Synthetic biology tools for production of insect pheromones in plants and filamentous fungi
- Autores: Elena Moreno Giménez
- Directores de la Tesis: José Francisco Marcos López (dir. tes.), Lynne Yenush (dir. tes.), Diego Vicente Orzáez Calatayud (dir. tes.)
- Lectura: En la Universitat Politècnica de València ( España ) en 2023
- Idioma: inglés
- Tribunal Calificador de la Tesis: Mª Lourdes Gómez Gómez (presid.), Nuria Andrés Colás (secret.), Antonio Di Pietro (voc.)
- Programa de doctorado: Programa de Doctorado en Biotecnología por la Universitat Politècnica de València
- Materias:
- Enlaces
- Tesis en acceso abierto en: RiuNet
- Resumen
The use of living organisms as biofactories have gained significant attention in the industry due to the increasing demand for sustainable production systems and the shortage of resources. Among their many applications, biofactories can be engineered to produce insect pheromones, which serve as eco-friendly alternatives to pesticides for pest management in agriculture. As a proof of concept, in this thesis we characterized Nicotiana benthamiana plants engineered with a multigene pathway to produce the moth pheromones (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc). The resulting transgenic plants produced modest amounts of both pheromones (111.4 µg g-1 FW and 11.8 µg g-1 FW for Z11-16OH and Z11-16OAc, respectively), and daily emission rates of ~10 ng g-1 FW for each pheromone.
Pheromone production in these plants significantly affected their fitness, likely due to the substantial metabolic burden and possible toxicity of lipid-derived products. One strategy to address these developmental abnormalities consists of engineering conditional transgene expression systems, thus allowing plants to grow normally before inducing the production of pheromones. To achieve this goal, in this thesis we developed a set of customizable synthetic promoters called GB_SynP, which can be activated by dCasEV2.1, a strong programable transcriptional activator recently developed for plant gene regulation. These GB_SynP promoters enabled tight regulation of single and multiple transgenes, with robust and tunable transcription levels in the ON state (presence of dCasEV2.1 loaded with the corresponding gRNA), and minimal or undetectable expression in the OFF state.
To implement a conditional expression system for pheromone production in plants, a newly engineered multigene pathway for the biosynthesis of moth pheromones was constructed under the control of GB_SynP promoters. In parallel, the dCasEV2.1 activator was transcriptionally regulated with the CUP2:GAL4 sensor for copper sulphate, an agronomically-compatible chemical trigger. The functionality of this system was tested transiently in N. benthamiana, resulting in estimated yields of 32.7 µg g-1 FW and 25 µg g-1 FW for Z11-16OH and Z11-16OAc respectively in the ON state, and negligible levels in the absence of copper. However, stable transformation of the same copper-regulated pheromone pathway in N. benthamiana plants resulted in significantly lower transgene expression levels, which translated into a great reduction of pheromone yields. This makes the system in its current form a non-viable pheromone biofactory in practical terms. Further optimization should focus on the improvement of the activation cascade, the use of alternative plant hosts with more biomass, and/or the enhancement of emission rates in planta.
As an alternative to pheromone production in plants, the interchangeability of DNA parts between plants and filamentous fungi could also be exploited to create fungal biofactories for pheromone production. In this regard, our research group previously adapted the GoldenBraid system for filamentous fungi, which we named FungalBraid. In this thesis, we expanded the FungalBraid collection by incorporating 27 new DNA parts, including different selection markers and several constitutive and inducible promoters, all of which were functionally characterized in Penicillium digitatum and P. chrysogenum. Furthermore, we successfully expressed the GB_SynP promoters developed for plants in P. digitatum, in combination with the non-integrative pAMA18-derived vector for the expression of a dCas9-based activator. Although further optimization of GB_SynP in filamentous fungi is required, as expression levels were lower than those previously observed in plants, this and the other tools available in the FungalBraid collection can be effectively employed in the future for the development of fungal biofactories that produce insect pheromones and other high value biomolecules.
