Expression cassette for CTX production in microbial cell factories, fused to GFP as a carrier protein.
During our research to mitigate the economical and social impact diseases like Greening, Green Mold and Sour rot have in Brazilian citriculture, we identified antimicrobial peptides (AMPs) as a promising solution. Through a literature review, we discovered CTX, a 21 amino acid cationic antimicrobial peptide secreted by the skin of anarboreal South American frog, Hypsiboas albopunctatus, with biological activity against both bacteria and fungi[1]. It has homology with the ceratotoxin peptide family, which permeabilizes membranes and forms pores via a 'barrel-stave' mechanism. Considering the potential of this molecule, we pursued two lines of investigation: evaluate CTX potential against the pathogens responsible for these diseases and scale up CTX production in a cost-effective way for agricultural applications.
CTX (BBa_25ONI4BC) an Antimicrobial peptide against citrus pathogens.
To conduct the experiments listed below we contacted the lead CTX researcher and received a chemically synthesized sample for testing. Further information about the procedures and results can be found in Engineering page.
Penicillium digitatum (Green Mold) and Geotrichum candidum (Sour Rot):
In this assay fungal spores were incubated with three different concentrations of CTX (10, 25, and 50 µM) at 25 °C, visual growth was monitored after 2 and 5 days. As a negative control we used MilliQ water, as a positive control we used Imazalil. Our preliminary results suggested that 50 μM of CTX is required for visible inhibition after 120 hours.
Assay testing CTX at different concentrations against two pathogens, P. digitatum and Geotrichum candidum. Growth inhibition of P. digitatum and G. candidum occurred at 50 µM after 120 hours.
Candidatus Liberibacter asiaticus (Greening): To investigate CTX antimicrobial potential against this non-cultivable pathogen, we incubated infected leaf tissue for 7 days in Falcon tubes containing CTX solutions (100 mM). After incubation, DNA was extracted from the petiole and sent to a specialized diagnostic clinic for qPCR quantification of Clas. As a negative control we used water, as a positive control we used tetracycline at 100 µg/mL, and it was used a baseline sample to compare bacterial loads and evaluate treatment effectiveness. The qPCR quantification results were delayed, preventing conclusions on whether CTX reduced bacterial load compared to controls.
Experimental design for testing the peptide against HLB
CTX Synthesis through Biofactories Using a Composite Part sfGFP-CTX (BBa_25SVFSZB).
The viability of CTX application in agriculture as a real and accessible solution depends on a cost-effective production of this molecule. With that in mind our plan was to develop microbial biofactories to produce CTX at scale. However, considering it has antimicrobial activity we performed a few tests against common expression hosts (Escherichia coli, Saccharomyces cerevisiae, and Aspergillus oryzae) to understand how they could be affected by this molecule.
Our results indicate that CTX at low concentrations inhibits the growth of both bacteria and fungi, while having only a slight effect on yeast. These findings highlight the need for a specialized strategy for its production.
Assay testing CTX against three pathogens, E. coli, S. cerevisiae and A. oryzae. Growth inhibition of E. coli occurred at 40 µM after 48 hours, growth inhibition of A. oryzae occurred at 50 µM after 72 hours, and S. cerevisiae presented resistance at 40 µM after 48 hours, but growth was still slightly affected.
To overcome this challenge, our scientific team developed a strategy in which CTX is initially fused to a highly expressed carrier protein. This fusion allows the peptide to be produced safely, as the carrier masks its antimicrobial activity during expression. After production, the fusion protein is specifically cleaved using a TEV protease site, releasing active CTX in its free form for downstream applications.
Composite part development: We selected sfGFP (BBa_I746916) as the carrier protein due to its well-characterized, high expression levels and its ability to enable easy detection of protein production. A TEV protease cleavage site (BBa_K20750037) was incorporated to allow precise separation of CTX from the carrier protein after expression. Additionally, a His-tag (BBa_K4422007) was added to the CTX sequence to facilitate purification, and a 3×GS linker (BBa_J18921) was included to prevent structural disruption of the carrier protein.
The coupling CTX in sfGFP strategy with 3x GS linker, TEV protease sites and 8x HIS tag.
Table 1 – Estimated CTX production for each strategy
Host
Carrier protein
Estimated production*
Escherichia coli
sfGFP
2 mg/L
Aspergillus oryzae
sfGFP
10 mg/L
We also explored two additional strategies to express CTX: fusing it to amylases in Aspergillus oryzae and anchoring it to the yeast cell wall. These approaches aimed to enhance production and stability of the peptide. However, both designs remained at the proof-of-concept stage and were not submitted as Parts to the iGEM Registry. The amylase-fusion system required more time for genetic construction and functional validation, while the yeast surface display strategy demanded additional optimization of cleavage and release conditions. Since iGEM Parts are expected to be fully characterized and reproducible, we decided to share these strategies in the Results section as preliminary designs rather than registered Parts. This ensures transparency while leaving clear opportunities for future teams to build upon and transform these concepts into standardized Parts.
