The NEFU-China 2024 team’s project provides a sustainable solution that aims to efficiently process coffee waste. This project can promote both the reutilization of food waste and the production of a new drug. Through genetic engineering, this project enables the conversion of caffeine in coffee grounds into 7-Methylxanthine (7-MX), a compound with a potential medicinal value, particularly in preventing myopia.

The project design includes three modules: Module 1 focuses on enzyme screening to identify key enzymes that efficiently catalyze the conversion of caffeine; Module 2 is dedicated to optimizing catalytic conditions to enhance reaction efficiency and stability; and Module 3 focuses on the reutilization of coffee grounds, ensuring resource recycling and environmental sustainability. These three modules work in synergy to ensure the efficiency and sustainability of the project, offering new perspectives on waste processing and drug development.

The goal of this module is to identify enzyme mutants capable of efficiently converting caffeine into 7-MX, enabling the development of high-performance engineered bacterial strains for the recycle of coffee grounds. The critical aspects of this design involve adjusting substrate specificity, altering enzyme activity , and improving the sensitivity and accuracy of screening methods. Traditional screening approaches, such asHPLC, are accurate, but mostly time-consuming and costly, which make them unsuitable for large-scale high-throughput screening. Therefore, our team designed a high-throughput screening method based on a biosensor that can quickly detect enzyme efficiency in converting caffeine to 7-MX.

Biosensor Design Strategy

1. guaB Gene Knockout:

The guaB gene in E. coli, responsible for synthesizing guanine nucleotides, was knocked out, leading to bacterial growth retardation [1], and the bacteria can well proliferate depending on external purine supplementation.

2. Introduction of Plasmid pDCE:

We introduced the plasmid pDCE, carrying genes from P. putida CBB5 responsible for 7-MX metabolism, into the E. coli ΔguaB strain [2]. In this system, caffeine is first converted to 7-MX, and then further metabolized into xanthine.

3. Utilization of gpt Pathway:

The endogenous gpt pathway in E. coli converts xanthine into guanine nucleotide, and subsequently restores bacterial growth. The extent of bacterial growth recovery directly reflects the efficiency of the enzyme in converting caffeine into 7-MX and xanthine.

Summary: This module is expected to achieve the goal of obtaining key enzymes that efficiently catalyze the conversion of caffeine into 7-MX.

To optimize the working conditions of the high-activity mutants generated in Module 1, systematic experiments were designed to investigate the effects of various factors on enzyme activity and stability, including biomass amount, reaction time, and substrate concentration. Our goal is to enhance the conversion efficiency while maintain enzyme stability.

1. Biomass Adjustment:
     Biomass levels significantly influence reaction efficiency, particularly in microbial fermentation and enzymatic conversion processes. Proper adjustment of biomass affects the enzyme yield and activity, as well as the system balance and stability.
2. Substrate Inhibition and Excess:
      Excessive substrate concentrations can inhibit enzyme activity, leading to abnormal kinetics and reduced reaction efficiency. Additionally, high substrate levels may have toxic effects on enzymes or bacteria, and consequently impair the overall system performance.
3. Enzyme-Cofactor Interactions:
      Many enzymatic reactions rely on cofactors (e.g., NADH, ATP) for efficient catalysis. The supply, regeneration, and stability of these cofactors can significantly affect the reaction [3]. Insufficient cofactor supply may halt the reaction or lead to low efficiencies. Varying cofactor concentrations also influence enzymatic catalysis.

4. Reaction Time and Enzyme Stability:
      Prolonged reactions increase the likelihood of enzyme deactivation due to protein denaturation, degradation, or other irreversible changes. Our team designed experiments to measure the time-dependent activity curve for these catalytic reactions.

Summary: This module is expected to improve the catalytic efficiency and stability of the key enzymes.

The goal of this module is to efficiently reuse food waste—coffee grounds—by converting the caffeine within them into 7-MX, which holds a great medicinal value. This module includes the collection of coffee grounds, extraction of caffeine, and practical applications, with the aim to establish a sustainable circular economy for resource utilization.

Summary: Through this module, we aim not only to address the reutilization of food waste, but also to establish an efficient resource recycling system that promotes sustainable waste management. Ultimately, this provides an effective model for a circular economy in resource utilization. This approach not only helps us reduce environmental pollution, but also paves the way for the development of high-value-added applications for waste in the future.

Each module of our project is designed to achieve a specific goal: Module 1 ensures the identification of key enzymes with high catalytic efficiency, Module 2 optimizes the reaction conditions to enhance the stability and efficiency of these enzymes, and Module 3 focuses on the reutilization of coffee grounds to ensure resource sustainability. Together, these modules contribute to the overall objective of efficiently converting caffeine from coffee waste into 7-MX, which both offers a sustainable solution for waste processing and contributes to potential medicinal applications for myopia prevention.

Please refer to the engineering page for detailed operations.

[1]Pimkin, M., Pimkina, J., & Markham, G. D. (2009). A regulatory role of the Bateman domain of IMP dehydrogenase in adenylate nucleotide biosynthesis. The Journal of biological chemistry, 284(12), 7960–7969. https://doi.org/10.1074/jbc.M808541200

[2]Yu, C. L., Louie, T. M., Summers, R., Kale, Y., Gopishetty, S., & Subramanian, M. (2009). Two distinct pathways for metabolism of theophylline and caffeine are coexpressed in Pseudomonas putida CBB5. Journal of bacteriology, 191(14), 4624–4632. https://doi.org/10.1128/JB.00409-09

[3]Design iGEM NEFU_China 2023