Due to the COVID-19 pandemic, the access to the laboratories and to the necessary material tend to be more restricted. So the planning of our experiments was limited by the regulation implemented at Instituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA) and some of the planned experiments had to be postponed.
Our project started by reviewing the literature to infer which species of bacteria are present in Pinus pinaster microbiota that are capable of metabolizing alpha-pinene1. This monoterpene tends to be overproduced in stress responses2, including the nematode infection of Bursaphelenchus xylophilus, so we designed a synthetic biology strategy dependent on the presence of this compound. Two bacteria were chosen for the analyses: Pseudomonas putida and Serratia marcescens. Both species have the ability to metabolize alpha-pinene1,3, so both possess regulatory mechanisms as a response to the presence of this compound. Nevertheless, the species Pseudomonas putida was selected to further proceed with the project since this microorganism is easier to acquire and work in the laboratory. In this phase of the project, we started to investigate which promoters are associated with the enzymes constituting the alpha-pinene degradation pathway. The main goal was to activate the production of a nematicidal compound as a response mechanism to a high concentration of alpha-pinene. After some research, we selected spectinabilin as our nematicidal compound since (i) it has been proven that it has a significant nematicidal effect4, (ii) the biosynthetic pathway for its production is fully disclosed (here) and (iii) its prokaryotic origin.
The utilization of alpha-pinene as an inductor was debated in the Biotechnology and Forest Scientific Meeting organized and hosted by us with the other scientists and researchers in this area. Alpha-pinene tends to be produced as a stress response in several situations, including infection by the nematode and tends to be present in both Pinus pinaster and Pinus pinea, with the last one not being as affected by Pine Wilt Disease (PWD). Furthermore, Pinus pinea tends to produce a lower quantity of alpha-pinene than Pinus pinaster. So, the utilization of this metabolite as an inductor must be considered if applied to other species. Probably, alternative compounds associated with the response of other species to the nematode infection must be researched to find other possible pathway triggers. Another important topic discussed in our meeting, was the existence of different chemotypes of Pinus pinaster that could lead to variations on the quantity of alpha-pinene produced in the tree. Nevertheless, despite all the different chemotypes and the consequent variations in the amount of alpha-pinene released, it is still observed an increase in its concentration after the infection and continues to be one of the most characteristic compounds associated with the PWD.
After considering all the challenges that have arisen from our debate, we concluded that the project was still feasible for combating the PWD in the Portuguese territory and we proceeded with alpha-pinene as our chosen compound to induce the expression system of the nematicidal compound.
After selecting the inductor, we had to select the respective inducible promoters. After some research in the literature and databases, we have selected two different promoters to regulate the expression of the genes constituting the spectinabilin biosynthetic pathway. We have focused on the promoters involved in the alpha-pinene metabolism in Pseudomonas strains1. Particularly, only a few genes belonging to this pathway were already annotated and well-characterized. We have selected two possible options: one promoter regulating the expression of the alpha-pinene oxide lyase gene from Pseudomonas rhodesiae CIP107491 and one putative promoter from the gene enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (fadB) from P. putida KT2440. The sequence of the first promoter was already annotated as a promoter region in NCBI. The last promoter was not yet annotated, so its potential role as a promoter was inferred by using the online bioinformatic tool BPROM from softberry. This tool allows recognizing potential transcription start positions of bacterial genes and it was suggested by other iGEMmers in the iGEM 2020 Global Slack (Thank you!).
Additionally, for this project we also used a terminator sequence that is suited for P. putida. This terminator with 158 bp, called T1, was already proven in previous research to efficiently terminate transcription in P. putida, Escherichia coli and other microorganisms5.
To validate the selected alpha-pinene inducible promoters, we constructed plasmids where the expression of the green fluorescent protein (GFP) is under the control of the previously chosen promoters. To do this, we designed two composite parts, each one containing three individual parts that were registered in the iGEM’s Part's Registry page. Our individual parts correspond to the enhanced green fluorescent protein (EGFP) (BBa_K3675002), the terminator (BBa_K3675003) and our promoters (BBa_K3675000 and BBa_K3675001). The composite parts were designed with one of the promoters, followed by the EGFP and ending with the terminator as depicted in Figure 2. The first composite part was the one with the promoter regulating the expression of the gene encoding the alpha-pinene oxide lyase (BBa_K3675004) and the second composite contains the promoter from fadB gene (BBa_K3675005). Both parts were cloned into the plasmid pSEVA234, a vector compatible with the selected expression strain Pseudomonas putida KT2440. Two different molecular biology strategies were designed using SnapGene to clone each promoter: one eliminating the original operon lac and trc promoter from the plasmid pSEVA234, and another one cloning the DNA constructs directly in the original multiple cloning site of the plasmid. For the first strategy, the restriction enzymes used were PacI and KpnI, while for the last design, EcoRI and KpnI were the selected enzymes. The resulting plasmid maps are presented in Figure 1.
The constructed vectors were finished, but we were not able to transform them into P. putidaand continue the experiment for validation of the selected promoters. Since our promoters are regulated by alpha-pinene, we assume that by supplementing this compound to the medium, the transcription of GFP would be activated with the consequent emission of fluorescence, easily visible under a UV light or measured by a fluorimeter. The same experiment without supplementing alpha-pinene would work as the negative control. The schematic representation of this experiment is present in Figure 2.
If the results of this experiment validate the use of alpha-pinene to induce gene expression with the chosen promoter(s), several experiments can follow. First, it is important to understand if the DNA construct(s) can be improved by measuring the emitted UV light or by running SDS-PAGE to evaluate the GFP levels of expression. Different plasmids and combinations of DNA parts can also be tested to improve the GFP expression levels. The minimal concentration of alpha-pinene to induce gene expression must be quantified to validate if it is sufficient to induce the gene expression in pine trees. We also intend to proceed with our main goal by expressing the spectinabilin biosynthetic pathway under the control of the validated promoter(s). By doing so, the spectinabilin production is only activated in the presence of alpha-pinene after the tree is infected by the nematode. Finally, we intend to co-culture our engineered Pseudomonas putida expressing GFP with nematodes to better understand the association behaviour between the two organisms. By adding alpha-pinene to the medium and consequently activating GFP expression, we would be able to detect the bacteria inside the nematode.
If the promoters fail to induce the gene expression in the presence of alpha-pinene, we have planned different strategies to address this problem. The first option would be to create genetic variability in the sequences of previously selected promoters by using techniques like random or site directed mutagenesis. For instance, we can design degenerate primers for regions that more likely affect the gene expression to create genetic diversity. The best mutants can be rapidly identified by measuring the intensity of the emitted UV light with a 96-well plate in a fluorescence microplate reader. Several rounds of optimizations can be performed until achieving the optimum GFP level of expression. In parallel, we can sequence the best performing promoters to identify the respective punctual mutations and implement a more rational strategy with site directed mutagenesis. In alternative, we can also test other promoters from different strains of Pseudomonas, or from other bacteria like Serratia marcescens, as mentioned earlier. Finally, we can broaden the research spectrum of potential promoters to other bacteria species from the pine microbiota, capable of metabolizing alpha-pinene. If all these strategies fail, we can use other compounds associated with the PWD to activate the gene expression.
- 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. 2019; 20, 911.
- Wright SJ, Caunt P, Carter D, Baker PB. Microbial oxidation of alpha-pinene by Serratia marcescens. Appl Microbiol Biotechnol. 1986; 23:224–227. doi:10.1007/BF00261919.
- 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;75(6):1585-1593. doi: 10.1002/ps.5272.
- Vanesa Amarelle, Ananda Sanches-Medeiros, Rafael Silva-Rocha, and María-Eugenia Guazzaroni. ACS Synthetic Biology. 2019;8 (4), 647-654. doi: 10.1021/acssynbio.8b00507.