The inspiration for our project stems from a deep engagement with Hubei WEL-SAFE Biotechnology Co., Ltd. We learned that antimicrobial lipopeptides, produced by soil microbes, are remarkably effective against plant pathogens and in high demand among farmers. However, the company faces a major bottleneck in scaling their production. This critical challenge directly sparked our iGEM project: to leverage synthetic biology to overcome this production barrier, thereby unlocking the full potential of these natural compounds to serve as powerful, sustainable guardians of global crop health.

A Dual Crisis in Crop Protection

Plant diseases pose a severe threat to global food security, leading to annual crop losses exceeding $220 billion ^[1]. While conventional chemical pesticides offer temporary relief, they fuel a vicious cycle of rising pathogen resistance and environmental degradation. These chemicals accumulate in soils, leach into water systems, and propagate through ecosystems, ultimately compromising the very foundation of agricultural sustainability ^[2,3].

Cyclic Lipopeptides: A Sustainable Alternative

In this context, biocontrol agents emerge as a necessary alternative. Among them, cyclic lipopeptides (CLPs)—such as surfactin, fengycin, and iturin—represent a breakthrough solution ^[4]. They exert their antimicrobial effect through physical disruption of pathogen cell membranes, a non-specific mechanism that greatly reduces the risk of resistance development ^[5,6].

Beyond Pathogen Control: Multifunctional Benefits

Highly specific in their action, CLPs preserve beneficial soil and plant-associated microbiota ^[7]. They are fully biodegradable, leaving no harmful residues. Beyond direct antimicrobial activity, certain CLPs also induce systemic resistance in plants and can enhance crop growth. By addressing both resistance and pollution challenges, CLPs offer a fundamentally greener pathway to sustainable crop protection^[8].

The yield of cyclic lipopeptides (CLPs) in wild-type strains is extremely low. For instance, fengycin production typically reaches only 10-17 mg/L in most strains ^[9], while yields of surfactin and iturin also remain below 1 g/L. Even with optimized culture media and fermentation conditions, improvements in yield have been limited.

Genetic engineering of the synthases involved in CLPs biosynthesis presents further challenges. CLPs biosynthesis is an intricate and meticulously controlled biochemical process. CLPs are synthesized through a unique nonribosomal peptide synthetase (NRPS) multi-enzyme system. The associated gene clusters are exceptionally large—over 26 kb in length—making heterologous expression highly challenging ^[10]. Moreover, CLPs production in the cell is controlled by multiple regulatory systems that tightly control synthesis, thereby restricting industrial-scale manufacturing and broader application ^[11]. As a result, most current engineering strategies rely on in-situ genomic modifications rather than full pathway transplantation ^[12].

Furthermore, the complex structure of lipopeptides, which incorporates both amino acids and fatty acids, involves intricate metabolic regulation, adding another layer of difficulty to strain engineering and yield enhancement^[13].

Our project aims to engineer Bacillus velezensis for the innovative production of lipopeptide biopesticides. The integrated workflow begins with ARTP mutagenesis and high-throughput screening to isolate high-yield mutants. We will then identify key mutations and employ CRISPR-Cas9 for gene editing in the wild-type strain, enabling us to build a regulatory model for lipopeptide synthesis. To meet our industrial partner's need for single-component lipopeptides, we will develop a dual-gRNA vector system to efficiently delete long fragment of NRPS gene cluster, thereby redirecting metabolic flux. Furthermore, guided by NRPS function prediction, we will reprogramming the COM domain to create novel fengycin variants. Collectively, these strategies form a comprehensive synthetic biology pipeline to advance the development and industrial application of next-generation biopesticides.

Our software platform addresses a fundamental challenge in synthetic biology: optimizing the modular assembly line of NRPS for efficient lipopeptide production. NRPS machinery synthesizes antimicrobial compounds through coordinated modules, each responsible for incorporating specific amino acid precursors. The communication (COM) domains between these modules serve as critical molecular interfaces that ensure proper intermediate transfer. However, natural COM domains often exhibit suboptimal pairing efficiency, limiting both production yield and structural diversity.

To overcome this bottleneck, we developed an integrated computational workflow that transforms NRPS sequence analysis into actionable engineering strategies. The platform processes input sequences through multiple bioinformatic modules: MAFFT-based multiple sequence alignment establishes evolutionary context, IQ-TREE phylogenetic reconstruction identifies conservation patterns, and InterProScan domain annotation maps functional domains (C, A, T, TE) while precisely locating COM interface regions.

The platform's core innovation enables deep analysis of COM domain interactions. It automatically extracts junction sequences spanning module interfaces and applies advanced algorithms to identify key residues governing inter-module recognition. By integrating structural modeling with evolutionary conservation data, the software predicts optimal COM pairing configurations and recommends specific mutations to enhance domain compatibility.

This computational guidance directly serves our project's objectives in three crucial aspects: First, it enables rational redesign of COM interfaces to improve Fengycin synthesis efficiency, addressing the production scalability challenge faced by our industrial partner. Second, it supports the creation of novel lipopeptide variants through COM domain reprogramming, expanding the spectrum of biopesticide candidates. Third, by optimizing the fundamental machinery of NRPS assembly lines, the software helps unlock the full potential of Bacillus velezensis as a microbial cell factory for sustainable agriculture.

The platform operates through a continuous learning cycle where experimental results from engineered strains refine prediction algorithms and expand our knowledge base of successful modification strategies. This iterative design-build-test-learn framework not only accelerates our current optimization efforts but also establishes a reusable resource for the synthetic biology community, demonstrating how computational tools can drive the development of next-generation biopesticides that balance agricultural productivity with environmental sustainability.

Our Human Practice activities are guided by the principle of "bidirectional shaping," where societal input continuously refines our project direction as we work to address real-world agricultural challenges.

Inspired by collaboration with WEL-SAFE Biotechnology Co., Ltd, we identified key bottlenecks in biopesticide production and ecological concerns of chemical pesticides. Through field research and stakeholder interviews, we confirmed the urgent need for sustainable alternatives.

Our project aligns with multiple UN Sustainable Development Goals: by developing green biopesticides, we contribute to Zero Hunger (SDG 2) and Good Health and Well-being (SDG 3). Through industrial application, we support Decent Work and Economic Growth (SDG 8).

We bridge science and society through inclusive science communication—adapting content for children, students, hearing-impaired communities, and the general public via social media and cultural products—promoting Quality Education (SDG 4) and Reduced Inequality (SDG 10).

By actively engaging in synthetic biology conferences and interdisciplinary collaborations, we strengthen Partnerships for the Goals (SDG 17). Moving forward, we remain committed to this bidirectional approach, ensuring our work creates tangible impact alongside scientific innovation.

[1]Food and Agriculture Organization of the United Nations. Plant production and protection. https://www.fao.org/plant-production-protection/about/en

[2]Hongoeb J, Tantimongcolwat T, Ayimbila F, Ruankham W, Phopin K. Herbicide-related health risks: key mechanisms and a guide to mitigation strategies. J Occup Med Toxicol. 2025;20(1):6.

[3]Kaur R, Choudhary D, Bali S, et al. Pesticides: An alarming detrimental to health and environment. Sci Total Environ. 2024;915:170113.

[4]Markelova N, Chumak A. Antimicrobial Activity of Bacillus Cyclic Lipopeptides and Their Role in the Host Adaptive Response to Changes in Environmental Conditions. Int J Mol Sci. 2025;26(1):336.

[5]Chen X, Lu Y, Shan M, Zhao H, Lu Z, Lu Y. A mini-review: mechanism of antimicrobial action and application of surfactin. World J Microbiol Biotechnol. 2022;38(8):143.

[6]Bucataru C, Ciobanasu C. Antimicrobial peptides: Opportunities and challenges in overcoming resistance. Microbiol Res. 2024;286:127822.

[7]Sabater C, Neacsu M, Duncan SH. Harnessing beneficial soil bacteria to promote sustainable agriculture and food security: a one health perspective. Front Microbiol. 2025;16:1638553.

[8]Sreedharan SM, Rishi N, Singh R. Microbial lipopeptides: Properties, mechanics and engineering for novel lipopeptides. Microbiol Res. 2023;271:127363.

[9]Zhou J, Wu G, Zheng J, et al. Research on the Regulation of Plipastatin Production by the Quorum-Sensing ComQXPA System of Bacillus amyloliquefaciens. J Agric Food Chem. 2023;71(28):10683-10692.

[10]Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J. Overview of the Antimicrobial Compounds Produced by Members of the Bacillus subtilis Group. Front Microbiol. 2019;10:302. 

[11]Qi X, Liu W, He X, Du C. A review on surfactin: molecular regulation of biosynthesis. Arch Microbiol. 2023;205(9):313.

[12]Qi X, Liu W, He X, Du C. A review on surfactin: molecular regulation of biosynthesis. Arch Microbiol. 2023;205(9):313.

[13]Evidente A. Bioactive Lipodepsipeptides Produced by Bacteria and Fungi. Int J Mol Sci. 2022;23(20):12342.

[14]Mahdi I, Allaoui A, Fahsi N, Biskri L. Bacillus velezensis QA2 Potentially Induced Salt Stress Tolerance and Enhanced Phosphate Uptake in Quinoa Plants. Microorganisms. 2022;10(9):1836. Published 2022 Sep 14. doi:10.3390/microorganisms10091836