Model

Design

Chlamydomonas reinhardtii :

Chlamydomonas reinhardtii is a photosynthetic green microalgae that has been massively studied and used as a model organism in plant biology. Its physiology and metabolism are well known, and both its nuclear and chloroplast genomes have been sequenced in 2007. Thanks to its structural and genetic characteristics, and to the fact that it is very easy to grow, it has been nicknamed “The green yeast”. That is why this photosynthetic organism is widely used for biotechnological applications. It has been used for instance for the development of biofuels, but also for recombinant proteins which can be used as cancer drugs.

Since it is very well known and rather easy to modify genetically, Chlamydomonas is an ideal chassis for synthetic biology. Moreover, its autotrophic metabolism allows us to create an energetically autonomous system. This is a key aspect of a sustainable water treatment system. Thus, we could imagine a system of water biofiltration by microalgae grown in a bioreactor with the products of CO2 fixation by photosynthesis as the only source of carbon. The use of phototrophic organisms in water treatment applications brings both the advantage of creating a low energy cost system, but also promotes the fixation of atmospheric carbon.

Modular cloning:

We used a standardized DNA strategy called Modular Cloning (referred to hereafter as MoClo). Both BioBricks and MoClo allow the assembly of basic parts such as promoters, CDS or terminators however the MoClo relies on the Golden Gate cloning strategy (Weber et al. 2011).

A- Type IIS restriction enzymes

Restriction enzymes are endonucleases which catalyze the hydrolysis of a double stranded DNA at a specific sequence resulting in a double strand break.

The classical restriction enzymes (e.g. EcoRI) cut inside their recognition site. They release a part of this recognition site and have to be ligated with a DNA sequence cut with a compatible restriction enzyme. After the ligation, the restriction site is reconstituted.

The type IIS restriction enzymes (BpiI, BsaI) cut downstream of their recognition sites and generate 4 bp 5’-overhangs (Engler et al. 2009) that are not specific to the restriction enzyme.

B- One-pot, one-step reaction (Engler et al. 2009)

The golden gate cloning allows a directional assembly of multiple DNA fragments in a one-pot, one-step reaction. Indeed, the restriction enzyme and the ligase are added at the same time as there are no scars of the restriction site after the ligation when type IIS enzymes are used, making the final product stable during the reaction. The two enzymes (for digestion and ligation) do not work at the same temperature (37°C for the restriction enzyme and 16°C for the ligase). To increase the cloning efficiency, several cycles (37°C-16°C) are done.

C- MoClo Assembly standard

In the MoClo assembly standard (Weber et al. 2011), the smallest units are basic genetic parts such as promoters, CDS, 5’UTR, tags. Each type of basic parts has a position defined by the fusion sites on both sides. Also, as all the same basic parts have a unique fusion site they are commutable at will. These basic units are cloned into plasmids with BpiI and are called "Level 0". A Level 0 acceptor plasmid contains BpiI restriction sites flanking a lacZ cassette, a pUC origin of replication, and the spectinomycin resistance gene. Upon digestion with BpiI, the plasmid releases the lacZ cassette and the part of interest hybridizes to the plasmid through complementarity of the flanking 4 bp overhangs, corresponding to the fusion site. This implies that there is a specific acceptor plasmid for each position within the MoClo standard for Chlamydomonas reinhardtii. These molecules are then ligated and the final products are used to transform E. coli (LacZdelta). The selection of the transformants carrying Level 0 plasmids is made by blue/white selection (pick white) on a LB medium with X-gal and spectinomycin.

All Level 0 plasmids also contain a BsaI recognition site that allows a second golden gate reaction to create an entire transcription unit (Level 1) from several Level 0 plasmids (Weber et al. 2011). It is the specificity of the overhangs which define the order of the parts into a transcriptional unit. Level 1 plasmids can be screened because they have the ampicillin resistance gene (and not spectinomycin like the Level 0).

A full transcription unit can be made by only choosing the basic units. It also allows the testing of a module library in the same transcription unit and thus the screening of the best module for a specific application (e.g. different promoter for a coding sequence). It is very useful for optimizing complex pathways in metabolic engineering.

For Chlamydomonas reinhardtii, a specific MoClo toolkit has been developed (Crozet et al. 2018), and we used their standard for the construction of the retrotransposon, as well as some parts of this kit.

D- Design of our standardized parts

In the case of the design of a coding sequence (CDS) of proteins of exogenous origin adapted to our chassis, we can reverse translate the protein sequence to generate cDNA. The redundancy of the genetic code means that the proteinogenic amino acids with the exception of methionine (AUG) and tryptophan (UGG) are encoded by at least 2 codons. This results in a variation in the frequency of use of the different codons that differs between organisms. The Chlamydomonas genome is very rich in GC which implies that the GC-rich codons are more frequently encountered. In order to maximize the expression of our transgenes, it is therefore necessary to adapt our sequences to the codon bias found in Chlamydomonas. For this, we used the codon usage table of Chlamydomonas which allowed us to obtain the most efficient sequence of our cDNA from the polypeptide sequence of our proteins of interest. The next step in the design of our standardized sequence was to add the fusion sites corresponding to the position of the brick relative to the standard MoClo plant and add restriction sites for type IIS restriction enzyme BbsI at the 5’ and 3’ ends. Finally, in order to avoid any internal hydrolysis of our gene parts during the digestion/ligation reaction, internal type IIS sites of BsaI and BbsI were removed.

Atrazine degradation:

At the start of our adventure, we learned from literature that the model microalgae Chlamydomonas reinhardtii harbours bioremediation abilities against hormone compounds (Hom-Diaz et al. 2015) and antibiotics (Rusch et al. 2019). Only one major class of pollutant was missing : pesticides. We chose to target atrazine, one of the most broadly detectable herbicides (Jablonowski et al. 2011).

We then imagined an engineered microalgae able to express the atrazine degradation pathway. This new function is the heart of our concept: making Chlamydomonas reinhardtii the ultimate tool for water depollution by enhancing its bioremediation spectrum. Because of the experimental difficulty to introduce a whole new pathway in a host organism, we limited ourselves to 3 genes: atzA, atzB and atzC, that encodes enzymes which metabolize atrazine into cyanuric acid, a less toxic compound.

The first gene in the degradation pathway atzA encodes for atrazine chlorohydrolase which catalyzes atrazine dechlorination to hydroxyatrazine. It was first identified in 1996 (de Souza et al. 1996)
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The second gene (atzB) encodes for hydroxyde-chloro-atrazine ethylaminohydrolase (atzB) and catalyzes the hydroxyatrazine deamidation, yielding N-isopropylammelide (Govantes et al. 2010)

Finally, N-isopropylammelide isopropyl amidohydrolase (atzC) transforms N-isopropylammelide to cyanuric acid and isopropylamine. (Sadowsky et al. 1998)

These enzymes are well described in the litterature and known since the nineties. They were isolated and characterized from the Pseudomonas sp. ADP strain, the most studied bacteria able to use atrazine as a carbon source (de Souza et al. 1996; de Souza et al. 1998). Our basic parts were constructed with the polypeptide sequences from this strain, adapted for the genome of Chlamydomonas reinhardtii.

A single multigenic plasmid containing this new metabolic pathway was created using the MoClo assembly standard.

Kill-switch:

For our project, developing a kill-switch is essential, as our modified algae is supposed to be in contact with the Seine’s water. To prevent the spread of our organisms in the river in an uncontrolled way and the creation of an imbalance in the ecosystem, the algae has to be contained in filters. However, these filters may not be completely infallible. For additional safety, we designed an ingenious kill switch system, causing the algae to die when it manages to pass through the filter. This system is based on light, especially UV-light. The algae will be cultivated and used under so-called ‘high-pass’ color filters that prevent UV light from passing through. When the algae is out of this chromatic filter, it is exposed to the entire light spectrum, including the UV-light that will induce its premature death.

In plants, the regulation and mechanism of programmed cell-death (PCD) is not yet fully understood, but similarly to all other eukaryotic organisms it is known that the fragmentation of genomic DNA inevitably leads to PCD.
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We have chosen the micrococcal nuclease as an effector to fragment genomic DNA. Since the genome is protected by the nuclear envelope, it is necessary to tag the nuclease with a nuclear localization sequence (NLS). As nucleases show their death-bringing activity at low concentrations, it is essential to put the kill-switch under tight spatio-temporal control and induce activity under specific conditions only.

How to prevent the action of the nuclease

To prevent the action of the nuclease, we have chosen to anchor it to the cytoplasmic membrane under basal conditions. In this form, the nuclease cannot activate its deadly potential as it is spatially separated from its site of action, the nucleus, more precisely inside the nuclear envelope.
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To make sure that the nuclease can be released, we used a long linker coupled with a TEV recognition/cleavage site between the transmembrane domain (TMD) and the NLS-tagged nuclease. In the presence of TEV protease activity, the nuclease is released from the membrane and is able to translocate into the nucleus thanks to its NLS tag. Once in the nucleus, it can fulfill its task as the executor of PCD by degrading the genomic DNA with its endo-exonuclease activity.
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The next step is to control the activity of the TEV protease. To do so we have chosen to induce its activity under UV-light.

How to control the activity of the protease

We have designed two fusion proteins, each one composed of the C-terminal or the N-terminal splitted part of the TEV protease, each coupled with either UVR-8 or COP1, which hetero-dimerize after UV-light exposure. As long as the two fusion proteins are separated, the protease cannot be active. When the fusion proteins dimerize, the TEV protease is reconstituted, resulting in a proteolytic activity for a specific TEV recognition site.
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The reconstituted TEV protease liberates the micrococcal nuclease from its membrane anchor by cleaving the TEV site and separating the nuclease and the transmembrane domain. The nuclear localization signal ensures nuclear translocation of the nuclease.
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Once in the nucleus, the nuclease will be able to unleash its deadly power by fragmenting genomic DNA and induce the death of C. reinhardtii.

The nuclease

For our kill switch, we have chosen a nuclease that presents the following characteristics:
- it is capable to digest all kind of DNA without any preference
- it has no disulfide bonds (because they do not form in the cytoplasm)
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So we have chosen the micrococcal nuclease of Staphylococcus aureus as our death effector (Heins et al. 1967). Indeed, it is an endo-exonuclease and it does not have any disulfide bonds. To anchor this protein to the cytoplasmic membrane, we are using the transmembrane domain of the HAPLESS2 membrane receptor of C. reinhardtii and the N-peptide signal of the carbonic anhydrase 1 of C. reinhardtii .
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To make sure that the nuclease is released from the membrane in presence of the TEV protease, we used a long linker with a TEV cleavage site. Our long linker is composed of 5 repetitions of the following amino-acid sequence : GlyGlyGlyGlySer with the TEV site in the C-terminal part of the linker.
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After the release of the nuclease by proteolysis of the TEV site, it will be guided inside the nucleus where it will act. To do so, we chose to use the nuclear localization sequence of SV-40 as it is well characterized and already available as a biobrick for MoClo.

The protease

To prevent the constitutive activation of the protease TEV and the release of the nuclease, we have decided to split it in two. Each half will be linked with a part of a protein complex that will dimerize under the action of an effector.
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To ensure this function, we have chosen the UV-light as an activator and UVR-8/COP1 as a protein complex. Indeed, after UV-light excitement, UVR-8 will hetero-dimerize with COP1. Without the UV-light, they will stay uncomplexed.
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So, we have two distinct constructs :
- The C-terminal part of the TEV protease linked to UVR-8
- The N-terminal part of the TEV protease linked to COP1
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Thanks to UV filters, UVR-8 is not excited and will not be complexed with COP1, so the TEV protease cannot be complete. When UV rays are not filtered, UVR-8 will monomerize in order to hetero-dimerize with COP1. This will complete the TEV protease which will cut the TEV site because of its restriction activity and release the nuclease (Wehr et al. 2006). And so, it will induce cell death.

How does UVR-8 work

UVR-8 (Ultraviolet-B resistance 8) is an UV-B-sensing protein, and a cryptochrome. It is present in some plants and initiates the plant stress response when it is exposed to ultraviolet light, especially in the 280 to 315 nanometer range with a sensitive pike at 285 nanometer which is the lower limit of UV-B. The ability of UVR-8 to be a photoreceptor seems to be linked to its tryptophans in the 285 and 233 positions (Ulm et al. 2015).
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This ability is really interesting for us since it allows us to activate an intracellular mechanism by modifying extracellular properties. Indeed, when this protein is stimulated by UV-B irradiation, the tryptophan residues absorb the light which disrupts the salt-bridges composed by the arginine residues adjacent to the tryptophans (Christie et al. 2012). UVR-8 will monomerize and interact with a protein called COP1 (constitutively photomorphogenic 1) (Cloix et al. 2012). As mentioned previously, we are basing our kill switch mechanism on this interaction by linking both halves of the TEV protease respectively to an UVR-8 and a COP1 protein. This mechanism does not naturally exist for our algae model but it does acclimate well (Tilbrook et al. 2016).

References:

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- Cloix C, Kaiserli E, Heilmann M, Baxter KJ, Brown BA, O'Hara A, Smith BO, Christie JM, Jenkins GI. C-terminal region of the UV-B photoreceptor UVR8 initiates signaling through interaction with the COP1 protein. Proc Natl Acad Sci U S A. (2012).
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Team:Sorbonne U Paris/Design