This year our team noticed the obesity problem in Chinese adolescents. In response to the growing issue, our team aims to develop an efficient biosynthesis strategy for producing low-calorie rare sugars, specifically D-tagatose, through synthetic biology approaches by genetically engineering Escherichia coli (E. coli). D-tagatose is a natural low-calorie sweetener with minimal impact on blood sugar levels, making it an ideal alternative for addressing this health concern. By developing the D-tagatose project, we have provided valuable information for the field of synthetic biology. We offer a more cost-effective method for fermentative synthesis of D-tagatose, which reduces the competition of intermediate products without hindering growth.The primary contributions of this project include:

Development and Validation of New Genetic parts: Designing and constructing novel genetic pathways that provide additional genetic tools for the scientific research community.
Innovative Gene Interference Techniques: Applying CRISPR interference (CRISPRi) technology innovatively to block competing metabolic pathways and enhance product synthesis efficiency.
Efficient Screening and Simplified Fermentation Device Design: Creating high-throughput screening methods and developing a cost-effective, user-friendly fermentation device, offering more efficient and accessible tools for researchers, educators, and the food and health industries.

· Beneficiaries and Impact:

- Scientific Community: Offers new genetic parts, improved gene interference methods, and optimized metabolic pathways, providing novel tools and methodologies for synthetic biology and metabolic engineering research.
- Food and Health Industry: Improves the production efficiency of D-tagatose, reducing production costs, and promoting its broader use in low-calorie, low-sugar products.
- Education Sector: The simple fermentation device provides a low-cost, easy-to-operate teaching tool for iGEM teams especially high school iGEM teams, which enable these teams to easily scale up microbial fermentation, thereby enhancing education and outreach in bioengineering.

- Contribution of New Genetic Parts -

This project successfully synthesized and expressed three key genes to establish a biosynthetic pathway for D-tagatose in Escherichia coli. The gatz gene from Caldilinea aerophila encodes fructose 6-phosphate 4-epimerase, which catalyzes the reversible conversion of fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P). The pgp gene from Archaeoglobus profundus encodes a phosphatase that converts D-tagatose 6-phosphate (D6P) into D-tagatose. Additionally, the overexpression of the pgi gene from Thermus thermophilus, which encodes phosphoglucose isomerase, enhances the conversion of glucose 6-phosphate (G6P) to F6P, improving the overall efficiency of the pathway.
Meanwhile, several different sgRNAs were designed to guide dCas9 to bind to key genes involved in competing pathways, which proved to be effective in enhancing tagatose synthesis. These sgRNAs were engineered with 1-2 mismatches in their seed region relative to the target genes, allowing for less stringent binding. This approach enabled variable levels of gene knockdown, optimizing the pathway for improved tagatose production.

- A gene interference method in E.coli -

In this project, we leveraged CRISPR interference (CRISPRi) technology to optimize metabolic flux and enhance D-tagatose production in Escherichia coli. Our approach combined a tunable mismatch CRISPRi system with a color-mediated screening methodology to identify high-production phenotypes. We used a programmable mismatch CRISPRi strategy capable of achieving multiple levels of gene knockdown through a one-pot sgRNA pool, consisting of 16 variants for each target gene, each containing two consecutive random mismatches in the seed region. This enabled us to finely control the knockdown of key competing genes, zwf (encoding glucose-6-phosphate dehydrogenase, G6PDH) and pfkA (encoding 6-phosphofructokinase I), thereby reducing the diversion of intermediates from the desired D-tagatose biosynthesis pathway (Fig. 1). Our method could also benefit other teams working with pathways that use F6P as a substrate, such as those involved in the production of glucosamine, sialic acid, or hyaluronic acid.
The innovative CRISPRi-based gene interference method developed in this project provides a versatile tool for fine-tuning metabolic pathways, which can be applied to a wide range of synthetic biology and metabolic engineering research. By demonstrating the effectiveness of a tunable mismatch CRISPRi system in optimizing metabolic fluxes for enhanced D-tagatose production, our approach offers a valuable framework for scientists seeking to improve the yield of other target metabolites. This strategy enables precise control over gene expression levels, facilitating more efficient pathway optimization and contributing to the advancement of microbial bioproduction technologies.
For iGEM teams, particularly those focusing on metabolic engineering projects, our method offers a practical and accessible approach to optimizing gene expression and pathway efficiency. The use of a programmable mismatch CRISPRi system allows for flexible, high-throughput screening of various gene knockdown levels, which can be easily implemented in any standard laboratory setting. This enables iGEM teams to quickly identify high-yield phenotypes and accelerate their project development, making the method an invaluable addition to the synthetic biology toolkit available to young researchers and student teams.

Fig. 1 The CRISPRi method used in this project. We used a programmable mismatch CRISPRi strategy capable of achieving multiple levels of gene knockdown through a one-pot sgRNA pool, consisting of 16 variants for each target gene, each containing two consecutive random mismatches in the seed region.

- High throughput and fast method for tagatose assay -

- The high-throughput screening method developed in this project enables the rapid identification of highly efficient D-tagatose-producing strains. By integrating automated absorbance measurement, our approach allows for the quick screening of optimal strains, such as DT-D9, significantly reducing experimental time and labor costs. This provides a swift and accurate method for characterizing tagatose production, offering a reliable tool for both industrial applications and further research.
Traditional methods like HPLC, which require a refractive index detector, are costly and time-consuming, with each sample taking around 20 minutes to analyze. This becomes impractical when screening large numbers of samples. Therefore, developing a faster, more cost-effective detection method for D-tagatose is essential.
To address this, we employed a microplate reader for detecting D-tagatose content, capable of testing 96 samples simultaneously with a detection time of only 1-2 minutes. The process involves diluting the supernatant and mixing it with a 0.75 mg/mL resorcinol solution, heating the mixture at 100°C for 20 minutes, and then cooling it at 4°C for 5 minutes. The reaction between D-tagatose and resorcinol produces a yellow color, whose intensity indicates the D-tagatose concentration. Initial screening is visually conducted by discarding samples with a faint yellow color, followed by precise quantification using a microplate reader at 400 nm.
This method allows for rapid and accurate quantification of D-tagatose, effectively meeting the needs of high-throughput screening without relying on expensive instruments such as flow cytometers. By using resorcinol-based detection, we achieve both speed and cost efficiency, thereby enhancing the practicality and scalability of tagatose production screening.
This technology offers significant benefits to other teams in two main ways:
1. Providing a Rapid Detection Method for D-Tagatose: The resorcinol-based colorimetric assay developed in our project offers a fast and cost-effective method for detecting D-tagatose. Unlike traditional methods like HPLC, which are time-consuming and expensive, this approach allows teams to quickly screen for D-tagatose concentrations with minimal equipment requirements. This rapid detection method can be easily adopted by other teams working on D-tagatose production or similar rare sugars, accelerating their experimental progress and reducing overall costs.
2. Offering a High-Throughput Screening Approach Using Microplate Readers: Our strategy introduces a high-throughput detection concept based on microplate readers, enabling the simultaneous analysis of 96 samples in a very short time. This method not only improves the efficiency of strain screening but also provides a scalable and adaptable framework for detecting various metabolites. Other teams can leverage this microplate-based approach to optimize their metabolic engineering projects, allowing for rapid data collection and better decision-making in pathway optimization and strain selection.

- A bioreactor suitable for high school students -

Our team has designed and manufactured a low-cost, small-scale fermentation device specifically tailored for educational purposes, particularly for high school iGEM teams conducting fermentation experiments. This compact fermentation bioreactor mimics the essential features of a laboratory 5-L bioreactor, including temperature control, stirring, pH adjustment, aeration, and feed addition. Made from readily available materials such as three-neck glass bottles, pH controllers, and PTFE tubes, the device is easy to assemble and operate, with a total manufacturing cost of only 3004 RMB.The device's affordable design meets the needs for D-tagatose production and other microbial fermentation processes. It is compact and portable, making it ideal for small-scale research and educational purposes in high schools, universities, and research institutions. By providing a user-friendly and practical tool for fermentation, the device offers students and junior researchers valuable hands-on experience, fostering talent development in synthetic biology and bioengineering.
Bioreactor design handbook
To enhance our understanding of the fermentation device and assist more high school teams in using this small fermentation device in the future, we have written this installation and user manual for the fermentation bioreactor.

- Conclusion -

This project provides a feasible and innovative solution for the efficient production of D-tagatose through genetic engineering, metabolic optimization, and hardware design. Its contributions are reflected in the advancement of scientific research, the optimization of industrial production, and the support of education and talent development. These contributions are expected to be further expanded and applied in the future.

- Reference -

(1) Wang, J., Li, C., Jiang, T. and Yan, Y., 2023. Biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening for over-production phenotypes. Metabolic engineering, 75, pp.58-67.

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⚪ Overview

⚪ Contribution of New Genetic Parts

⚪ A gene interference method in E.coli

⚪ High throughput and fast method for tagatose assay

⚪ A bioreactor suitable for high school students

⚪ Conclusion

⚪ Reference