Team:NOVA LxPortugal/Implementation

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Next Steps

Due to the short duration of the iGEM competition and to the COVID-19 pandemic, it was impossible to perform all the desired experiments and analysis required for the full implementation of our idea. During the course of the project, only half of the initially planned experiments were performed due to the limitations on the access to the lab. Particularly, the tests in greenhouses, which would present a more realistic perspective of the efficiency and safety of the utilization of spectinabilin in pine trees, would take several months to be implemented. Nevertheless, we envision to continue working on our project in the future, so we have planned short; middle and late-term objectives to guarantee the successful implementation of our project in the real world.
In the short run, we would like to test some modifications in the protocols already implemented (see here) and to perform a new set of experiments to increase the probability of our project’s success:

  • Dry Lab: perform simulations and optimizations of the spectinabilin-producing Pseudomonas putida model using as the growth media the compositions of xylem and the transport tissue of vascular plants. During our research, we could not find the quantitative constitution of the Pinus pinaster xylem. So, a quantitative and qualitative metabolic analysis of the pine xylem is required to characterize the environmental conditions and to better simulate the real-life conditions.

  • Wet Lab: perform the other two experiments initially planned: the production of spectinabilin in a heterologous expression system (Figure 1) and the subsequent integration of the desired genes in the Pseudomonas putida chromosome (Figure 2). Lastly, we would like to perform the knockouts obtained through optimizations of the in silico analysis to maximize the production of spectinabilin.

Design of the heterologous expression system
Figure 1 - Design of the heterologous expression system. The genes of both spectinabilin and (S)-methylmalonyl-CoA production pathways will be expressed in three different plasmids. The quantification of the produced spectinabilin will be performed by Liquid Chromatography-Mass Spectrometry (LC-MS) after induction with alpha-pinene.

Molecular biology strategy for the integration of the heterologous genes
Figure 2 - Molecular biology strategy for the integration of the heterologous genes required for spectinabilin production in the Pseudomonas putida chromosome. The lambda Red recombinase system will be used to insert the pathway genes through homologous recombination. The genes will be introduced by successive recombinations of small fragments due to the large amount of DNA parts required. The inserted sequences contain antibiotic resistance genes, such as clm (chloramphenicol) and spc (spectinomycin) to select the positive transformants, and are going to be transformed together with a plasmid that expresses the recombinase and CRISPR associated protein 9 (Cas9). The final sequence will not have any antibiotic resistance genes to be implemented in trees complying with the European Union directives.

The next step would be testing our engineered strain in an environment that resembles more closely the pine tree ecosystem. To guarantee the non-toxicity of spectinabilin when released into the environment and the actual efficiency of the production of the compound by Pseudomonas putida, our project would be tested in Pinus pinaster trees in controlled systems like greenhouses.

Lastly, if the greenhouse tests prove to be successful, which would mean that spectinabilin is non-toxic to pine trees and its production capable of reducing the nematode population, we will try to implement it in the Pinus pinaster natural environment. To achieve this, we designed three different strategies:

  1. The inoculation of the modified Pseudomonas putida directly in Pinus pinaster in the Portuguese forests;

  2. The release of the insect vector M. galloprovincialis previously inoculated with the modified Pseudomonas putida;

  3. The reforestation of Portuguese forests with young pine trees inoculated with the modified bacteria.

The third strategy is currently the most promising one since it can be implemented together with reforestation programmes. Usually, these programmes take place after the summer fires that ravage Portuguese forests every year, this way we can prevent the spread of this disease in young pine trees. It is also important to note that we intend to perform an economic analysis of the impact and costs of our project in order to guarantee its financial viability against the existing strategies.

We also envision a more global solution to treat infected pines in other countries. After validating our solution in the Portuguese territory, we will evaluate if our engineered bacteria is compatible with the pine species more prevalent in the most affected regions by PWD. Otherwise, we will modify our strategy accordingly, in particular, the strain and the inductor can be altered to develop a more fitted solution.

Economic Analysis and Future Plans

How much would the producers be willing to pay for our product?

Pine industry in Portugal represents 35% of all forest exports with a business volume of over 4 million Euros and over 55.000 jobs.

The impact of PWD is difficult to estimate, as it involves all direct costs in trying to control the disease, the decrease in the value of the forest materials, sanitary restrictions in exports and sales and overall disinvestment in the sector by the private investors, which, coupled with the problem with fires in Portugal, justifies a constant decrease in the overall maritime pine area, with 27% decrease in the number of ha from 1995 to 2015.

The sole direct costs involving measures such as felling and traps have been estimated in over 4 Million Euros/year (numbers provided by one of our partners).

Thus, the estimation of the value the customers would be willing to pay for our product is difficult to assess, since they involve not only the cost of losing the infected trees but mainly the potential costs of the spread of the disease, which would affect the producer and the overall chain. It is thus reasonable to expect that the costs of our product, once in the market, would be shared by the affected producer and the government through subsidies. In fact, the same already happens for most of the pests and diseases in other cultures such as chestnut trees.

In terms of what the producer would be willing to pay, and using one tree as the unit, we estimate a value similar to the value of one tree, which is around 4-10 Euros for an adult pine tree (average from the market values, considering 1 tree with around 0.2-0.3 m3/tree. This value is in the same order of magnitude of the cost of the product PURSUE by Syngenta, which would be around 15 Euros/tree.

It should be emphasized, as mentioned before, that although our unit is 1 tree, the treatment of 1 tree will impact a much wider area, by preventing the spread of the disease.

If we consider the subsidies provided for other diseases, that range from 0,01 Euros/tree (processionary in maritime pine trees) to 30 Euros/tree (cancer in chestnut trees) and considering the economic impact of PWD, we expect the incentives to be significantly high, more in the range of the later values.

Therefore, we can estimate a value of 4-15 Euros for treating 1 tree as the value a producer is willing to pay.

What are the ideal doses of spectinabilin for curing 1 tree?

According to the article by Liu et al1, an effective dose of spectinabilin is of 2-4 mg/tree. Assuming a production of 100 mg/L of spectinabilin in a biomass concentration of 5 g/L, this would imply 20 mg of spectinabilin/g of biomass. In this assumption, we are only using normal conservative values of production of heterologous compounds in our PI’s Lab (100 mg/L) and normal biomass concentrations for E. coli or P. putida (5 g/L).

This would mean we would have to inject a maximum of 0,2 g of biomass in each tree.

How much does it cost to produce spectinabilin for 1 tree?

Since biomass production costs can be estimated in 50-60% raw materials and the remaining the operating costs (Ritala et al)2, and calculating the costs of the cultivation medium for bacteria in less than 1 Euros/kg (accounting that sugar costs are lower than 0,4 Euros/kg and considering the industrial costs in the market for all other M9 medium components), and assuming a yield of 0.3 Kg of biomass/kg of sugar (also a normal value in our PI’s Lab), we obtain that the production cost for 1 Kg of Pseudomonas Biomass is less than 0.3 Euros.

Thus, the production of 1 dose for 1 tree would cost less than 0,0001 Euros, already considering all the operating costs.

These costs are normal for a phytopharmaceutical product, implying that the production costs are not significant when compared with the other costs.

What other costs should we consider?

Thus, we should be mainly concerned with the investment costs, that will be mainly:

-The R&D costs and the costs of approval in the Regulatory agencies such as FDA.

-The investment costs for the equipment for producing the bacteria (fermentation equipment and downstream equipment)

Assuming that, instead of acquiring the fermentation and downstream equipment we could rent it, these costs would be added to the production costs and most probably, would still not make them significant.

Regarding the R&D and regulatory investment costs, and after talking with people in the market, we estimate this to be several Million Euros.


Therefore, we can conclude:
-The operating costs of producing the spectinabilin are very low, allowing to attribute a price to the product that is only dependent on the initial investment and market analysis;
-We expect a maximum price tag (maximum the producers would be paying) for the doses for treating 1 tree to be 4-15 Euros, considering the direct costs with the disease and the economic costs of its spread. We also expect that part of this value would be paid by governmental subsidies;
-Considering that at least 100 000 trees need treatment per year in Portugal (number provided by one of our partners), we could expect to sell 1,5 Million Euros maximum in Portugal. If we expand to the rest of the world, we could expect to have maximum revenues of several tens to few hundreds of Million Euros;
-This would clearly be attractive to our initial investors that would have to invest several millions for R&D and regulatory processes in a 5-10 years time window before the product is approved for the market;
-The exact cost of R&D and regulatory processes still needs to be estimated.

Our Next steps would then be:
-Perform the experiments mentioned above;
-Perform a detailed market and business model analysis, estimating the investment needs and time;
-Launch a startup to perform the remaining R&D and submit the regulatory dossier


  1. Liu, M. et al. Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus. Pest Manag. Sci. 75, 1585–1593 (2019)
  2. Ritala A, Häkkinen ST, Toivari M, Wiebe MG. Single Cell Protein-State-of-the-Art, Industrial Landscape and Patents 2001-2016. Front Microbiol. 2017;8:2009. Published 2017 Oct 13. doi:10.3389/fmicb.2017.02009