Algenuity was part of a four-year EU funded project with 11 partners called TriForC that officially ended last week on 31st October 2017. Mike Yates caught up with Dr. Sarah D’Adamo, Algenuity’s Lead Scientist, to learn more about TriForC and some of the concluding outcomes.
Starting from the beginning, what does TriForC stand for and what was the goal of the project?
TriForC stands for “ Tri terpenes for C ommercialisation”. The goal was to create a pipeline and platform to discover, sustainably produce, and commercialise known and novel high-value triterpenes. Several chassis platforms were explored: microalgae, yeast, and tobacco. Algenuity led the microalgal expression platform work.
What are triterpenes and why are triterpenes important?
Triterpenes are secondary metabolites that are often found in plants, and plants uses them as natural self-defence mechanisms from pests.
Plant triterpenes have been studied as novel bio-pesticides in the agri-tech space or unique anti-inflammatory agents in the pharmaceutical space. If you do a search on the US clinical trials database or a patent search under specific triterpenes, you will find several drugs that are in clinical trials and that use triterpenes as an active pharmaceutical ingredient (API). There is a lot of potential for these natural solutions.
Why would you need a platform like microalgae, yeast, or tobacco to express triterpenes?
One of the challenges is that certain triterpenes are structurally complex and are difficult to create via chemical synthesis.
Triterpenes are often found as complex mixtures in their natural hosts of plants, and the desired triterpene is usually only present in small amounts. This makes the isolation and large-scale extraction very costly.
One would think – why not increase the yields of triterpenes in the natural host of plants and use plants as the platform? The problem is that most plants do not have an established transformation procedure, and it is difficult to scale up and grow plants on a suitable commercial scale. Furthermore, there are sustainability and possible ethical concerns of cutting down trees and plants to source a chemical.An industrial biotech platform based on microalgae would provide a consistent, continuous (not limited to harvests), sustainable, and homogenous supply.
Was the team successful in producing triterpenes in microalgae? And if so, what microalgal strain?
Yes! We successfully expressed selected triterpenes in the diatom and brown microalga, Phaeodactylum tricornutum . However, the triterpene yields were still low and more work needs to be done to optimise the pathway in order to go towards industrial scale. We did, however, accomplish our deliverable targets of triterpene expression in microalgae.
Oh brilliant. Did you try any other microalgal species?
Yes, we tried Chlamydomonas reinhardtii and Nannochloropsis oceanica , but we were unsuccessful with those strains. We stopped work on Chlamydomonas early on. Nanno and Phaeo were the two main work horse strains for the project.
What do you think was special about Phaeodactylum tricornutum ?
There could be a lot of reasons. One possibility is that the basic biosynthetic pathway for a triterpene (generally cytosolic) relies on a isopentenyl diphosphate (IPP) precursor. In higher plants, IPP production involves both the cytosolic mevalonate (MVA) and the chloroplastic non- mevalonate (MEP) pathway.
As a higher plant, Phaeodactylum tricornutum has the machinery for both pathways. Other microalgal species (like Chlamydomonas, Nannochloropsis ), instead, have only one pathway (MEP), and this pathway is often localised in the chloroplast, making the precursor IPP availability possibly different.
Interesting. What are some of the innovations that came out of the project?
There were several innovations that drove forward microalgal synthetic biology – some were minor and foundational and others were larger.
The engineering of a complex plant metabolic pathway into a diatom for the first time and getting enzyme expression and decent yields were big outcomes. We also performed protein engineering and built a synthetic chimeric protein that came from the fusion of two distinct plant enzymes. This fused protein was successfully and functionally expressed in P. tricornutum , and it was a truly valuable accomplishment.
We followed a synthetic biology approach of grounding all our work in modular parts. We chose a Golden Gate approach, and we created a hierarchical design structure where modular parts systematically built on each other. The standardised parts also allowed us to iterate through different combinations of promoters and enzymes rapidly.
We also developed some unique promoter parts where expression could be switched on and off via low cost inputs. Inducible promoters are valuable in engineering a controllable metabolic pathway.
Lastly, we developed a parallel yeast platform where we could rapidly iterate through enzyme candidates and then shuttle successful hits into Phaeodactylum . This made us go so much faster through the Design-Build-Grow-Test-Learn synbio cycle.
Wow, that’s amazing. I love the speed and efficiency and systematic design of the work.
Yeah, going fast in solving problems is something we pride ourselves here at Algenuity. Even with our RNA-extractions and qPCR work, we let the liquid-handling robot perform those functions. Modular parts and controllable parts are so important to making progress.
Did you get to use the Algem labscale photobioreactor in the research?
Yes, the Algem was a big help. A lot of people forget that in the microalgal synthetic biology cycle of Design-Build-Grow-Test-Learn, “Grow” is a vital part of the cycle.
You can have the most sophisticated “Design” and “Build” technology, but if your “Grow” part is archaic and not reproducible and with large error bars, you are letting the whole process down.
The Algem allowed for reproducible growth conditions with tight error bars, and it allowed us to limit the amount of noise and focus on the true modifications from our design and build work. The Algem was especially helpful to measure (via qPCR) the gene expression of a selected set of sterol biosynthetic enzymes from the chloroplast localized MEP pathway and cytosolic MVA pathway. The Algem helped to give us reproducible qPCR data and clear insights into the plant transgene influence on the native sterol biosynthesis expression in the transformant line compared to wild type line.
How did microalgae compare to yeast and tobacco?
When comparing on a “per cell” basis, yeast, tobacco, and microalgae showed very similar triterpene titres. At the moment, yeast provides higher biomass productivity compared to microalgae, has an established heterotrophic fermentation platform, and has more advanced genetic tools available. However, microalgal synthetic biology is quickly progressing, and with improved culturing and bio-refinery techniques, microalgae could prove to be just as competitive in the near future. This is our hope and goal!
What were some of the challenges of the project?
Boosting yields is probably the biggest challenge. Further optimisation with enzymes and metabolic switches could possibly improve product titres.
The biomass productivity of Phaeodactylum and microalgae in general is a challenge. We looked at introducing a glucose-transporter to allow heterotrophic growth. This would likely boost productivity.
Looking back now, what is your favourite part of the project?
The unique nature of the 11-partner EU-funded consortium was very special. It is amazing to interact with leaders in their respective fields (academia and industry) all across Europe and access the deep research heritage of each country. We had partners from the UK, France, Denmark, Spain, Italy, Belgium, Greece, and Israel.
Are there any papers that will come from Algenuity’s contribution?
We submitted a paper that is under review at the moment, and we are currently working on a few others. You should hopefully see some papers in the near future.
One quick non-serious question – what is your favourite microalgae and why?
Phaeo, of course! Engineering P. tricornutum was a satisfactory and straight-forward process. But I also like Tetraselmis . I have worked with some halophilic isolates in the past, and I appreciated their robustness in growth and adaptivity to multiple conditions. Recently, I also fell in love with Haslea ostrearia , a diatom that produces a very peculiar blue pigment called marennine. I have yet to work with this strain but would love to check it out in the near future!
Is there a project website or another resource where we can learn more about TriForC?
The TriForC website is http://triforc.eu/
Another great source is http://www.ingentaconnect.com/content/sil/impact/2017/00002017/00000001/art00013
Any other last comments?
I would like to thank our fantastic lab team of Michiel Matthijs, Henry Taunt, Joanna Szaub-Newton, Gino Schiano di Visconte, Gavin Lowe, Sam Sizer, Denise Pallister, Patrick Hickland, and Andrew Spicer. The EU-consortium partners were brilliant too.
Thanks so much for your time Sarah.
Algenuity is looking into releasing selected synthetic biology tools for Phaeodactylum tricornutum and yeast to the academic community under an "academic research only" license and for a reasonable and competitive fee. If you would like to register or express your interest in these synbio tools, please email email@example.com.