Part BBa_K5007000 is our first composite. It’s a combination of the coding sequences of the three enzymes responsible for olivetolic acid (OA) synthesis, fused head-to-tail in a seamless fashion, intercalated by the self-cleaving T2A peptide CDS. The enzymes’ CDS have already been catalogued by Waterloo’s 2022 iGEM team, and the self-cleaving peptide CDS we chose has already been catalogued by SYSU-MEDICINE team from iGEM 2016.

It's worth noting that the inclusion of the self-cleaving peptide leaves fragments on the N-, C- or both terminals of our peptides. Even though they are not particularly large fragments, some of our enzymes are also quite small, so we did a thorough modeling and evaluation of each one of our enzymes with these fragments in order to evaluate how these fragments interfered with our proteins’ structure, stability and possible functions.

Talking a little bit more about the specifics of this part: it contains the CDS for Hexanoyl-CoA synthase (CsHCS), Olivetol Synthase (CsOS) and Olivetolic Acid Cyclase (CsOAC), intercalated by the T2A (part BBA_K1993019) CDS (fig. 1).

Figure 1. Part BBa_K5007000 diagram.

Source: Authors, 2023.

Hexanoyl-CoA synthase (slightly modified from part BBA_K4393005) is a CoA-ligase, responsible for the activation of hexanoic acid. It is quite similar to other acyl-activating enzymes, as well as many CoA synthases in structure, function and conservation. The main differences are around the catalytic pocket, where hexanoic acid must enter in order to be ligated to CoA (Stout et al., 2012).

Olivetol synthase (slightly modified from part BBA_K4393006) is an interesting enzyme: dimeric, somewhat large and takes a lot of substrates and ligands. It is responsible for the synthesis of olivetol from hexanoyl-CoA and three malonate molecules, having a reaction mechanism that is considerably elucidated. There are several similar enzymes throughout the Cannabaceae family, which allowed us to develop a robust phylogenetic analysis to understand better the possible catalytic and ligand positions (Taura et al., 2009).

Olivetolic acid cyclase (from part BBA_K4393003) is a cute one - tiny, only a hundred amino acids long (in its monomeric form), forms a heart-like shape when it’s dimerized, and catalyzes a very important reaction: it turns olivetol into olivetolic acid, the direct precursor to cannabinoid synthesis (Gagne et al., 2012). Being so small, we had to be very careful with the addition of the T2A fragments – we cannot have it so modified that it doesn't fold properly, so we positioned it at the very end, where the T2A fragment would be the smallest possible. There are a few comparable enzymes along the Cannabaceae family, and these similarities helped us a lot when we needed to validate docking results, which was a little difficult on the docking servers we used, its size being a factor in the issues we faced..

Part BBa_K5007006 is our second composite, and it consists of only two enzymes: Prenyltransferase 4 (CsPT4) and Cannabidiolic acid synthase (CsCBDAS), with a single T2A between them, fused with the same logic from before (fig. 2). This composite contains the necessary information to synthesize cannabidiolic acid (CBDA) from olivetolic acid, going through the necessary prenylation step. Both part A and part B are composite parts optimized for Saccharomyces cerevisiae, but they can easily be optimized for other organisms and cloned into diverse plasmids for expression in various chassis.

Figure 2. Part BBa_K5007006 diagram.

Source: Authors, 2023.

Prenyltransferase 4 (part BBa_K5007001, improved from part BBA_K4393004) is a prenyltransferase from the UbiA family of enzymes (Luo et al., 2019). It is the most complex of our parts, due to it being a membrane protein, directed to Cannabis plants chloroplasts through a large (77 amino acid residues) transport peptide, not having its catalytic mechanism elucidated and not having a crystallized structure (up until the Wiki freeze date) published. Even though it is a protein that presented us with a myriad of challenges, we needed to understand it better, and not because there were no other options, but because we knew we could find out some interesting things about it. The reaction this enzyme catalyzes is the prenylation of olivetolic acid with geranyl diphosphate, which is synthesized by S. cerevisiae along its mevalonate pathway, generating cannabigerolic acid (CBGA), the precursor to most plant cannabinoids. In order to improve the part previously catalogued in the iGEM registry, we removed the transit peptide, making it possible for this protein to be translocated to the plasmatic membrane or stay in the cytosol instead. This embedding in the cytoplasmatic membrane may cause the generated products to permeate the cell membrane and be secreted to the periplasm or the extracellular space. This, however, may not be an issue, due to the relatively small size of CBGA making it possible for the molecule to go from one cell to the other, and the predicted secretion of CsCBDAS complementing CsPT4’s action. Adding to the challenges mentioned before, there are not a lot of well characterized prenyltransferases, even less so in the Cannabaceae family. However, we managed to develop a phylogenetic analysis for this enzyme, with which we were able to identify several sites of interest for the coupling of substrates, cofactors and ions, which were not previously cataloged anywhere in literature. These results allowed us to develop a very well characterized prediction model for this enzyme, allowing its use in diverse synthetic biology contexts.

Cannabidiolic acid synthase (from BBA_K4393007) is the last enzyme in our pathway, responsible for the very last conversion of CBGA into CBDA (Taura et al., 2007). This enzyme is, just as the other four, not very well characterized, which led us to perform the same bioinformatic analysis we performed before, in order to better understand and evaluate this protein. We found that this protein contains not only a secretion signal peptide, but also contains several N-linked glycosylations [5]. Not coincidentally we chose S. cerevisiae – it has all the necessary machinery and tolerance for the synthesis and needed post translational modifications of all of our enzymes. With our bioinformatic analysis, we were able to unravel lots of important aspects of this and all of our enzymes (see Modeling and Engineering success), and this is reflected in the very rational design of our biological circuits.

Parts BBa_K5007007 and BBa_K5007008 are our devices. They’re expression cassettes designed to promote the biosynthesis of olivetolic acid (BBa_K5007007) in our yeast cells from hexanoic acid (supplied externally, or hijacked from the cell’s lipid synthesis pathway) and cannabidiolic acid (BBa_K5007008) from olivetolic acid (Park & Kim, 2006). It consists of either part BBa_K5007007 or part BBa_K5007008, fused to the Gal1 promoter on the 5’ end, and the ADH1 terminator on the 3’ end (fig.3), both parts being efficient in starting and ceasing transcription of our cassettes in yeast cells. These devices are under control of the Gal1 promoter (part BBA_J63006), which is a strong inducible yeast promoter, activated by galactose. ADH1 (part BBa_K392003) is the terminator for the homologous, constitutive, alcohol dehydrogenase gene. We expect these parts to perform smoothly in the expression of our pathway enzymes, with the needed post translational modifications.

Figure 3. Diagrams for devices BBa_K5007007 (top) and BBa_K5007008 (bottom).

Source: Authors, 2023.

We’ve designed all of these parts to be highly interchangeable – each CDS, fragments or the entire parts can be amplified individually through PCR with the submitted primers, and further amplified for cloning, with different primers, into diverse plasmids in order to study the expression of CBDA through our base strategy in diverse chassis. The designed cassettes may be only suitable for yeasts and the T2A sequence may only work in eukaryotes, but we encourage other teams and groups to create different cassettes for expression, e.g., in plant cells through agroinfiltration assays or in filamentous fungi, based on our cassettes. We still haven’t tested the efficiency and capabilities of our circuits in-vitro, but if our bioinformatics is anything to go by, we are fairly secure that our strategy is viable and reduces largely many of the challenges encountered by other groups studying the heterologous production of CBD – one of the main ones being the difficult cloning and transformation steps required.

We also submitted two plasmids - pRS423s (BBa_K5007028) and pRS425s (BBa_K5007025), which were constructed from pRS426, substituting the auxotrophic mark URA3 from the original pRS426 by LEU2 (on pRS425s) or HIS3 (on pRS423s). The original plasmids pRS423 and pRS425 are a little different from our versions, and we built our own similars for a simple reason: we had pRS426, and we had access to the HIS3 and LEU2 marker cassettes, so why not amplify the markers and clone them into pRS426, effectively transforming the plasmid we HAD into the plasmids we NEEDED? Even though the developed plasmids are a little different from the original ones, they have the same needed characteristics - they have selection marks and replication origins for E. coli, replication origins for S. cerevisiae and a very robust cloning region we can use for white-blue selection on our available E. coli strains.

Parts from BBa_K5007009 to BBa_K5007024, BBa_K5007026 and BBa_K5007027 are primers designed for amplification of all of our parts. Most of them were designed for cloning via Gibson Assembly, so they are long, larger than 34bp, and their annealing temperature is on the high side. There shouldn’t be any problems, however, if these primers are annealed at around 70 to 72°C. We do recommend running a screening on them to find their optimum PCR temperatures for each lab conditions, though.

Our other parts are slightly modified versions of parts previously deposited by other teams, or cloning primers. The designed primers and composites allow not only for the replication of experiments by various teams, but also the design of new cassettes and isolation of parts we developed by other teams, making it possible for this project to be further developed and built upon.

We are running for best part collection, and for a good reason: the full synthesis of cannabinoids has been investigated by several groups, all around the world, with varying degrees of success. In order for these medications to be accessible, available and affordable for patients-in-need, we need to develop their synthesis processes via synthetic biology to not only be as open-source as possible, but all of the needed parts need to be easily understandable, workable, retro- and forward compatible, optimizable and exchangeable between plasmids, chassis and other systems. The part collection we published has this goal: it allows groups to study cannabidiol obtention via transformant yeast fermentations, it provides tools for cloning parts and isolating the coding sequences for the metabolic pathway. It also provides tools for modifying and cloning into different yeast plasmids. It contains parts that are optimized AND optimizable, and, most importantly, parts that were validated through several steps to ensure they will function as we expect them to.

In summary, this part collection is running for best part collection because it facilitates the entire workflow for the introduction of the CBDA biosynthesis pathway, or even other cannabinoids, into a chassis, since one of our composites promotes the synthesis of one the main precursors to phytocannabinoids. This is about facilitating access and research, shortening manipulations and helping to make it possible for people in need to finally be able to access this medication without having to worry about the extraordinarily expensive processes involved in importation of the medication.

Gagne, S. J., Stout, J. M., Liu, E., Boubakir, Z., Clark, S. M., & Page, J. E. (2012). Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides. Proceedings of the National Academy of Sciences of the United States of America, 109(31), 12811–12816. https://doi.org/10.1073/pnas.1200330109

Luo, X., Reiter, M. A., d’Espaux, L., Wong, J., Denby, C. M., Lechner, A., Zhang, Y., Grzybowski, A. T., Harth, S., Lin, W., Lee, H., Yu, C., Shin, J., Deng, K., Benites, V. T., Wang, G., Baidoo, E. E. K., Chen, Y., Dev, I., … Keasling, J. D. (2019). Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature, 567(7746), 123–126. https://doi.org/10.1038/s41586-019-0978-9

Park, K. S., & Kim, J. S. (2006). Engineering of GAL1 promoter-driven expression system with artificial transcription factors. Biochemical and Biophysical Research Communications, 351(2), 412–417. https://doi.org/10.1016/j.bbrc.2006.10.050

Stout, J. M., Boubakir, Z., Ambrose, S. J., Purves, R. W., & Page, J. E. (2012). The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes. Plant Journal, 71(3), 353–365. https://doi.org/10.1111/j.1365-313X.2012.04949.x

Taura, F., Sirikantaramas, S., Shoyama, Y., Yoshikai, K., Shoyama, Y., & Morimoto, S. (2007). Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa. FEBS Letters, 581(16), 2929–2934. https://doi.org/10.1016/j.febslet.2007.05.043

Taura, F., Tanaka, S., Taguchi, C., Fukamizu, T., Tanaka, H., Shoyama, Y., & Morimoto, S. (2009). Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway. FEBS Letters, 583(12), 2061–2066. https://doi.org/10.1016/j.febslet.2009.05.024