In recent years, the problem of antibiotic resistance has become increasingly severe. According to a landmark study by the Global Research on AntiMicrobial resistance (GRAM) project, it is estimated that by 2050, the number of deaths globally due to drug-resistant infections will rise dramatically, with a staggering 39 million people potentially losing their lives (University of Oxford, 2024). This makes the development of novel and effective products that can replace antibiotics an urgent priority (Zeth & Sancho-Vaello, 2021). To address this challenge, the Jiangnan University iGEM team proposes a synthetic biology-based solution: utilizing Saccharomyces cerevisiae as a "cell factory" to express the human-derived antimicrobial peptide LL-37 with broad-spectrum antibacterial activity. LL-37 is a small molecule peptide composed of thirty-seven amino acid residues (sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), and is the only known Cathelicidin-class antimicrobial peptide present in the human body, formed from the precursor protein hCAP-18 after signal peptide removal and hydrolysis to produce the active peptide (Fig. 1) (Burton & Steel, 2009). Previous research indicates that LL-37 and its mouse ortholog CRAMP can suppress pancreatic β-cell inflammation in type 1 diabetes models and enhance islet function to promote insulin secretion. Furthermore, LL-37 can alleviate neuroinflammation by inhibiting Aβ42 amyloidosis (Armiento et al., 2020). Compared to traditional antibiotics, LL-37 is safe, less likely to develop resistance, and exhibits broad-spectrum antibacterial effects against Gram-positive bacteria, Gram-negative bacteria, fungi, and certain viruses (Cao et al., 2014). The Jiangnan University team reduces synthesis costs and enhances LL-37 production through selecting yeast strain background, knocking out multiple protease genes, and optimizing protomer sequences, etc. Combined with dry lab predictions of LL-37 variants and optimized culture medium formulations, we are the first team successfully achieved endogenous production of LL-37 from S. cerevisiae Our project not only provides new insights for combating antimicrobial resistance but also offers new pathways for the biosynthesis of functional peptides and the achievement of the United Nations Sustainable Development Goals (SDGs), promoting sustainable and accessible global health.
Fig. 1 Basic inforamtion of LL-37
(a)Active conformation of LL-37 (b) Molecular structure of LL-37 (c) Humanized synthesis of LL-37
Synthesis challenges: The broad-spectrum antimicrobial properties of LL-37 conflict with the strict protein quality control system of S. cerevisiae, making heterologous expression and secretion enormously challenging, with almost no prior attempts in this field. Cost and performance issues: Compared to traditional antimicrobial drugs, biosynthetic LL-37 has higher costs, and its antimicrobial performance still has room for improvement. Resistance issues: Certain bacteria and fungi have already evolved multiple resistance mechanisms against native LL-37, urging for new variants of LL-37 to be developed.
Our project is named Cytopia: Cell-to-Yield Factory Producing Innovative Antimicrobial Peptides. Our goal is to construct a "cell factory" capable of efficiently expressing novel antimicrobial peptides. The concrete aspects include:
The inspiration for our project Cytopia comes from the urgent global need to combat antimicrobial-resistant bacteria, and our desire to build a sustainable "cell factory" that utilizes microbial chassis to efficiently synthesize antimicrobial peptides. Project Cytopia is the embodiment of this vision: we aim to achieve efficient production of LL-37 and its variants in a highly optimized S. cerevisiae chassis, bringing antimicrobial peptides toward a low-cost, industrializable future.
To support this goal, we designed the dry lab platform CytoFlow. CytoFlow provides a holistic architecture to guide our project's "Design-Build-Test-Learn" (DBTL) cycle. Under the CytoFlow architecture, we can achieve molecular design predictions of antimicrobial peptides, fermentation condition optimization, and guide expression element engineering.
Chassis optimization: We screened suitable yeast chassis strains and reduced protease degradation through gene knockout.
Localization and extraction: Through fluorescence localization, we discovered that LL-37 adsorbs to the cell membrane, so we successfully washed it off through high salt, low pH, and surfactant treatments, ultimately achieving efficient purification using magnetic beads and semi-preparative liquid chromatography.
Expression optimization: We first attempted to secrete LL-37 through signal peptides and chaperone proteins, modified promoters to relieve galactose dependency and glucose repression. Then we progressively improved LL-37 expression levels from single gene knockout to multi-gene combination knockouts, while further enhancing yield through multi-copy integration. Additionally, dry lab designed the CytoGrow module, including yeast growth kinetics model, glucose consumption model, and culture medium ratio optimization model. These three models coordinate with each other to systematically improve the S. cerevisiae fermentation growth environment and increase LL-37 production.
Variant design: Dry lab used computational modeling to predict more potent LL-37 variants, combined with D2P technology for synthesis, and screened out the best-performing candidate molecules through improved antibacterial assay methods.
Fig. 2 Overview of the engineering cycle, illustrating the problem input, intermediate solution steps, and the target output.
Through optimization with the CytoGrow model in dry lab, the biomass (OD value) of S. cerevisiae improved by approximately 6% compared to the experimentally determined optimal culture medium. Wet lab experiments achieved an increase in LL-37 production from nanogram to milligram levels. In the future, we hope to extend the LL-37 cell factory to the production of more types of antimicrobial peptides and functional short peptides. We are not limited to applying LL-37 in food applications but aim to combine it with drug development, public health, and other fields, providing practical and feasible solutions for the global fight against AMR.
Armiento, V., Hille, K., Naltsas, D., Lin, J. S., Barron, A. E., & Kapurniotu, A. (2020). The Human Host-Defense Peptide Cathelicidin LL-37 is a Nanomolar Inhibitor of Amyloid Self-Assembly of Islet Amyloid Polypeptide (IAPP). Angewandte Chemie International Edition, 59(31), 12837-12841.
Burton, M. F., & Steel, P. G. (2009). The chemistry and biology of LL-37. Natural Product Reports, 26(12).
Cao, X., Zhang, Y., Mao, R., Teng, D., Wang, X., & Wang, J. (2014). Design and recombination expression of a novel plectasin-derived peptide MP1106 and its properties against Staphylococcus aureus. Applied Microbiology and Biotechnology, 99(6), 2649-2662.
University of Oxford. (2024). Antibiotic resistance has claimed at least one million lives each year since 1990. https://www.ox.ac.uk/news/2024-09-17-antibiotic-resistance-has-claimed-least-one-million-lives-each-year-1990
Zeth, K., & Sancho-Vaello, E. (2021). Structural Plasticity of LL-37 Indicates Elaborate Functional Adaptation Mechanisms to Bacterial Target Structures. International Journal of Molecular Sciences, 22(10).
