Antimicrobial resistance is one of the top global public health and development threats. It is estimated that bacterial AMR was directly responsible for 1.27 million global deaths in 2019 and contributed to 4.95 million deaths (Murray et al., 2022). In addition to death and disability, AMR has significant economic costs. The World Bank estimates that AMR could result in 1 trillion dollars additional healthcare costs by 2050, and 1 trillion to 3.4 trillion dollars gross domestic product (GDP) losses per year by 2030.
Antimicrobial peptides are small (usually <50 amino acids), cationic, and amphiphilic molecules. In view of their potent and rapid broad-spectrum antimicrobial activity (even against multidrug-resistant ESKAPEE pathogens— Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.), antimicrobial peptides have shown great promise as potential therapy for tackling AMR, with minimal risk of inducing AMR (Ho et al., 2025; Magana et al., 2020; Mookherjee et al., 2020).
The Jiangnan-China team has innovatively constructed Cytopia, a versatile cell factory capable of efficiently producing LL-37, an antimicrobial peptide, offering a novel solution to the global challenge of antibiotic resistance while significantly reducing production costs. We employed Saccharomyces cerevisiae as the chassis for antimicrobial peptide expression, since continuous biosynthesis in yeast provides yields far superior to chemical synthesis. To further enhance cost-effectiveness, we engineered S. cerevisiae to synthesize antimicrobial peptides directly from glucose—an inexpensive and renewable substrate—rather than costly raffinose and galactose. In addition, Cytopia is equipped with the Cytoflow model, an intelligent framework that can predict antimicrobial peptide performance, generate peptide variants tailored to specific needs, and optimize medium composition to maximize yield. This highlights Cytopia as a truly smart cell factory.
Ultimately, Cytopia not only addresses antibiotic resistance effectively, but also achieves the ultimate goal of intelligent cell factories: the low-cost, efficient, and green biosynthesis of antimicrobial peptides and even other small functional peptides.
Fig 1. Human synthesis of LL-37
Antimicrobial peptides (AMPs) possess potent and rapid broad-spectrum antimicrobial activity, with a minimal risk of inducing AMR. Their favourable properties are attributed to multitargeting of membrane integrity, cell wall biosynthesis, cytoplasmic membrane, or a combination of these. Antimicrobial peptides also have immunomodulatory, wound healing, and a host of other properties.
LL-37 is a small peptide composed of 37 amino acid residues (sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), and it is the only known cathelicidin antimicrobial peptide present in the human body. It is generated from the precursor protein hCAP-18 by removal of the signal peptide and subsequent proteolytic cleavage, yielding the active peptide (Ridyard & Overhage, 2021). Compared with conventional antibiotics, LL-37 is safe, less prone to resistance development, and exhibits broad-spectrum antimicrobial effects against Gram-positive bacteria, Gram-negative bacteria, fungi, and certain viruses.
Fig 2. Heterologous expression of LL-37 in Saccharomyces cerevisiae
We selected Saccharomyces cerevisiae as the chassis cell because it is a generally recognized as safe (GRAS). As an eukaryotic microorganism with a complex cellular structure, S. cerevisiae enables proper protein folding and post-translational modifications (PTMs) during protein production (Liu et al., 2013). Moreover, its translation and post-translational processing are rapid, straightforward, and cost-effective, making it an ideal prototype for a cellular factory.
Fig 3. Engineering diagram of Saccharomyces cerevisiae cell factory
To further enhance the production of LL-37 in our cellular factory, we adopted strategies including promoter engineering, protease gene knockout, and multi-copy integration. In promoter engineering, the strong constitutive promoters TEF1 and GAP were used to replace the original GAL1 promoter. During protease gene knockout, both rational and semi-rational screening revealed that deleting protease genes such as PEP4, PRB1, YSP3, YPS1, and UBP9 significantly increased LL-37 yield. Additionally, increasing the gene copy number through multi-copy integration further boosted LL-37 production (see Results).
Fig 4. Framework overview of dry lab experiments
Since the antimicrobial activity of LL-37 is suboptimal, we aimed to obtain LL-37 variants with enhanced antibacterial performance through sequence mutations. To this end, our dry lab team developed the CytoFlow model, composed of CytoEvolve, CytoGuard, and CytoGrow. Specifically, CytoEvolve generates LL-37 variant sequences with improved antibacterial properties through targeted site-directed mutations, while CytoGuard accurately predicts the antimicrobial performance of these variants based on their sequences. Using the CytoFlow model, we successfully identified variant sequences with antibacterial efficacy that surpasses that of wild-type LL-37 (see Model).
Fig 5. Predicted structural model of LL-37 based on computational modeling.
Our goal is to achieve cost-effective and efficient expression of the antimicrobial peptide LL-37. To this end, we focused on replacing costly carbon sources with more economical alternatives and optimizing medium composition to maximize production efficiency. Regarding carbon source replacement, we knocked out GAL80 to eliminate the galactose-dependent induction effect, which allowed us to identify glucose as the most cost-effective option among eight non-galactose carbon sources, thereby significantly reducing production costs. For medium optimization, our CytoGrow model is capable of predicting yeast growth rate, glucose consumption, and the optimal medium composition. Ultimately, this strategy increased the biomass of S. cerevisiae by 25.6% (see Model). Ultimately, the purified LL-37 was obtained and ready for further study or practical use.
Fig 6. Comparison of Biomass (OD Value) Across Different Medium Compositions
Beyond the synthesis of antimicrobial peptides, our smart cell factory is also capable of low-cost production of various small peptides, including flavor-enhancing peptides and bioactive peptides used in cosmetic ingredients. With applications spanning pharmaceuticals, food, and cosmetics, it drives the development of future industries and serves as a truly versatile platform.
Ho, C. S., Wong, C. T. H., Aung, T. T., Lakshminarayanan, R., Mehta, J. S., Rauz, S., & Ting, D. S. J. (2025). Antimicrobial resistance: a concise update. The Lancet Microbe, 6(1). https://doi.org/10.1016/j.lanmic.2024.07.010
Liu, F., Wu, X., Li, L., Liu, Z., & Wang, Z. (2013). Use of baculovirus expression system for generation of virus-like particles: successes and challenges. Protein Expression and Purification, 90(2), 104-116. https://doi.org/10.1016/j.pep.2013.05.009
Magana, M., Pushpanathan, M., Santos, A. L., Leanse, L., Fernandez, M., Ioannidis, A., & Tegos, G. P. (2020). The value of antimicrobial peptides in the age of resistance. The Lancet Infectious Diseases, 20(9), e216-e230. https://doi.org/10.1016/s1473-3099(20)30327-3
Mookherjee, N., Anderson, M. A., Haagsman, H. P., & Davidson, D. J. (2020). Antimicrobial host defence peptides: functions and clinical potential. Nature Reviews Drug Discovery, 19(5), 311-332. https://doi.org/10.1038/s41573-019-0058-8
Murray, C. J. L., Ikuta, K. S., Sharara, F., Swetschinski, L., Robles Aguilar, G., Gray, A., & Naghavi, M. (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet, 399(10325), 629-655. https://doi.org/10.1016/S0140-6736(21)02724-0
Ridyard, K. E., & Overhage, J. (2021). The Potential of Human Peptide LL-37 as an Antimicrobial and Anti-Biofilm Agent. Antibiotics, 10(6). https://doi.org/10.3390/antibiotics10060650
