Parts

Introduction

For the development of the project, it was necessary to work with basic parts different from those already present in the iGEM part registry. The main parts presented here are coding sequences of the candidate enzymes for our piperamide biosynthetic pathway, as shown in the following figure.

There are 11 required enzymes in the designed pathway. Eight of these enzymes were obtained from the Piper nigrum genome, selected with the differential expression analysis performed to discover the genes overexpressed the most in the mature fruit, the part of the plant with the highest piperamides concentration. The eleventh enzyme was directly obtained from Schnabel, 2021, where it was found to perform the expected reaction.

Enzyme number 3 was obtained from the NCBI database with ID: WP_015103234.1 (Berner, 2006). For enzyme 7, the sequence was obtained from the NCBI database with ID: WP_226326691.1, which codes for a lysine decarboxylase CadA in E. coli. The tenth enzyme was taken from the DpkA enzyme found in Pseudomonas syringae, obtained from Goto, 2005.

Modified Promoter

The last basic part was a designed promoter modified from the BBa_J63006 iGEM part. The promoter is inducible, specifically GAL1, and will be incorporated into both constructs. It will be modified relative to the wild-type version by deleting the binding regions for the MIG1 protein, a repressor in the presence of glucose, while preserving the four binding sites for GAL4, the inducer in the presence of galactose.

This modification enables activation of both constructs' expression through galactose addition during the stationary growth phase, while glucose remains the primary carbon source. The endogenous GAL1 promoter, with the MIG1 regions intact, will still be inhibited by glucose presence, ensuring proper metabolic activity.

Part Registry

The parts mentioned above were registered in the iGEM part registry for the iGEM Mexico team participating in the 2024 iGEM competition. The parts are registered under the codes from BBa_K5479000 to BBa_K5479011. The following table provides a summary of the part registry.

References

• Berner, M., et al. (2006). Genes and enzymes involved in caffeic acid biosynthesis in the actinomycete Saccharothrix espanaensis. Journal of Bacteriology, 188(7), 2666-2673.
• Goto, M., et al. (2005). Crystal structures of Δ1-piperideine-2-carboxylate reductase: Conformational change and stereochemistry of the reaction. Journal of Biological Chemistry, 280(49), 40875-40884.
• Moreno, C. J., et al. (2024). Expanding Synthetic Applications of Δ1‐Piperidine‐2‐carboxylate/Δ1‐pyrroline‐2‐carboxylate Reductase from Pseudomonas syringae (DpkAPsyrin). Biocatalytic Asymmetric Synthesis of (S, E)‐2‐hydroxy‐4‐arylbut‐3‐enoic Acid Derivatives. Advanced Synthesis & Catalysis, 366(4), 990-1000.
• Schnabel, A., et al. (2021). Identification and characterization of piperine synthase from black pepper, Piper nigrum L. Communications Biology, 4(1), 445.