Contribution: Overview

Our 2025 iGEM Korea-HS project aims to develop a bio-upcycling platform that transforms fruit peel waste into valuable products using an engineered probiotic bacterium. Specifically, we are engineering Lactobacillus reuteri to express a codon-optimized polygalacturonase (PGXc) gene derived from Aspergillus niger. This enzyme enables L. reuteri to efficiently degrade pectin, a major polysaccharide found in citrus and other fruit peels.

Our long-term goal is to reduce food waste accumulation and valorize agricultural byproducts by converting fruit peels into useful biomaterials such as bioethanol, bioplastics, or prebiotic oligosaccharides. This project demonstrates a sustainable synthetic biology solution to address global food waste challenges and promote circular bioeconomy principles. This year, we added new documentation to an existing registry and contributed five new parts to the Registry.

1. ermB Promoter (BBa_K3183000)

New documentation added: registry.igem.org/parts/bba-k3183000

Sequence Information

ACCAGGAATGAATTACTATCCCTTTTATCAAGAAGCGCACAAAAAGAAAAACGAAATGATACACCAATCAGTGCAAAAAAAGATATAATGGGAGATAAGACGGTTCGTGTTCGTGCTGACTTGCACCATATCATAAAAATCGAAACAGCAAAGAATGGCGGAAACGTAAAAGAAGTTATGGAAATAAGACTTAGAAGCAAACTTAAGAGTGTGTTGATAGTGCAGTATCTTAAAATTTTGTATAATAGGAATTGAAGTTAAATTAGATGCTAAAAATTTGTAAT

General Information & Literature Review

The ermB promoter originates from the erythromycin resistance gene (ermB), commonly found in Gram-positive bacteria.

  • Constitutive expression: Drives continuous transcription without external inducers.
  • Robust expression in LAB: Widely used in Lactobacillus and related lactic acid bacteria.
  • Food-grade context: Safe to use in non-pathogenic LAB with appropriate biosafety practices.

Why This Promoter?

  • Avoids dependence on costly/unstable inducers (nisin/IPTG).
  • Enables stable enzyme production under variable, low-cost conditions for waste degradation.
  • Supported by prior studies expressing enzymes and therapeutic proteins in LAB.

Application in Our Project

The ermB promoter constitutively drives the signal peptide–modified pgxC gene in L. reuteri, ensuring continuous polygalacturonase production for pectin degradation in fruit peels, independent of environmental inducers.

References

  1. Lizier, M., et al. (2010). FEMS Microbiology Letters.
  2. Teh, B. S., et al. (2016). Frontiers in Microbiology, 7, 928.
  3. Mugwanda, K., et al. (2023). Bioscience Reports, 43(1).

2. N-terminal Secretion Signal Peptide of Usp45 (BBa_25KVZJA0)

New part: registry.igem.org/parts/bba-25kvzja0

Sequence Information

ATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCT

General Information & Literature Review

The Usp45 signal peptide from Lactococcus lactis efficiently directs proteins to the Sec-dependent secretion pathway and is widely used in LAB systems.

Application in Our Project

We fused the Usp45 signal to pgxC to ensure extracellular secretion, enabling direct action on pectin-rich fruit peels.

References

  1. van Asseldonk, M., de Vos, W. M., & Simons, G. (1993). Molecular & General Genetics, 240(3), 428–434. https://doi.org/10.1007/BF00280397
  2. Ng, D. T. W., Goh, J. K., & Ning, W. (2013). Applied and Environmental Microbiology, 79(14), 4316–4325. https://doi.org/10.1128/AEM.02667-12

3. pgxC Coding Sequence (BBa_252AGPHV)

New part: registry.igem.org/parts/bba-252agphv

Sequence Information

ATGTCTGTCTTCAAGGCATCATTCCTATTTCTTCTTTCCTCCTCACTAGTCCACGGGGTTCCACACTCCAGCAGAGCATCTCGGAGCCAACAATGCGTGGTTCCGTCCAAATACCAGGCATCGAATGGGACGGCTGATGACTCGGCTGCTGTCTCCCAGGCCTTTGCACAATGCGCGACTGACTCGGTTATTATTTTCGAGGAGGGTGTCAACTATAACATCTTTCAGCCGATCACCGCCACCAACCTCAGCAATGTGGAAATCCGGATGCACGGCAACCTGCATCTGCCACAGAATATCACTGCGGTGCAGAATATAGTCAGTGACGGTACTTCTACATGGTTTACCCTAGAAGGACCAAAAGTGGACTGGATTGGTCCTGAAGACGTGAACAATGGTTGGATTGACTCGTACGGACAACCGTGGTGGGATGCGAACCCTGCAGGTAGTTCAGGCATCGATAACCGTCCGCATCTCATGAGCTTCAAGTCTAGCCAAGCCACTATGAAATACTTCAGGTCTAGGAAGCCCATCGCCTGGAATGTCAAACTGCATGGACAAGACATTACAGTCAGCCACGCTATTATCGACGCTACCTCGACAGGTAGCTTCCCATTCAACACTGACGGTTTCGATGTTGAGGGTACCAATATCCAGATCACCGACAGTATCATGTACAACGGCGACGATGCGATTGCAGTAGGCGCGGACTCGCACGACATACTTTTCACAAGAAACACCATCGGCTACCAGACTCACGGCATGAGCATTGGATCGCTGGGAAAGGATCCCACAGACTTCGCCAATATCAGCAACATCCGCTTTGACGATGTGACTGTTGTCGATGGGCTCTACGCAGCACGCTTCAAGTCATGGAGCGGAGGGACCGGACTCGTCAAGAATGTGACCTGGAACAATATTAGAGTCTTCAACGTGACGTTCCCGATCTTTGTGACTCAGAGTTATAGCGACCAAGGCGCCTCCCGGTCTGGAACTGTCAATGCTAGTTCGGCTGTGATGATGGAGGATTTTACCTGGTCTGACTTTGCTGGCTCGATCAATACGTACCAGCCTGGTGACGGTTCTTGCGTTTCCGACCCTTGCTGGTACAACGTTGGGCTGCCAAATTTGAAACATACGGAGGCCCTCATTATCGAATGCCATACTGCTCAATCTTGTAAGAACTTTGTGACGGACAACATCCAGCTATACCCGCAGGTTCTGGAACCAACGAGTGTGATCTGCATGAACGCAACGGCAGCCCTCAATCCTGATCTTGGATTTACATGTAAAAACGGGACCTACAGCCCATTATCTAATTAA

General Information & Literature Review

The pgxC gene encodes polygalacturonase, a pectinolytic enzyme that hydrolyzes α-(1,4)-glycosidic linkages in polygalacturonic acid, releasing galacturonic acid monomers. It is found across bacterial and fungal species and is widely used in juice clarification, textiles, paper processing, and food-waste degradation.

Application in Our Project

In our construct, pgxC is controlled by the ermB promoter and fused to a secretion signal to achieve extracellular enzyme release in L. reuteri.

Expected Results

  • Secretion of active polygalacturonase into the medium.
  • Increased reducing sugars (galacturonic acid) by DNSA assay.
  • Faster fruit peel degradation vs. wild type.

References

  1. Jayani, R. S., Saxena, S., & Gupta, R. (2005). Process Biochemistry, 40(9), 2931–2944. https://doi.org/10.1016/j.procbio.2005.03.026
  2. Soriano, M., Diaz, P., & Pastor, F. I. J. (2006). Applied Microbiology and Biotechnology, 73(4), 861–869. https://doi.org/10.1007/s00253-006-0545-3
  3. Klug-Santner, B. G., et al. (2006). Enzyme and Microbial Technology, 39(4), 890–897. https://doi.org/10.1016/j.enzmictec.2006.01.020

4. Engineered N-terminal Secretion Signal Peptide pgxC (BBa_2596ET3M)

New part: registry.igem.org/parts/bba-2596et3m

Sequence Information

ATGAAAAAAAAGATTATCTCAGCTATTTTAATGTCTACAGTGATACTTTCTGCTGCAGCCCCGTTGTCAGGTGTTTACGCTGTTCCACACTCCAGCAGAGCATCTCGGAGCCAACAATGCGTGGTTCCGTCCAAATACCAGGCATCGAATGGGACGGCTGATGACTCGGCTGCTGTCTCCCAGGCCTTTGCACAATGCGCGACTGACTCGGTTATTATTTTCGAGGAGGGTGTCAACTATAACATCTTTCAGCCGATCACCGCCACCAACCTCAGCAATGTGGAAATCCGGATGCACGGCAACCTGCATCTGCCACAGAATATCACTGCGGTGCAGAATATAGTCAGTGACGGTACTTCTACATGGTTTACCCTAGAAGGACCAAAAGTGGACTGGATTGGTCCTGAAGACGTGAACAATGGTTGGATTGACTCGTACGGACAACCGTGGTGGGATGCGAACCCTGCAGGTAGTTCAGGCATCGATAACCGTCCGCATCTCATGAGCTTCAAGTCTAGCCAAGCCACTATGAAATACTTCAGGTCTAGGAAGCCCATCGCCTGGAATGTCAAACTGCATGGACAAGACATTACAGTCAGCCACGCTATTATCGACGCTACCTCGACAGGTAGCTTCCCATTCAACACTGACGGTTTCGATGTTGAGGGTACCAATATCCAGATCACCGACAGTATCATGTACAACGGCGACGATGCGATTGCAGTAGGCGCGGACTCGCACGACATACTTTTCACAAGAAACACCATCGGCTACCAGACTCACGGCATGAGCATTGGATCGCTGGGAAAGGATCCCACAGACTTCGCCAATATCAGCAACATCCGCTTTGACGATGTGACTGTTGTCGATGGGCTCTACGCAGCACGCTTCAAGTCATGGAGCGGAGGGACCGGACTCGTCAAGAATGTGACCTGGAACAATATTAGAGTCTTCAACGTGACGTTCCCGATCTTTGTGACTCAGAGTTATAGCGACCAAGGCGCCTCCCGGTCTGGAACTGTCAATGCTAGTTCGGCTGTGATGATGGAGGATTTTACCTGGTCTGACTTTGCTGGCTCGATCAATACGTACCAGCCTGGTGACGGTTCTTGCGTTTCCGACCCTTGCTGGTACAACGTTGGGCTGCCAAATTTGAAACATACGGAGGCCCTCATTATCGAATGCCATACTGCTCAATCTTGTAAGAACTTTGTGACGGACAACATCCAGCTATACCCGCAGGTTCTGGAACCAACGAGTGTGATCTGCATGAACGCAACGGCAGCCCTCAATCCTGATCTTGGATTTACATGTAAAAACGGGACCTACAGCCCATTATCTAATTAA

General Information & Literature Review

Engineered signal peptides can improve secretion efficiency by tuning positive N-region charge, hydrophobic core, and linkers for folding/stability. We designed a synthetic leader fused to pgxC to maximize extracellular yield in L. reuteri.

Application in Our Project

  • Rationale: Native signals may be enzyme-dependent; engineering can enhance translocation and activity.
  • Advantages: Higher secretion yield, greater activity on extracellular pectin, improved reproducibility and scalability.

References

  1. Mathiesen, G., et al. (2009). BMC Genomics, 10, 425. https://doi.org/10.1186/1471-2164-10-425
  2. Ng, D. T. W., Sarkar, C. A., & Goh, J. K. (2016). Applied and Environmental Microbiology, 82(24), 7128–7138. https://doi.org/10.1128/AEM.01913-16

5. pTRKH3 Plasmid Backbone (BBa_25GUIQHH)

New part: registry.igem.org/parts/bba-25guiqhh

General Information & Literature Review

pTRKH3 is a shuttle vector for E. coli and LAB (e.g., Lactobacillus, Lactococcus) with dual replication origins (p15A and pAMβ1) and erythromycin selection, enabling cloning in E. coli and expression in LAB.

Application in Our Project

  • Promoter: ermB (constitutive).
  • Gene of Interest: pgxC (polygalacturonase).
  • Secretion Elements: Usp45 or engineered signal peptide.
  • Terminator: LAB-compatible terminator for stable transcription.

References

  1. O’Sullivan, D. J., & Klaenhammer, T. R. (1993). Gene, 137(2), 227–231. https://doi.org/10.1016/0378-1119(93)90011-Q
  2. Lee, J. H., & O’Sullivan, D. J. (2006). MMBR, 70(3), 585–603. https://doi.org/10.1128/MMBR.00004-06
  3. De Vos, W. M. (1999). Current Opinion in Microbiology, 2(3), 289–295. https://doi.org/10.1016/S1369-5274(99)80051-8

6. pTRKH3 + Secretion Signal + pgxC Composite Part (BBa_252WDOMF)

General Information & Literature Review

A single construct combining promoter, secretion signal(s), coding sequence, terminator, and pTRKH3 backbone enables stable expression and extracellular secretion of polygalacturonase in a food-grade LAB chassis.

Design Elements

  • ermB promoter (constitutive in LAB).
  • Usp45 or engineered secretion signal (Sec-pathway export).
  • pgxC coding sequence (polygalacturonase for pectin degradation).
  • LAB terminator + pTRKH3 backbone (shuttle vector).
Schematic of final composite construct.
Figure 1. Schematic representation of the final construct.
Plasmid map of composite part with modified pgxC.
Figure 2. Final construct plasmid map with modified pgxC sequence.

Application in Our Project

This composite part is the core innovation enabling engineered L. reuteri to act as a fruit peel biodegradation agent, suitable for waste management, composting, and bioreactor contexts.

References

  1. O’Sullivan, D. J., & Klaenhammer, T. R. (1993). Gene, 137(2), 227–231. https://doi.org/10.1016/0378-1119(93)90011-Q
  2. Jayani, R. S., Saxena, S., & Gupta, R. (2005). Process Biochemistry, 40(9), 2931–2944. https://doi.org/10.1016/j.procbio.2005.03.026
  3. Le Loir, Y., et al. (2005). Microbial Cell Factories, 4(1), 2. https://doi.org/10.1186/1475-2859-4-2

Additional Notes

Besides the five new parts we added to the Registry, we added two additional parts for engineering purposes:

*The pgxC signal peptide was modified by a silent PstI site removal to restore RFC 10 compatibility while maintaining the original amino acid sequence and secretion functionality.