Presented by Team Aix-Marseille
Samuel CARIEN1, Camille HERROU1, Lucy JACQUES-VUARAMBON1, Laurie JAYET1, Younes MESKINI1, Maud MIOT1, Jeanne PREEL1, Boukari DIALLO2, Joy GIRAUD2, Lucie RAGO2, Nadjiba MARES2, Julien GIRAUD2, Camille CLAMAGIRAND2, Marion JOSSE2, Saïd IBRAHIM2, Elisa DI LELIO2, Clara HUSSER2, Nabil AIT TABASSIR2, Jennifer ARRINDELL3, Megan CHARLES-ALFRED3, Sebastien NIN3, Marie GRANDJEAN3, Jonas DESJARDINS3, Yorgo EL MOUBAYED3, Valérie PRIMA3, Hakim MEDJOUEL3, Julie VIALA4, Laetitia HOUOT4 and James STURGIS5
1iGEM Student Team Member, 2Former iGEM Student Team Member, 3iGEM Team Advisor, 4iGEM Team PI, 5iGEM Team Primary PI.
All our PI work at the LISM, a joint research unit of the CNRS (UMR7255). Our PI are also teachers at the Aix-Marseille University.
Abstract
Since the 70's, the green algae have been proliferating and forming what are called ¨the green tides¨. They are found on certain beaches in Europe, but also in China and the United States. This is mainly due to its high level of nitrogen fertilizers that are used in intensive agriculture. The green algae on the beaches decompose themselves and produce hydrogen sulfide, which is a deadly gas for animals and humans. To avoid the toxic gas emanation in the air, there is one very promising way: Bioethanol! This can be achieved by producing bioethanol from the green algae, that can be blended with the gasoline and be used by adapted vehicles.
The objective of our project is to define an efficient process to transform ulvan into bioethanol. Ulvan is a sugar that is in a polymeric form, that is found in large quantities on the wall of Ulva (which are green algae). By using the enzymes from Formosa agariphila bacterium in Saccharomyces cerevisiae we will be able to produce rhamnose and other fermentable sugars from ulvan. Thanks to the fermentation capacity of S.cerevisiae and Pichia stipitis we will finally be able to transform the rhamnose into bioethanol.
Before beginning our project on S.cerevisiae, it is crucial to verify the enzymatic activity of our proteins and to prove that our engineering is workable.This is why we decided to break down the project into two successive engineering phases. In the first phase, presented to the iGEM 2020 competition, we designed a proof-of concept system where an engineered Escherichia coli W3110 strain would secrete the enzymes, allowing rhamnose production from ulvan directly into the culture medium.
Team:Aix-Marseille/Poster
Poster: Aix-Marseille
Green tides are a big issue for the affected areas. They cause ecological, financial, touristic, and sanitary problems. We want to provide a solution to solve this phenomenon. During our research, we understood that we will not be able to decrease algae proliferation directly. So, we decided to act by developing their revalorization into bioethanol.
Our goal is to turn the economical, ecological and sanitary burden that are greentides into an eco-friendly source of income for the regions impacted by this phenomenon.
A proof of concept: We want to test the capability of enzymes from F.agariphila to degrade ulvan into rhamnose. This strain is naturally able to degrade ulvan polysaccharides with the enzymes inside its PUL genome. For that we have thought to create an E.coli ∆rhaB/rhaT strain transformed with 4 selected genes that are supposed to degrade specifically ulvan into rhamnose.
Validation and characterisation: The protein activity and the concentration of product degradation will be analyzed and will allow us to verify that the enzymatic pathway designed is functional. The rhamnose concentration will be measured thanks to our biosensor strain design using the Paris Bettencourt 2012 model.
Adaptation to industrial production: Then, our objective will be to transpose our system of ulvan degradation into yeasts for an industrial production. S.cerevisiae would be transformed with the four enzymes to transform ulvan into rhamnose. And then, rhamnose fermentation will be done by a consortium of S.cerevisiae and P.stipitis to produce bioethanol.
Our project this year carried out proof of concept of productions and activities of enzymes after processing in E.coli. We choose 4 enzymes of interest: two ulvan lyases, a glycoside hydrolase and a sulphatase. Those enzymes come from the PUL of the bacterium F.agariphila.
Unfortunately, we could not carry out experiments in the laboratory due to Covid-19 and we were therefore only able to design our construction that we would like to use.
The four enzymes we selected for the ulvan degradation are as follows:
P30_PL28: Endo-acting ulvan lyase: This gene codes for an enzyme which catalyzes rapid dissolution of insoluble ulvan: this enzyme cleaves the glycosidic linkage between Rha3S and GlcA or IduA, producing oligosaccharides with unsaturated uronic acid at the non-reducing end.
P10_PLnc: Endo-acting ulvan lyase: the enzyme coded by this gene dissolves soluble ulvan oligomers at the cell surface. This enzyme cleaves also the glycosidic linkage between Rha3S and GlcA or IduA, producing oligosaccharides with unsaturated uronic acid at the non-reducing end.
P33_GH105: Unsaturated 3S-rhamnoglycuronyl hydrolase: The enzyme coded by this gene is periplasmic and works together with ulvan lyases to fully degrade the ulvan polymer. It catalyzes specifically the cleavage of the unsaturated uronic acid at the non-reduced end of the oligosaccharides generated by the ulvan lyase.
P36_ S1_25: Bifunctional sulfatase/alpha-L-rhamnosidase: The enzyme coded by this gene is periplasmic and is a bifunctional enzyme containing sulfatase and alpha-L-rhamnosidase activities involved in ulvan degradation. The sulfatase is the part of this protein that interests us.
The hypothesis behind this selection is that these four enzymes are sufficient to degrade ulvan into rhamnose.
Diagram representing the enzymatic activity of our proteins of interest on ulvans (sulphated polysaccharides composing the ulves). The first step is performed by the two ulvans lyases (P30_PL28 and P10_PLnc) which cleaves the glycosidic linkage between Rha3S and GlcA or IduA. The second step performed by the glycosyl hydrolase is the cleavage of the unsaturated uronic acid at the non-reduced end of the oligosaccharides. The last step is the desulfatation of L-rhamnose at the 3-position. So, finally thanks to our enzymes we can obtain monomers of rhamnose.
Firstly, we want to test the production and activity of our 4 enzymes of interest in E.coli.
Each of the 4 genes of interest must be integrated one by one into the E.coli strain in order to see if each of the proteins is well produced and functional. To verify this, a tag for purification (by Western-Blot) has been added as well as a secretion domain, based on the iGEM 2018 Pasteur Paris team's project. This secretion domain is composed of alpha-haemolysin signal sequence (hylA) and TEV cleavage sequence to secrete enzymes out of the cells.
Figure illustrating the construction of the 4 plasmids for further analysis of each gene. Only the part before the dotted line would be changed in each plasmid. A different tag is added at each gene's ends. A His Tag, a HA Tag, a Flag Tag and a Strep Tag in the C-terminal side of P30_PL28, P10_PLnc, P36_GH105, P33_S1_25 respectively.
After, we wish to quantify protein concentration thanks to HPLC-MS and test the enzyme activity on ulvan polysaccharides.
Then, the 4 biobricks tested individually are transformed into E.coli using two plasmids because of the length of the sulfatase gene which needs to be alone on a plasmid. The production of the 4 enzymes and their activities would be tested as previously done for individual enzymes.
Figure illustrating our proof of concept. We thought about inserting our four enzymes into two plasmids (P36_S1_25 is too big to be inserted with the three others). Constructed plasmids will be inserted into an E.coli W3110 ∆rhaT/rhaB strain.
Thus, the degradation of ulvans by our chassis possessing the 4 genes of interest can be tested by our biosensor. Indeed, we have designed a rhamnose sensitive biosensor inspired by the iGEM 2012 Paris Bettencourt team. L-rhamnose metabolism relies on the following genes:
the rhaT gene, that encodes a symporter proton-rhamnose which imports rhamnose into the cell.
the rhaBAD operon, which encodes for proteins that metabolize L-rhamnose.
the rhaST operon, which encodes for 2 transcriptional activators involved in the regulation of rhaBAD expression.
This biosensor is composed of a PrhaBAD promoter inducible by the L-rhamnose, a gfp and rhaS gene and finally an antibiotic resistance gene. So, we will have a green fluorescence dose-dependant of the amount of rhamnose produced. The fluorescence emission will be measured using a fluorescence microplate titer.
Figure illustrating the mechanism of biosensor strain. The RhaS sensor, when complexed with rhamnose, induces GFP emissions. The ampR gene gives the ampicillin resistance. Thus, the GFP emission is proportional to the rhamnose concentration.
We found 3 articles bringing new knowledge about this biobrick. So, to contribute to iGEM, we added recent literature information into the biobrick page to improve the characterization of the BBa_K914003 biobrick. We found that:
Adding a constitutive rhaS gene will give a stronger signal to the rhamnose biosensor1. In addition, using a plasmid with a constitutive rhaS which enables the expression of this biobrick in unusual bacteria, such as cyanobacteria2.
It can be interesting to use this biobrick in a rhaB mutant to have an independent response of the metabolism of rhamnose, since E.coli metabolizes the L-rhamnose and so decreases its quantity with time3. In addition, we can have a on/off response in a rhaB strain mutant.
Using a rhaT/rhaB mutant bacteria allows to have a real dose-dependent induction of the promoter, which is independent of the rhamnose metabolism3.
L-mannose is a non-metabolizable analogue able to bind RhaS to induce efficiently the PrhaBAD promoter without being metabolized by E.coli1. It avoids having a transient response of the system but has the same advantages that the L-rhamnose. It is the same mechanism if we use L-lyxose instead of L-rhamnose. (see below)
Schema showing the GFP expression in function of the inducer. Left: the emission in presence of rhamnose, showing a transient emission. Right: the GFP emission in presence of L-mannose and L-lyxose and overexpression of RhaS, showing a sustained emission.
Kelly et al, 2016 Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the PrhaBAD Promoter
Kelly et al, 2018 A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria
Hjelm, 2017 Tailoring Escherichia coli for the L‑Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins
As we did not have access to the laboratory this year, we are counting on next year to put our project into practice.
Using our E.coli chassis, we will validate our minimal ulvan degradation pathway to produce rhamnose. Next, because E.coli is not optimum for large-scale fermentation, we plan to switch the design to a yeast chassis: S.cerevisiae. This will allow us to reach the next step in ulvan valorization, by using the rhamnose-fermentation capacity of the yeast to produce ethanol.
To do so, we are planning on establishing a consortium between S.cerevisiae and P.stipitis, another yeast previously described for its ability to improve fermentation and to produce bioethanol.
Moreover, we will be able to analyze the concentration of rhamnose at each step of the process thanks to our biosensor designed in 2020.
Our industrial set-up involves the use of GMO to convert ulvans into rhamnose and bioethanol. In order to prevent any environmental risk, we will also include a genetic containment circuit into our final chassis.
Finally, one of the challenges for next year will be to transpose our project into the business world: we will conduct a market study, document economical aspects of the project, but also other technical aspects such as the best way of harvesting the algae present on the beaches, and possibly the culture of algae to assure continuous access to raw material even during periods when there is no green algae on the shores.
First of all, our Advisors and Instructors, for their help through the competition and their support.
- Sylvain BALLU (CEVA)
- Pierre RENAN (CEVA)
- Amr CHAMAA (LIGER)
- Philippe POTIN (CNRS)
- Ann Sophie BURLOT (Olmix)
- Erwann LORET (CNRS)
- Nicolas TERRAPON (AFMB)
- Frederic BESSON (CEA)
- Agassi WONG
- Sebastien NIN
Visual art and logos:
- Charlotte HOCQUAUX
- Justine FAUSTINO
We had a great time participating to the art project of Nantes’ team with help from:
- Charlotte HOCQUAUX
- Florie LE HELLEY
- Emile TORNIOR
- François ISNARD
Promotion video:
- Litchi video
- François Reimbold
- Bill Dewes
