalytic ability of the bacterial solution, while E. coli may not be the best species. Other prokaryotes, yeasts, molds, etc. are also candidate chassis organisms. However, due to time limits, these experiments can only be carried out in the future.

Strategy to optimize production cost using a constitutive promoter

In addition, an expression system with induction can better control the expression of enzyme at the right timing. But we learned in Human Practices interviews that iPTG, as an inducer, introduces high costs in industrial production. Therefore, constitutive expression may be more suitable for industrial production, and constitutive expression system does not need to consider the choice of induced reagents, inducing reagent concentration, and other variables, and thus the system is conducive to the reduction of inter-batch difference and to increase stability. So we plan to create a system of constitutive expression system for the enzyme.

In the BL21 strain, both the T7 RNA polymerase and the β-glucuronidase enzyme on the pet28a vector contain lacO sequences in their upstream and therefore any expression needs to be induced. In the future, we plan to remove the LacO sequence before the T7 RNA polymerase gene in the BL21 genome through gene editing, as well as the LacO sequence in the pet28a vector to obtain a system for the expression of β-glucuronidase enzymes in a constitutive way.

However, due to the time limit, it is not realistic for us to realize genome editing during the 2021 iGEM season, and we are not currently doing this experiment. Alternatively, as a proof of concept, we designed a gene circuit in DH5a bacteria, using the E. coli constitutive promoter J23101 in upstream of the β-glucuronidase enzyme coding sequence. We tested for enzyme activity of the bacteria without any addition of the inducer IPTG.

Examine the enzyme activity with HPLC detection and glycyrrhizic acid as substrate

Unfortunately we had received a negative but inconclusive result.

In the above experiments, we used Phenolphthalein-β-D-glucuronide instead of glycyrrhizic acid to conduct experiments to reduce the time cost. Finally, we also used HPLC to verify some of the above experimental results. HPLC results show that β-glucuronidase enzyme can indeed convert glycyrrhizic acid. However, the experiment did not detect a signal of glycyrrhetinic acid after the reaction. Except for the possibility that better HPLC measurement conditions might help to detect the glycyrrhetinic acid signal, there is still the possibility that some new product was formed but escaped from the detection. It is believed that after continuous optimization, this method can be used to make glycyrrhetinic acid cheaper and more environmentally friendly in the industry for use in medical, food, and other industries.

HPLC now shows a negative result. First of all, HPLC is very time-consuming, costly, and labor-intensive. We sent a batch of samples very early, in July, at the beginning of our experiment. Then the other party gave us this result in October. Our July system may not be very mature yet, and then we did not have a chance to optimize it. Our conditions may not be the most suitable, so this does not prove that our system is definitely not able to produce glycyrrhetinic acid. We will optimize the conditions later, hoping to have more time and do more attempts to determine whether this can produce glycyrrhetinic acid.

Sometimes an enzyme is indeed normal, but it can’t be induced because the bacteria are in a poor condition. So it is possible that when we first started the experiment, the status of our bacteria was not well controlled. We did not do SDS-PAGE to check for protein expression at the same time as adding glycyrrhizic acid, which is our mistake. So we now speculate that this may be one of the possible reasons. It is also possible that we added glycyrrhizic acid too early at the beginning. In this case, it will put too much pressure on the growth of bacteria, so we may have to try different conditions in the future when the OD600 reaches a certain level (0.6~0.8), and then add inducer and glycyrrhetinic acid.

Future work

First up in the future, we would have to gain more chances of HPLC measurements to confirm the enzyme activity on glycyrrhizic acid. This might require optimization of HPLC experiment conditions, optimization of enzyme expression conditions, and hydrolysis conditions.

Then we can explore the optimization of glycyrrhetinic acid (GA) production and yield, and then explore conditions for better scale-up tests, conditions for extraction of GA and conditions for immobilization of β-glucuronidase enzyme.

Up to this stage, the lab results can support successful manufacturing of the product, which will be used for downstream pharmaceutical, food additive and cosmetic industry at lower cost and potentially better performance.

References

[1]. Jones, J., Vernacchio, V., Lachance, D. et al. ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci Rep 5, 11301 (2015). https://doi.org/10.1038/srep11301
[2]. Adams, A. M., Kaplan, N. A., Wei, Z. et al. In vivo production of psilocybin in E. coli. Metabolic Engineering, vol 56 p111-119, (2019). https://doi.org/10.1016/j.ymben.2019.09.009.
[3]. Afkhami-Poostchi A., Mshreghi, M., Iranshahi M., et al. Use of a genetically engineered E. coli overexpressing β-glucuronidase accompanied by glycyrrhizic acid, a natural and anti-inflammatory agent, for directed treatment of colon carcinoma in a mouse model. International Journal of Pharmaceutics, Vol 579, 119159 (2020). https://doi.org/10.1016/j.ijpharm.2020.119159.
[4]. Wang, X., Feng, X., Lv, B., et al. Enhanced yeast surface display of β-glucuronidase using dual anchor motifs for high-temperature glycyrrhizin hydrolysis. AIChE Journal. Vol 65, e16629 (2019).
[5]. Wang, Z., Pei, J., Li, H., and Shao, W. Expression, Characterization and Application of Thermostable β-glucuronidase from Thermotoga maritima. Chin J Biotech Vol 24(8), 1407-1412 (2008).
[6]. Wang, Guojing. Enzymatic Production of Glycyrrhetinic Acid and the Separation Process Design. Master Thesis, Beijing Institute of Technology. (2016)
[7]. Brittona, J., Majumdar, S., and Weiss, G. A. Continuous Flow Biocatalysis. Chem Soc Rev. Vol 47(15): 5891–5918. (2018) doi:10.1039/c7cs00906b

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