A tale of two unicellular organisms

Why Chlamydomonas?

Chlamydomonas reinhardtii is a well-studied, photosynthetic, unicellular eukaryote, making it an attractive candidate for developing an autonomous photosynthetic system capable of converting PET into vanillic acid. Microalgae, including Chlamydomonas, offer a promising platform for recombinant protein synthesis, contrasting with traditional heterotrophic organisms like E. coli, yeast, or CHO cells. The low-cost phototrophic cultivation of microalgae, coupled with the "generally recognized as safe" (GRAS) status of several algae strains, including Chlamydomonas reinhardtii, makes it an ideal choice for our study.

Our project is divided into two main steps:
Step 1 involves introducing a gene encoding the PETase-MHETase fusion protein into the genomes of E. coli and Chlamydomonas reinhardtii, enabling these organisms to convert PET into TPA (terephthalic acid).
Step 2 focuses on transforming the obtained TPA molecule into PCA (protocatechuic acid), and subsequently converting PCA into vanillic acid (VA). To achieve the full transformation of TPA into VA, we will introduce genes encoding the necessary enzymes responsible for each step of the conversion.


Why Bacteria?

While our ultimate goal is to create a photosynthetic organism capable of converting plastic into a valuable molecule, we decided to conduct the same experiments in both bacteria and Chlamydomonas for comparison. By testing the process in both systems, we aim to evaluate the efficiency of each approach. Based on the outcomes, combining the two systems may prove advantageous for achieving optimal PET degradation and vanillic acid production.

Creating our CDS and Plasmid

With Bacteria:


PET to TPA


This stage appears to be the simplest, as the plasmid encoding the PETase-MHETase enzyme is commercially available under the name pCJ190 (reference). Therefore, for this step, we do not need to construct the coding sequence (CDS) ourselves. All that is required is to order the plasmid and perform the bacterial transformation. The plasmid pCJ190 is represented below:

TPA to PCA


For this purpose we engineered à fusion protein named tphall ( contains proteins tphaAa,tphAb,tphAc,tphB linked together via 12 AA Gly/Ser linker) and adapted this sequence for MoClo technique and to create a unique plasmid.
The techniques and main steps of this process are inspired by the work of the 2020 iGEM team from Sorbonne University.
For plasmid construction, we rely on the MoClo (Modular Cloning) technique, which facilitates the assembly of basic genetic parts such as promoters, coding sequences (CDS), and terminators. This process follows the principles of Golden Gate cloning (Weber et al., 2011).
In the MoClo assembly system, the smallest functional units are fundamental genetic elements like promoters, CDS, 5’ UTRs, and tags. Each element is assigned a specific position, determined by unique fusion sites on both ends. These basic parts are cloned into "Level 0" plasmids using BbsI restriction sites. A Level 0 acceptor plasmid typically contains BbsI restriction sites flanking a lacZ cassette, a pUC origin of replication, and an antibiotic resistance gene. When digested with BbsI, the lacZ cassette is excised, allowing the part of interest to hybridise to the plasmid via complementary overhangs. The molecules are then ligated, and the resulting plasmids are used to transform E. coli.
Subsequent assembly steps, such as creating Level 1 and Level M constructs, follow the same principle but use the BsaI restriction enzyme to combine multiple units into a final, more complex plasmid.



Level 1:


During the MoClo process, type IIS restriction enzymes (BbsI and BsaI) are used. These enzymes, unlike traditional restriction enzymes, cut outside of their recognition sites, generating overhangs that can be ligated to compatible sequences. Importantly, the original recognition site is not reconstructed after ligation, ensuring efficient and seamless assembly. In our Level 0 plasmid, the initial BbsI recognition site within the CDS is no longer present after ligation with the backbone. Same principle works for Level 1 et Level M.
We added appropriate fusion sites at the ends of the sequence. At the 5' end, we incorporated a B3 scar (AATG) along with the BbsI restriction site, and at the 3' end, a B5 scar with a BbsI restriction site.
Also, we adapted our CDS of tphall fusion protein for the position B3-B5, so we added appropriate fusion sites at the ends of the sequence. At the 5' end, we incorporated a B3 scar (AATG) along with the BbsI restriction site, and at the 3' end, a B5 scar with a BbsI restriction site.
Additionally, to prevent any internal cleavage of our gene during the digestion/ligation process, we removed internal type IIS restriction sites (BsaI, BbsI, and SapI).
The final construct is shown below.

Level 1:


The goal of Level 1 is to combine several parts into a single plasmid using the same principles as Level 0. Here, we aim to assemble the following plasmids:

pCM0-011-PAR (Pro + 5’UTR_A1-B1)
Plasmid TPH-ALLl
pCM0-119-TRPL23 (3’UTR + Ter_B6-C1)


Positions A1, B1, B2, B3-B5, B6-C1 correspond to the MoClo positions (Crozet et al., 2018).
The plasmids:

pCM0-011-PAR (Pro + 5’UTR_A1-B1)
pCM0-119-TRPL23 (3’UTR + Ter_B6-C1)
are compatible with the bacteria and are derived from the MoClo kit (Crozet et al., 2018).
For the assembly, we proceed in the same manner as for constructing Level 0, except this time using the BsaI enzyme instead of BbsI.

PCA to VA


The same steps were followed to construct the HsOMT gene sequence in order to adapt it for the MoClo technique and to create a Level 0 and Level 1 plasmid.
We decided to adapt HsOMT for the B3-B5 positions. At the 5' end, we incorporated a fusion site (AATG) along with the BbsI restriction site, and at the 3' end, a fusion site (GCTT) with a BbsI restriction site. For the positioning of the sites, see the table from Crozet et al.
Additionally, to prevent any internal cleavage of our gene during the digestion/ligation process, we removed internal type IIS restriction sites (BsaI, BbsI, and SapI).

Finally, we have three different plasmids:


pCJ190 encoding PETase-MHETase (ampicillin resistance)
Tphalll encoding the tph proteins (kanamycin resistance)
HsOMT level 1 encoding HsOMT (streptomycin resistance)
We can now proceed with the triple transformation of bacteria.

With Chlamydomonas:



PET to TPA


The process with Chlamydomonas is slightly more complex than with bacteria. The genome of Chlamydomonas is highly GC-rich, meaning that GC-rich codons are more commonly used. Since PETase-MHETase is originally a bacterial protein, it is essential to adapt its sequence to the codon bias of Chlamydomonas. To achieve this, we used the PETase-MHETase sequence from the pCJ190 plasmid (which includes a 12-amino-acid linker and a 6x His Tag) as a base and optimised it for Chlamydomonas using the codon usage table from the Kazusa Codon Usage Database. This resulted in a CDS of PETase-MHETase adapted to the codon preference of Chlamydomonas.
To introduce this CDS into Chlamydomonas, we will employ the MoClo technique to construct the necessary plasmid for transformation. Our CDS needs to be adapted for MoClo assembly, so we plan to insert our gene into positions B3-B5 of the MoClo system. To do this, we added appropriate fusion sites at the ends of the sequence. At the 5' end, we incorporated a B3 scar (AATG) along with the BbsI restriction site, and at the 3' end, a B5 scar with a BbsI restriction site (see Chlamy Guide made by 2019 iGEM Sorbonne University team for more details).
Additionally, to prevent any internal cleavage of our gene during the digestion/ligation process, we removed internal type IIS restriction sites (BsaI, BbsI, and SapI).
The final construct of PETase-MHETase, optimised for MoClo assembly, is shown below.

To achieve the first step of converting PET into TPA, we need to transform organisms with a plasmid containing the gene for the PETase-MHETase fusion protein, responsible for the reaction sequence PET -> MHET -> TPA.



Level 0:

This Level 0 plasmid is derived from the insertion of the PETase-MHETase CDS, optimised for Chlamydomonas, into the acceptor plasmid pICH41308 (from the MoClo toolkit). Transformants carrying Level 0 plasmids are selected by blue/white screening and by antibiotic resistance ( streptomycin)


Level 1:


The goal of Level 1 is to combine several parts into a single plasmid using the same principles as Level 0. Here, we aim to assemble the following plasmids:
pCM0-011-PAR (Pro + 5’UTR_A1-B1)
pCM0-049-mVenus (CDS_B2)
p0_PETase-MHETase (CDS_B3-B5)
pCM0-119-TRPL23 (3’UTR + Ter_B6-C1)
Positions A1, B1, B2, B3-B5, B6-C1 correspond to the MoClo positions (Crozet et al., 2018).



The plasmids:


pCM0-011-PAR (Pro + 5’UTR_A1-B1)
pCM0-049-mVenus (CDS_B2)
pCM0-119-TRPL23 (3’UTR + Ter_B6-C1)
are derived from the MoClo kit. For the assembly, we proceed in the same manner as for constructing p0_PETase-MHETase (CDS_B3-B5), except this time using the BsaI enzyme instead of BbsI.
The theoretical results from SnapGene are shown below:

TPA to PCA


The same steps used to adapt the CDS of PETase-MHETase for Chlamy and MoClo were followed to adapt the sequences of the proteins tphAa, tphAb, tphAc, and tphB. We will assemble the following:
pCM0-017-PAR (Promoter + 5’UTR_A1-B1)
P0_thpall-level0 (same as TPHALL for bacteria but with codon optimization, CDS_B3-B5)
pCM0-119-TRPL23 (3’UTR + Ter_B6-C1)
This will in turn give us the Result: p1_tphall - chlamy


PCA to VA:


The same steps used to adapt the CDS of PETase-MHETase for Chlamy and MoClo were followed to adapt the sequences of the protein HsOMT
We will assemble the following:
pCM0-017-PAR (Pro + 5’UTR_A1-B1)
P0_HsOMT (,CDS_B3-B5)
pCM0-119-TRPL23 (3’UTR + Ter_B6-C1)
In order to have the following Result: p_1plasmid HsOMT lvl 1-chlamy

Level M in Moclo:


At Level M, we assemble plasmids from Level 1 by combining them with a backbone, a resistance gene, and linkers (sequences that connect DNA fragments). The goal of this assembly is to create a functional plasmid for the organism Chlamydomonas, which includes the genes of interest and allows selection of the transformed cells through the resistance gene.
In this case, we will be assembling the following:


p1_PETase-MHETase: A genetic sequence combining two enzymes, PETase and MHETase, involved in plastic degradation.
P1_tphall - chlamy: A Level 1 plasmid for Chlamydomonas, which likely contains the tphall gene (coding for tphAa,Ab,Ac,B enzymes involved in a metabolic pathway of TPA to PCA).
p1_plasmid HsOMT - chlamy: Another Level 1 plasmid for Chlamydomonas, containing the HsOMT gene to transform PCA in VA.
The goal is to create a single plasmid that contains:


A resistance gene (to select Chlamydomonas cells that have integrated the plasmid),
Three genes of interest (PETase, MHETase, tphall, and HsOMT), each controlled by its own specific promoter (Pr), a 5'UTR sequence to regulate translation, and a 3'UTR sequence with a terminator to stop gene expression.
In the end, the assembled plasmid will have the following structure:


Resistance gene – PETaseMHETase – tphall – HsOMT – Final linker.
Then we make the transformation and this construction enables Chlamydomonas to receive a functional set of genes with all the necessary elements for their expression in the organism.

Testing our work

To test our theoretical ideas, we conducted laboratory experiments, with detailed results shown in the Results section. In summary, we successfully produced the PETase-MHETase fusion protein in bacteria, but unfortunately, due to time constraints, we couldn’t progress further. However, this doesn't stop us from planning the next experiments, which are also described in the link Results section!

References

1. Tanasupawat S, Takehana T, Yoshida S, Hiraga K, Oda K. Ideonella sakaiensis sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate). Int J Syst Evol Microbiol. 2016 Aug;66(8):2813-2818. doi: 10.1099/ijsem.0.001058. Epub 2016 Apr 5. PMID: 27045688.
2. Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, Pardo I, Topuzlu E, Anderson JJ, Austin HP, Dominick G, Johnson CW, Rorrer NA, Szostkiewicz CJ, Copié V, Payne CM, Woodcock HL, Donohoe BS, Beckham GT, McGeehan JE. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc Natl Acad Sci U S A. 2020 Oct 13;117(41):25476-25485. doi: 10.1073/pnas.2006753117. Epub 2020 Sep 28. PMID: 32989159; PMCID: PMC7568301.
3. Sasoh M, Masai E, Ishibashi S, Hara H, Kamimura N, Miyauchi K, Fukuda M. Characterization of the terephthalate degradation genes of Comamonas sp. strain E6. Appl Environ Microbiol. 2006 Mar;72(3):1825-32. doi: 10.1128/AEM.72.3.1825-1832.2006. PMID: 16517628; PMCID: PMC1393238.
4. ​​Brochado, Ana & Matos, Claudia & Møller, Birger & Hansen, Jørgen & Mortensen, Uffe & Patil, Kiran. (2010). Improved vanillin production in baker's yeast through. Microbial cell factories. 9. 84. 10.1186/1475-2859-9-84. [5]Kim, Hee & Kim, Jae & Cha, Hyun & Kang, myung Jong & Lee, Hyun & Khang, Tae & Yun, Eun & Lee, Dae-Hee & Song, Bong & Park, Si Jae & Joo, Jeong Chan & Kim, Kyoung. (2019). Biological Valorization of Poly(ethylene terephthalate) Monomers for Upcycling Waste PET. ACS Sustainable Chemistry & Engineering. XXXX. 10.1021/acssuschemeng.9b03908.
5. https://www.addgene.org/162667/
6. https://2020.igem.org/Team:Sorbonne_U_Paris/Description
7. Crozet, Pierre & Navarro, Francisco & Willmund, Felix & Mehrshahi, Payam & Bakowski, Kamil & Lauersen, Kyle & Pérez-Pérez, María & Auroy, Pascaline & Gorchs Rovira, Aleix & Sauret-Gueto, Susana & Niemeyer, Justus & Spaniol, Benjamin & Theis, Jasmine & Trösch, Raphael & Westrich, Lisa & Vavitsas, Konstantinos & Baier, Thomas & Hübner, Wolfgang & de Carpentier, Félix & Lemaire, Stéphane. (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology. 7. 10.1021/acssynbio.8b00251.