Overview

Our project focuses on addressing the environmental challenge of spent coffee grounds (SCG) by developing a biological decaffeination system using engineered E. coli. We have made several key contributions to the iGEM community, including troubleshooting enzymatic efficiency, characterizing novel enzyme constructs, providing detailed hardware modeling documentation, and analyzing policy frameworks for biofertilizer applications. These contributions are designed to support future teams working on related metabolic pathways, bioreactor design, and environmental biotechnology applications.

1 Troubleshooting for Heterologous Expression of Multi-Enzyme Systems for Enhanced Functionality

In the process of establishing a heterologous caffeine degradation pathway in E. coli, our team encountered a common yet often overlooked challenge in synthetic biology: high protein expression levels do not always correlate with high enzymatic activity. Our initial attempt to express the full ndmA-E gene cluster from P.putida in E.coli BL21(DE3) under the T7 promoter system resulted in strong protein expression confirmed by SDS-PAGE. However, the caffeine degradation efficiency remained suboptimal, with only approximately 35% reduction. We hypothesized that the high-level, rapid expression in BL21(DE3) could lead to improper protein folding or the formation of inclusion bodies, thereby compromising enzymatic activity.

To address this, we evaluated the same constructs in E.coli DH5α, a host known for lower basal expression. Notably, DH5α achieved a significantly higher degradation efficiency of approximately 60%, despite the absence of protein bands on SDS-PAGE. This suggests that moderate expression levels in DH5α favor correct protein folding and functional assembly of the multi-enzyme Ndm complex. This finding underscores that the choice of expression host is critical, and for complex heterologous pathways, high protein expression does not necessarily correlate with high functional activity. We recommend that future teams consider screening other host strains and employ tunable expression systems (e.g., pBAD) to optimize the balance between protein yield and catalytic efficiency for similar multi-enzyme systems. By documenting this host-dependent activity discrepancy and proposing a practical workflow for strain selection, we provide a reusable troubleshooting strategy that can help future iGEM teams avoid common pitfalls and accelerate the development of reliable biological systems.

2 Independent Validation and Characterization of NdmDA, and NdmDCE part

The NdmA-E enzymes, initially registered by the NEFU-China team in the pYB1s plasmid system, required further characterization in more common expression vectors to enhance their versatility for the iGEM community. Our project systematically addressed this by subcloning these parts into a standardized pET28a backbone and conducting parallel functional analyses in two widely used E. coli chassis: BL21(DE3) and DH5α.

This side-by-side comparison provided critical insights. We confirmed protein expression in both strains but discovered a striking host-dependent discrepancy between expression levels and functional efficiency. While BL21(DE3) showed strong expression via SDS-PAGE, it yielded lower caffeine degradation. In contrast, DH5α—despite lower detectable protein expression—achieved significantly higher degradation activity, underscoring the importance of host selection for complex enzyme complexes.

Key Contribution to the iGEM Community:

· Cross-Platform Validation in Standard Vectors: We have transferred the existing Ndm parts from the original pYB1s system into the widely adopted pET28a vector, providing functional validation in two common lab strains (BL21 and DH5α). This offers future teams a reliable, ready-to-use alternative and a direct comparative dataset for these popular chassis.

· Practical Guidance on Host Selection: Our work delivers a practical, data-backed guideline: for complex multi-enzyme systems, high-expression hosts like BL21 may not be optimal, and screening alternatives like DH5α is crucial. This finding can save future teams significant time during troubleshooting.

· Characterization Data: All characterization data, including the comparative performance in both hosts and the optimized conditions for the pET28a constructs, have been submitted to the corresponding Registry pages. This greatly expands the practical knowledge base for these BioParts, enabling more robust and informed design choices for all future teams.

Registered parts：

BBa_K5313027 NdmDA

BBa_K5313029 NdmDCE

3 An Open-Source, Modular Bioreactor for Enzymatic Caffeine Degradation

To address the challenge of biodegrading caffeine in spent coffee grounds, we have designed and constructed an open-source, modular benchtop bioreactor that provides a stable and controllable environment for multi-enzyme systems such as Ndm. This reactor is also suitable for other iGEM projects involving biological fermentation, and its modular architecture allows teams to iteratively adapt and optimize the system according to their specific needs.

Key Contributions:

- Modular & Reproducible Design

The system consists of independent modules - temperature control, mixing and aeration, control unit, and sampling - enabling easy customization, maintenance, and upgrades.

Comprehensive design files, code, and assembly guidelines are provided to significantly lower the barrier for future teams to rebuild and adapt the system.

- Clear Iterative Development Path

Through three distinct versions - from a basic prototype (V1) to a stable glass water-jacket reactor (V2), and further to an intelligent closed-loop system (V3) - we have defined clear design priorities and optimization strategies at each stage. This offers a valuable engineering framework for subsequent hardware development in iGEM.

- Real-World Application

Designed specifically for processing organic waste such as coffee grounds, the reactor demonstrates the potential of synthetic biology hardware in distributed bio-upcycling and sustainable waste valorization.

- Open-Source Hardware Platform

All technical documentation will be fully publicly accessible. The reactor is not only applicable to caffeine degradation but also serves as a versatile enzymatic catalysis platform, allowing future iGEM teams to readily adapt it for a broad range of biocatalytic projects.

4 Regulatory Insight: Policy and Market Access for Biofertilizers in China

To ensure that our caffeine-detoxified SCG biofertilizer can be safely applied and potentially commercialized in China, we conducted systematic research on national biofertilizer regulations and certification requirements. This effort bridges synthetic biology innovation with real-world agricultural compliance and provides a clear route planning for future iGEM teams exploring similar applications.

Our research summarized the current regulatory framework, including:

● Law of the People‘s Republic of China on the Prevention and Control of Environmental Pollution by Solid Wastes (2020 revised): Supports the resource recovery and recycling of household waste.

● Circular Economy Promotion Law of the People's Republic of China (2018 revised): Encourages waste-to-resource innovation, aligning with our project’s circular economy goals.

● Regulations on the Administration of Safety of Agricultural Genetically Modified Organisms (2017 revised): Defines biosafety standards for using engineered microorganisms, guiding the strain design and containment strategy.

● Organic Fertilizer Standard (NY/T 525-2021): Specifies product quality and safety requirements for market-approved fertilizers.

We also summarized the biofertilizer registration pathway managed by the Ministry of Agriculture and Rural Affairs (MARA), which includes:

1 ) Provincial preliminary review of production conditions;

2 ) Strain safety assessment and inactivation verification for engineered microbial products;

3 ) Optional toxicity evaluation depending on strain type;

4 ) Product quality inspection and field trials to confirm efficacy;

5 ) Comprehensive evaluation and certificate issuance by MARA.

Based on this framework, we clarified that engineered biofertilizers intended for market use in China must ensure the complete inactivation of engineered strains after fermentation, provide validated evidence of strain safety, and conduct standardized field efficacy tests under national organic fertilizer standards.

By summarizing and sharing our insights on China’s biofertilizer regulatory framework on our Wiki, we aim to help future iGEM teams understand how biosafety and market compliance are integrated into agricultural biotechnology. This analysis offers a reference for teams interested in developing microbial or environmental applications, encouraging early consideration of policy feasibility and safety design in their synthetic biology projects.