DESIGN

Computational design

Synthetic Biology Design

Pseudomonas putida

Alpha-pinene

Spectinabilin

Dry Lab Design

Experimental design

References

Computational design

This project is supported by an in silico analysis to validate and guide the synthetic biology design. The details of the project modeling can be found here.

Synthetic Biology Design

We aim at designing a genetically modified Pseudomonas putida strain to produce a strong nematicidal compound, spectinabilin, when induced by alpha-pinene, a monoterpene produced by the tree as a stress response. To do this, we will construct three plasmids containing the necessary genes to produce spectinabilin under the control of an alpha-pinene inducible promoter. Then, these DNA constructs will be introduced in a Pseudomonas putida strain, leading to the production of the nematicidal compound as a response to a higher concentration of alpha-pinene.

Figure 1- Schematic representation of the synthetic biology design of our project. A strain of Pseudomonas putida will be engineered to produce spectinabilin induced by alpha-pinene.

Pseudomonas putida

Pseudomonas putida is one of the most abundant and frequently detected bacteria in association with Bursaphelenchus xylophilus (nematode). Several authors have proposed that this bacterium establishes a mutualistic symbiotic relationship with the nematodes relevant in the PWD mechanism. The P. putida strains help the nematode in the degradation of xenobiotic compounds found inside the tree host such as alpha-pinene, contributing to the tree weakening¹. These bacteria were selected due to their ability to degrade alpha-pinene, a stress response hormone². Taking advantage of this mechanism, our aim is to genetically engineer the bacteria to trigger the production of spectanibilin in the alpha-pinene.

Alpha-pinene

α-pinene is a monoterpene present in the pine resin that tends to be overproduced when the trees are infected with Bursaphelenchus xylophilus³. The chosen microorganism for the project, Pseudomonas putida, is capable of degrading this molecule². We intend to use alpha-pinene to induce the expression of the genes constituting the spectinabilin production pathway. So, when the tree is infected by the nematode, the increment in the alpha-pinene levels activates the expression of the heterologous pathway genes, leading to the formation of spectinabilin.

Figure 2 - Alpha-pinene's chemical structure.(® Marvin JS ChemAxon)

Spectinabilin

Spectinabilin is a chemical compound produced by Streptomyces spectabilis⁴. This compound has shown high activity against PDN with an LC₅₀ of 0.84 μg.mL^-1 ⁴. Although the detailed mode of action of spectinabilin remains to be determined, this compound is capable of inhibiting hatching rate and of decreasing the population numbers by the disorder of the nematode’s tissue and vacuole formation⁴. Moreover, under greenhouse controlled and field conditions, spectinabilin has shown to effectively control the disease in Pinus densiflora⁴.

Figure 3 - Spectinabilin's chemical structure.(® Marvin JS ChemAxon)

Dry Lab Design

The in silico analysis of the project will be performed using Optflux, a metabolic engineering software created in Portugal.

The spectinabilin biosynthesis pathway will be added to the Pseudomonas putida KT2440 genome-scale metabolic model iJN1462 to first validate the possibility of using this bacterium to heterologously produce spectinabilin. Then, we will conduct several metabolic simulations and optimizations to find an optimum genotype to maximize the production of spectinabilin, without compromising the biomass.

Our goal is to support our experimental work by the results obtained from the computational analysis so we can manipulate the metabolism of our engineered bacteria to maximize the production flux of spectinabilin.

Experimental design

In our project, we plan to validate our experimental design by performing the following activities:

Validation of the alpha-pinene inducible promoters. Since Pseudomonas putida strains have the ability to degrade alpha-pinene, we intend to test some gene promoters from this pathway to regulate the production of spectinabilin as a response to the presence of this metabolite. In order to validate the selected promoter(s), we will build a plasmid where the Green fluorescence protein (GFP) has its expression regulated under the control of the alpha-pinene regulatory zone. This vector will be transformed into a P. putida strain to test the expression of GFP as a result of the addition of alpha-pinene to the culture. This is a rapid experience since the expression of GFP emits fluorescence that can be easily accessed under a UV light or measured by a fluorimeter. If successful, this experiment will be repeated using a co-culture of the engineered Pseudomonas strain and nematode to observe the association between both organisms. In Figure 4, it is depicted a schematic representation of the planned experiment.

Figure 4- Schematic representation of the experiment for validating the alpha-pinene inducible promoters. A plasmid containing the Green fluorescence protein (GFP) under the control of the selected promoters r will be inserted into a Pseudomonas putida strain, alpha-pinene will be supplemented to the culture and the fluorescence emission under UV light will be assessed.

Construction of the spectinabilin biosynthetic pathway. In order to produce spectinabilin, three plasmids will be constructed containing the necessary genes, from both spectinabilin⁵ and (S)-methylmalonyl-CoA⁶ production pathways, under the control of an alpha-pinene inducible promoter, determined by the previous experience. The (S)-methylmalonyl-CoA pathway allows the connection of the spectinabilin pathway with the P. putida metabolism via the Krebs cycle. Then these plasmids will be transformed into a Pseudomonas putida strain, creating a heterologous expression system for spectinabilin production. The quantification of spectinabilin will be performed by Liquid Chromatography-Mass Spectrometry (LC-MS).

Figure 5- Spectinabilin and (s)-methylmalonyl-CoA formation pathways

Figure 6- 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.

Integration of the spectinabilin biosynthetic pathway into the genome of Pseudomonas putida. The genes constituting the spectinabilin production pathway will be inserted into the Pseudomonas putida genome to avoid the possible loss of the inserted genetic material. The genes constituting the pathway will be inserted through homologous recombination using the lambda Red recombinase system . Due to the large amount of DNA parts required, the genetic material will be inserted by successive recombination of small fragments. The initial inserts will also include genes conferring antibiotic resistanceto select the positive transformants. In the final step, a plasmid expressing the recombinase and CRISPR associated protein 9 (Cas9) will be also transformed. The last sequence will have no antibiotic resistance and will be transformed together with a plasmid that expresses a single guide RNA designed for a specific zone of the previously inserted sequence that is not present in it. It is expected that bacteria that did not undergo this last recombination will see this zone cut off, with only bacteria with the complete sequence growing.

Figure 7- 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.

Spectinabilin toxicity assay. Since there is not much information about spectinabilin’s toxicity and mechanism of action when introduced in the trees, we intend to perform a toxicity assay with Arabidopsis thaliana to validate the safety of this compound to plants. This plant was chosen due to the fact that it is easy to germinate and the growth rate is relatively high. The seeds of A. thaliana will be inoculated with successive increasing concentrations of spectinabilin. The goal of this experiment is to observe if there is any change in the phenotype - like growth arrest or the inhibition of germination - when compared with the control experiment (without the addition of spectinabilin). In Figure 8, a schematic representation of this assay is presented.

Figure 8- Arabidopsis thaliana wild-type seeds will be plated on plates containing increasing concentrations of spectinabilinto evaluate the effects of this compound on the plant’s phenotype.

References

Alves, MS, Pereira, A, Vicente, C, Mota, M, Henriques, I. Pseudomonas associated with Bursaphelenchus xylophilus, its insect vector and the host tree: A role in pine wilt disease?For Path. 2019; 49:e12564. https://doi.org/10.1111/efp.12564
Tudroszen NJ, Kelly DP, Millis NF. alpha-Pinene metabolism by Pseudomonas putida. Biochem J. 1977;168(2):315-318. doi:10.1042/bj1680315
Li, Y. et al. Comparative Transcriptome Analysis of the Pinewood Nematode Bursaphelenchus xylophilus Reveals the Molecular Mechanism Underlying Its Defense Response to Host-Derived α-pinene. Int. J. Mol. Sci. 20, 911 (2019)
Liu MJ, Hwang BS, Jin CZ, Li WJ, Park DJ, Seo ST, Kim CJ. Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus. Pest Manag Sci. 2019 Jun;75(6):1585-1593. doi: 10.1002/ps.5272.
Choi YS, Johannes TW, Simurdiak M, Shao Z, Lu H, Zhao H. Cloning and heterologous expression of the spectinabilin biosynthetic gene cluster from Streptomyces spectabilis. Mol Biosyst. doi:10.1039/b923177c
Gross F, Ring MW, Perlova O, et al. Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation. Chem Biol. 2006;13(12):1253-1264. doi:10.1016/j.chembiol.2006.09.014