Team:Sorbonne U Paris/Design


Our chassis : Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a freshwater unicellular green microalga.
Further information about Chlamydomonas reinhardtii are available on our collaboration page.
We chose C. reinhardtii as our chassis for the purpose of developing a new way of producing oil compounds for several reasons: Firstly, it already produces the two main compounds of palm oil: palmitic and oleic acid. C. reinhardtii already has the tools needed to produce these components, so the challenge will be to tweak its metabolism to produce more of these specific compounds and also prevent its insaturation as C. reinhardtii produces polyunsaturated fatty acids from them.
Secondly, C. reinhardtii is a photoautotrophic organism that can produce its own food using light as a source of energy. Thereby, it needs much less nutrients than heterotrophic organisms like bacteria or yeasts. Moreover, C. reinhardtii can be grown in marine water or photobioreactors and thus does not compete with arable lands. It is therefore an ecological organism consistent to our project philosophy.
Lastly, there are a lot of tools and resources to do molecular biology on this organism. Its genome has been sequenced in 2007 (Merchant et al. 2007) and it has several tools required to perform genetic engineering such as the Chlamydomonas Modular Cloning kit (Crozet et al. 2018).

Design iterations

For the first design iteration of our engineered C. reinhardtii strain, we will be integrating several enzymes from african oil palm, Elaeis guineensis. These enzymes were selected because they are either key components in fatty acid composition in triglycerides or limiting steps in triglyceride synthesis.

Fatty acid synthase (FAT) is an enzyme involved in the fatty acids elongation pathway. It catalyzes, the hydrolysis of the bond between the acyl carrier protein (ACP) and the fatty acid, releasing a free fatty acid which can no longer be elongated as the cycle requires the fatty acid to be bound to the ACP. Different isoforms of the FAT enzymes are known to have different affinities depending on the fatty acid length. We are using this property to create two strains of C. reinhardtii that will have two different FAT isoforms of E. guineensis : FAT-A and FAT-B2 (Dussert et al. 2013), which have a higher affinity for fatty acids C18:X, like oleic acid (C18:1), and palmitic acid (C16), respectively. This will allow us to tweak the fatty acid composition of triglycerides by enriching in a certain fatty acid, which in turn will be more incorporated in the triglyceride synthesis pathway, also called Kennedy pathway.

Lysophosphatidic acid acyltransferase (LPAAT) is responsible for the incorporation of the second fatty acid at the position sn-2 of the glycerol in the Kennedy pathway. This step has been shown to be limiting (Voelker et al. 1996) for the modification of the fatty acid composition in triglycerides after incorporating an heterologous FAT (Voelker et al. 1992). A combined expression of both the FAT and LPAAT have been shown to produce the most drastic change in fatty acid composition in triglycerides (Knutzon et al. 1999). Cloning E. guineensis LPAAT-A, which is highly expressed in its mesocarp (Dussert et al. 2013), will allow us to further increase the level of palmitic and oleic acid in triglycerides.

Diacylglycerol acyltransferase (DGAT) is responsible for the incorporation of the third and last fatty acid at the position sn-3 of the glycerol in the Kennedy pathway. Heterologous expression of this enzyme has been shown to drastically increase the quantity of triglycerides produced by the modified organism. As it is known to be highly expressed in the mesocarp of the oil palm (Dussert et al. 2013), we chose to express E. guineensis variant DGAT-1-2 in C. reinhardtii to improve the overall production of triglycerides in our modified strains.

To conclude, we will have two modified C. reinhardtii strains: The first one, aimed to produce palmitic acid, will have a E. guineensis FAT-B2 - LPAAT-A - DGAT-1-2 multigenic construct. The second one, aimed to produce oleic acid, will have a E. guineensis FAT-A - LPAAT-A - DGAT-1-2 multigenic construct.




Golden Gate Modular Cloning : MoClo

We want to engineer our microalga by modifying its genome to create a new phenotype. This is why we have chosen the Golden Gate Modular Cloning (MoClo) technology that allows fast track assembly of multigene constructs (Werner et al. 2012).

Type IIS restriction enzymes

Restriction enzymes are endonucleases that cut double stranded DNA. They are classified in two types :

The classic restriction enzymes cut DNA substrate at the recognition site : the cleavage site and the recognition site are the same. The fragment released from the plasmid using a pair of classical restriction enzymes contains parts of the recognition sequence at its ends, so when it ligates to a piece of DNA that has compatible ends, the recognition site is present between the two fragments (Figure 2).
The type IIS restriction enzymes cut outside of their recognition sequence: the cleavage site and the recognition site are separated. Contrary to classic restriction enzymes, no part of the recognition site is present in the fragment released from the plasmid using type IIS enzymes and it can be ligated to other fragments that have compatible ends. Moreover, there is no part of the recognition site between the two fragments after the ligation (Figure 2).


Moclo assembly standard

In 2009, a new subcloning strategy based on the use of type IIS restriction enzyme has been reported (Engler et al. 2009). This method, named Golden Gate Cloning, is as efficient as recombination-based cloning technologies and has two main characteristics :
Firstly, it is scarless. Indeed, type IIS restriction enzymes cut outside of their restriction site so there is no recombinaison site sequences left in the final construct. In other words, there are no “scars” of the restriction sites after the ligation. Secondly, it is a one pot, one step reaction. Indeed, this method allows subcloning one or several DNA fragments from donor plasmids to an acceptor vector in one tube and one step reaction (Engler et al. 2009).

The MoClo strategy relies on the Golden Gate Cloning except that there is no need for construct-specific cloning strategies because the MoClo uses a defined set of pre-made vectors and a defined assembly strategy (Werner et al. 2012).

The MoClo kit is composed of libraries of sequenced genetic elements such as promoters, 5’ untranslated regions, coding sequences, terminators etc. These elements are flanked by BpiI recognition sites and element-specific fusion sites that allow cloning via BpiI into a pUC19 backbone called level 0 acceptor plasmid. This one contains a spectinomycin resistance gene and BpiI restriction sites flanking a lacZ cassette which is removed during the cloning process, allowing blue/white selection after transformation in E. Coli (Weber et al. 2011). There are called level 0 modules (Figure 3).

As all level 0 plasmids contain BsaI restriction sites, the different elements can be removed from the level 0 vectors and assembled to form a transcription unit into a level 1 destination vector via BsaI (Figure 3). The element-specific fusion sites located at the overhangs allow the different parts to be assembled in the correct order in one step. Contrary to the level 0 modules, the level 1 destination vectors contain an ampicillin resistance gene allowing counter selection against level 0 module backbones (Weber et al. 2011).


A MoClo toolkit for plants and Chlamydomonas reinhardtii

The limiting step of the MoClo technology is a process called “domestication”. Indeed, during the designing step you have to remove the type IIS recognition sites from all starting elements (promoters, 5’UTR etc.). To allow exchange of DNA parts between independent laboratories and make the assembly faster and easier, a Golden Gate Modular Cloning Toolbox for plants, containing domesticated standard parts such as plant promoters, untranslated region, tags etc. has been developed in 2014 (Engler et al. 2014).

In the same way, a Chlamydomonas MoClo toolkit has been reported (Crozet et al. 2018). We used this toolkit for our design iterations.

Proof of concept

To demonstrate the validity of our metabolic engineering strategy, we will analyse and compare the fatty acid profile of triglycerides in our wild type and modified C. reinhardtii strains using gas chromatography. We hope to see an increase in the percentage of palmitic or oleic acid in triglycerides of our modified strain, with also an overall increase in triglyceride concentration compared to a wild type strain.

HiBiT for MoClo

In parallel of our main project, we will implement the HiBiT technology in the MoClo kit by standardizing and integrating it into the C. reinhardtii MoClo kit. This 11 amino acid tag developed by Promega will allow a quick and effective measurement of protein expression. The HiBiT tag is essentially one part of the NanoBiT® enzyme. When the HiBiT-tagged protein is incubated with the LgBiT, which is the other part of the NanoBiT, and its substrate, a functional NanoBiT will be assembled and emit a measurable and quantifiable luminescence signal (Figure 4).



We want to develop the HiBiT tag in order to measure the expression of our enzymes. This tag will greatly facilitate the detection of protein expression. There are currently no antibodies for our enzymes, and detecting them by immunoblotting using a tag (e.g. 6His tag) is quite a long experiment as it requires performing a western blot. Tagging with fluorescent tags such as GFP could be a solution but their large size could potentially affect the activity of our enzymes. Therefore this tag will allow for an easy detection without hindering our enzyme’s function as much as other bulkier tags.



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