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To address the issue of adolescent obesity, we identified excessive sugar intake as a major contributing factor. Microbial fermentation for the production of D-tagatose, a low-calorie sugar substitute, offers significant potential for practical application. We selected Escherichia coli as the chassis organism and constructed a biosynthetic pathway for D-tagatose. Our design focuses on reducing production costs and minimizing competition from endogenous metabolic pathways in E. coli to enhance D-tagatose yield.
Figure 1: The pathway for the synthesis of tagatose from glucose in Escherichia coli.

The molecular formula of D-tagatose is C6H12O6, and it is an isomer of D-galactose. It is a rare naturally occurring monosaccharide. Its sweetness is similar to sucrose, but it provides only one-third of the calories, which is why it is considered a low-calorie sweetener.
(1) During heating, D-tagatose undergoes Maillard reactions and caramelization, which enhance flavor, making it suitable for use in bread, sweets, and yogurt.
(2) D-tagatose cannot be metabolized by oral bacteria, effectively reducing acidity levels and minimizing tooth decay. Therefore, it can be added to toothpaste and mouthwash.
(3) Ingesting D-tagatose does not significantly affect blood sugar or insulin levels, making it suitable for incorporation into foods for individuals with type II diabetes.
(4) Upon entering the body, D-tagatose is absorbed in the small intestine, and the remainder is fermented by gut microbiota. The fermentation process stimulates the growth of lactic acid bacteria and Lactobacillus, inhibiting pathogenic bacteria, thus promoting gut health through its prebiotic properties.
Here is a comparison between D-tagatose and other sweeteners:
Table 1: A comparison of the sweetness, price, applications, and potential health hazards of common sugar substitutes in the market.

D-Tagatose is naturally found in heated milk, apples, and pineapples, but its scarcity makes natural extraction highly challenging. Currently, D-tagatose is primarily synthesized through chemical or enzymatic methods. The chemical synthesis method uses galactose as a raw material, which reacts with metal hydrogenation to form a precipitate, later neutralized with acid to yield D-tagatose. However, this approach generates numerous by-products, involves complex separation processes, and incurs high costs. The enzymatic method utilizes expensive substrates such as lactose, galactose, or galactitol, requiring one or more enzyme-catalyzed reactions to produce D-tagatose. This method also suffers from high production costs, low yields, and limitations in scalability for industrial production.
To address these challenges, we identified glucose as a low-cost, readily available substrate and developed a multi-step enzyme-catalyzed biosynthetic pathway for D-tagatose. Additionally, we employed metabolic engineering techniques to modify key genes and regulate competing pathways, aiming to enhance D-tagatose production while reducing costs.
Figure 2: A summary of chemical and biological pathways for the synthesis of tagatose.

In this study, we constructed three key plasmids to enable D-tagatose biosynthesis in E. coli. Since the endogenous pathways in E. coli do not produce fructose 6-phosphate (F6P) and tagatose 6-phosphate (T6P), which are critical intermediates for D-tagatose production, we searched the NCBI database for relevant genes encoding high-activity enzymes. After comparing data, we synthesized genes that encode these key enzymes.
We first designed primers specific to the target gene fragments and amplified them using PCR. These fragments were then inserted into the pYB1c empty vector using the Gibson assembly method. We subsequently transformed E. coli DH5α to facilitate plasmid validation and amplification, resulting in two key plasmids: pYB1c-Gatz-PGP and pYB1c-Gatz-PGP-PGI. This vector incorporates a high-copy replication origin (P15A_ORI) and a PBAD promoter, which promotes efficient and rapid expression of the target genes while minimizing the formation of inclusion bodies. Additionally, the PBAD promoter mitigates the toxic effects commonly associated with conventional inducers, significantly enhancing the strain's efficiency.
For the CRISPRi operation, we targeted the pfkA and zwf genes in E. coli's competing metabolic pathways and constructed sgRNA-based plasmids. Given the size discrepancy between the gene and vector fragments, and because only the sgRNA needed to be replaced or added, we employed the Golden Gate assembly method to ensure accurate plasmid construction and high positive rates. We designed specific primers for the target fragments, amplified them by PCR, and incorporated BsaI recognition and cleavage sites flanking the sgRNA to ensure the correct order of fragment assembly. The plasmids were then chemically transformed into E. coli DH5α, facilitating subsequent validation and amplification, leading to the successful construction of the key plasmid R6k-dcas9-pfkA-zwf-M.
Table 2: All plasmids constructed during the experimental process.

Utilizing the CRISPR-Cas9 system, we mutated the Cas9 protein to remove its cleavage ability while preserving its capacity to target specific DNA sequences through the guidance of sgRNA sequences. The resulting dCas9 protein binds to the target DNA sequence within the genome, creating steric hindrance that prevents RNA polymerase from binding to the promoter of the target gene, thereby inhibiting transcription and effectively suppressing gene expression.
To further refine this system, we introduced mutations at the 7th and 8th base pairs of the sgRNA spacer region using degenerate primers to create mismatched sgRNAs. These mismatches altered the binding efficiency of dCas9 to the target gene, allowing for varying levels of inhibition. This approach enables a more balanced trade-off between bacterial growth and increased product yield.
To assess the effectiveness of this system, we targeted sgRNA for the egfp gene and introduced mutations at the 7th and 8th base pairs. We tested the inhibition efficiency of 16 different sgRNA mutants and observed a range of inhibition levels from 14.6% to 95.6%. Thus, by introducing specific mutations at these positions, our system can achieve a controlled spectrum of gene inhibition, providing a flexible tool for optimizing metabolic pathways.

Figure 3: The principle of CRISPRi technology.
Figure 4: Functional validation of CRISPRi technology.

(1) Guerrero-Wyss, M., Durán Agüero, S. and Angarita Dávila, L., 2018. D‐Tagatose is a promising sweetener to control glycaemia: A new functional food. BioMed Research International, 2018(1), p.8718053.
(2) Tandel, K.R., 2011. Sugar substitutes: Health controversy over perceived benefits. Journal of Pharmacology and Pharmacotherapeutics, 2(4), pp.236-243.
(3) Oh, D.K., 2007. Tagatose: properties, applications, and biotechnological processes. Applied Microbiology and Biotechnology, 76, pp.1-8.
(4) Miao, P., Wang, Q., Ren, K., Zhang, Z., Xu, T., Xu, M., Zhang, X. and Rao, Z., 2023. Advances and Prospects of d-Tagatose Production Based on a Biocatalytic Isomerization Pathway. Catalysts, 13(11), p.1437.
(5) Bilal, M., Iqbal, H.M., Hu, H., Wang, W. and Zhang, X., 2018. Metabolic engineering pathways for rare sugars biosynthesis, physiological functionalities, and applications—a review. Critical reviews in food science and nutrition, 58(16), pp.2768-2778.
(6) Li, J., Li, H., Liu, H. and Luo, Y., 2023. Recent Advances in the Biosynthesis of Natural Sugar Substitutes in Yeast. Journal of Fungi, 9(9), p.907.
(7) 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.