Abstract

An engineering cycle should be established from the beginning and undergoes continuous improvement with the results of experiments, thus, giving birth to a successful engineered product. And that is why and how we document our engineering below.

In the first stage, we reviewed the literature to summarize the metabolism of THC, and tentatively determined that we would engineer E.coli Nissle 1917 to express a human-derived degradative enzyme line to transfer Δ9-THC into non-psychoactive substance. After we assessed the difficulty of directly constructing a plasmid containing three enzymes simultaneously, we decided to seperate them into three vectors to obtain a higher operability in our experiments, but still under the sensing of Δ9-THC.

THC-sensing module

Imagine 1: What can we use to receive the signal?

At the very beginning, we came up with using G-protein-coupled receptor called CB1 that can sense the Δ9-THC and its analogues. After a series of signal transduction, the adenylate cyclase is activated and cAMP is produced. Thus, we planned to find a kind of promoter that can be activated by cAMP to reach the visualization of the sensing of Δ9-THC by expressing special proteins. However, we then found the method uncontrollable since there are other ways that can influence the content of cAMP in the cell. Besides, we could not find the suitable promoter which can accurately and specifically sense the cAMP.

It was then that the sensing system of 2021 BNU-China popped into our mind. Last year, they replaced the iron(III)-binding motif of Pmr system with an anti-mCherry motif and successfully achieved sensing mcherry and coding proteins. Considering that they have already proved the feasibility of the system, we decided to modify their design.

Design 1: How can we sense the THC?

The main focus of our design is on sensing the Δ9-THC. PmrB receptors of the Pmr system, which is located in the cell membrane, use the iron (III) -binding motif to sense iron levels in the physiological environment . We replaced the iron (III) binding motif with an anti-THC motif(2019 Queens_Canada), which specifically binds Δ9-THC and then promotes PmrB to phosphorylate PmrA protein. Phosphorylated PmrA protein further activates the PmrC promoter, initiating the expression of reporter protein and enzymes to degrade THC.

Imagine 2: How can we visualize the sensing results?

After sensing the Δ9-THC by the Pmr system, we tried to find a kind of reporter protein to distinctly remind the people who have taken in Δ9-THC by mistake.

At the very beginning, we considered the most common reporter protein--GFP. We imagined users testing their excrement to know if they have any mis-ingestion of cannabis. Soon we found that testing fluorescence is not convenient. Luckily, a piece of news inspired us--Eating edible coloring pigments results in colorful excrement. Because of that, we chose to express a kind of workable edible coloring pigment to visualize the results.

Design 2: What can we use to achieve visualization?

We decided to make our engineering bacteria secrete C-phycocyanin to verify if they have successfully finished the sensing pathway . C-phycocyanin is a natural blue pigment extracted from Spirulina. It is one of the rare pigment proteins in nature, which allows us to make a distinction between daily food and cannabis intake. It is not only bright in color, but also a harmless food.

Fig.1 Schematic representation of the α (brighter color) and β (darker color) subunits of phycocyanin^[1].

Build 1: How can we build it?

We got the full sequence of PmrA, PmrB, PmrC and C-phycocyanin from NCBI. While replacing the original iron (III) -binding motif of PmrB, we put C-phycocyanin downstream of the PmrC promoter and connected all of them with plasmid PUC57. We also added His-tag linked with the C-terminal of the C-phycocyanin for purification and OmpA signal sequence with the C-terminal of the C-phycocyanin for extracellular secretion.

Test 1

To confirm the correct structure and expression of C-phycocyanin, we linked phycocyanin to lacO which can be activated by IPTG in plasmid PET-Duet1. We transfected PET-Duet1 into E.coli(DH5α) for amplification. Then we extracted the plasmid for sequencing. The result met our expectations generally. So we transfected the plasmids into E.coli(BL21) to express the C-phycocyanin. After inducing with IPTG, we centrifuged and broke the bacteria to acquire the lysate. Finally we used Nickel column to adsorb C-phycocyanin and verified it.
Following that, we began to verify our pathway by using 11-OH-Δ9-THC instead of the controlled drug △9-THC. We induced our engineering bacteria by adding 11-OH-△9-THC. The experiment is still on going……

Learn & Improve 1

During our experiment, we surprisingly found that the C-phycocyanin might be oxidized to brown but not blue in the air. ^[1]This may cause interference for visualization. Therefore, we tried to find some feasible methods to ensure the stability of C-phycocyanin. Finally, the whey protein came into our sights, which was proved to result in blue color maintenance.^[2] We designed to pack the whey protein powder and our engineering bacteria in a kind of suitable capsule, so as to release them together and protect the C-phycocyanin. Besides, if this way cannot work well, we will turn back to fluorescin like mCherry and improve the hardware.

Fig.2 We tried to find some feasible methods to ensure the stability of C-phycocyanin.

THC metabolism module

Imagine 1: How to develop a THC-metabolizing pathway that works upon sensing Δ-9-THC?

The focus of our design is to help people degrade addictive cannabis that they have ingested in their intestine in a safe way, which means the addictive substance is converted to non-addictive substance. In human body, Δ9-THC is hydroxylated to 11-OH-Δ9-THC, which is still psychoactive and is further oxidized to psychoactive Δ9-THC-COOH. In the course of our in-depth review of the literature, we introduced three enzymes, CYP2C9, CYP2C19^[3][4], which are necessary for the metabolism of Δ9-THC , and UGT1A3^[5], which converts Δ9-THC-COOH into non-addictive cannabinoid glucuronic acid.

Fig.3 Cytochrome P-450(CYP-450) metabolic pathways for cannabinoids and investigated metabolites based on in vitro data.^[4]Supporting data (Bland et al., 2005; Bornheim et al., 1992; Chimalakonda et al., 2012; Jiang et al., 2011; Matsunaga et al., 2000; Richardson et al.,1995; Watanabe et al., 1995, 2002, 2007).
Fig.4 Glucuronidation activity screening. Selected recombinant UGT isoforms and human liver microsomes were screened for activity toward CBN, THC-OH, THC-COOH, and CBD. Glucuronidation activities were measured by incubating microsomal protein 5 μg of recombinant UGT; 50 μg of human liver (HL)] with 500 μM substrate and 4 mM UDP-GlcUA. All reactions were normalized as described under Materials and Methods.^[5]

Design 1: How do we ensure the Δ9-THC conversion in the body?

At first, we hoped to introduce the above pathway into the intestinal flora to metabolize Δ-9-THC by expressing three enzymes, so as to avoid the harm and influence caused by ingestion of cannabis. However, in the actual construction of plasmids, we found that the introduction of three enzyme genes into a plasmid would make the plasmid too large, and CYP2C9 and CYP2C19 have homologous sequences, which would make the construction of plasmids difficult. Therefore, we decided to introduce the gene sequences of the three enzymes into three plasmids respectively, and all of them were induced and detected by PET-DUET as the chassis.

Build 1: How can we build it?

We obtained the sequences of CYP2C9, CYP2C19 and UGT1A3 from NCBI. We designed the sequence of these enzymes by codon optimization and added the OmpT signal peptide sequence with the N-terminal of each gene sequence for surface display. We introduced these three enzyme genes into three plasmids separately.

Test 1: Do we get what we want?

The results generally met our expectations. After culturing E.coli for 5h in a shaking bed, we took ultrasonic fragmentation to break the cells, purified the protein through his-tag protein extraction kit and verified it by SDS-PAGE. We finally obtained the correct bands.

In subsequent experiments, we will use Western Blot to detect protein expression. Moreover, we found Enzyme immunoassay for human cytochrome P450 family member 2C19 (CYP2C19) ,Enzyme immunoassay for human cytochrome P450 family member 2C9 (CYP2C9)kit and Human uridine diphosphate glucuronosyltransferase (UGT) enzyme immunoassay kit to verify the biological activity of three enzymes.

Learn & Improve 1

For the idea that we express three enzymes separately, we were questioned in subsequent communication with professors and other teams - how to adjust the proportion of the three bacteria carrying the enzyme separately? Is there an interaction between them? These talks really inspired us that in future improvements, we would consider emerging technologies like the Crispr-Cas9. We hope to express sgRNA and Cas9 through a plasmid, perform fusion PCR on the genes of the three enzymes to construct Donor DNA, transfer the plasmid substituted with sgRNA and Cas9 and Donor DNA into Escherichia coli at the same time, and guide Cas9 protein through the targeted localization of sgRNA. Hopefully the genes for three enzymes will be inserted into the genome of E.coli.

(1)The host strain is prepared into competent cells, and λ-Red recombination system is integrated in the genome of the host strain in advance to induce the expression of recombinase during the preparation of competent cells;

(2)Plasmid pCas9cur, plasmid psgRNA and linear Donor DNA are co-transferred into host cells, or plasmid psgRNA and linear Donor DNA are co-transferred into host cells containing plasmid pCas9cur;

(3)Linear Donor DNA recombines with genome target sequences mediated by recombinase, and the constitutively expressed Cas9 and sgRNA form a complex to recognize and cut the genome sequences that have not been recombined.

Fig.5 We will take the Crispr-Cas9 technology to solve the problems of expression of three enzymes.

References

1. Pez Jaeschke D, Rocha Teixeira I, Damasceno Ferreira Marczak L, Domeneghini Mercali G. Phycocyanin from Spirulina: A review of extraction methods and stability. Food Res Int. 2021 May;143:110314. doi: 10.1016/j.foodres.2021.110314. Epub 2021 Mar 17. PMID: 33992333.
   Full text     Back to text
2. Zhang S, Zhang Z, Dadmohammadi Y, Li Y, Jaiswal A, Abbaspourrad A. Whey protein improves the stability of C-phycocyanin in acidified conditions during light storage. Food Chem. 2021 May 15;344:128642. doi: 10.1016/j.foodchem.2020.128642. Epub 2020 Nov 16. PMID: 33223303.
   Full text     Back to text
3. Watanabe K, Yamaori S, Funahashi T, Kimura T, Yamamoto I. Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes. Life Sci. 2007 Mar 20;80(15):1415-9. doi: 10.1016/j.lfs.2006.12.032. Epub 2007 Jan 17. PMID: 17303175.
   Full text     Back to text
4. Stout SM, Cimino NM. Exogenous cannabinoids as substrates, inhibitors, and inducers of human drug metabolizing enzymes: a systematic review. Drug Metab Rev. 2014 Feb;46(1):86-95. doi: 10.3109/03602532.2013.849268. Epub 2013 Oct 25. PMID: 24160757.
   Full text     Back to text
5. Mazur A, Lichti CF, Prather PL, Zielinska AK, Bratton SM, Gallus-Zawada A, Finel M, Miller GP, Radomińska-Pandya A, Moran JH. Characterization of human hepatic and extrahepatic UDP-glucuronosyltransferase enzymes involved in the metabolism of classic cannabinoids. Drug Metab Dispos. 2009 Jul;37(7):1496-504. doi: 10.1124/dmd.109.026898. Epub 2009 Apr 1. PMID: 19339377; PMCID: PMC2698943
   Full text     Back to text