Engineering Success

Traditional sugar substitutes, such as artificially synthesized sweeteners and some natural extracts, often have taste defects, health controversies, or functional limitations. For example, aspartame is unstable at high temperatures and unsafe for specific populations; excessive intake of sugar alcohols easily causes gastrointestinal discomfort; and natural products like stevioside often have a distinct aftertaste. Long-term intake of high sugar or inappropriate synthetic sweeteners may increase metabolic burden and even disrupt the balance of intestinal flora. With the global prevalence of obesity and related metabolic diseases, there is an urgent need to develop safer functional sugar substitutes with additional health benefits.

As novel functional oligosaccharides, agarooligosaccharides (AOS) and fructooligosaccharides (FOS) not only possess favorable sweetening properties but also exhibit prebiotic activity—they can promote the proliferation of beneficial intestinal bacteria and demonstrate potential physiological functions such as antioxidant and anti-inflammatory effects. However, traditional production processes are limited by low efficiency, high cost, and uneven product quality. This study aims to construct efficient expression systems using synthetic biology approaches to produce α-agarase (AgaE) and endoinulinase (Inulinase), respectively. These enzymes catalyze the hydrolysis of agar and inulin to generate high-purity AOS and FOS, providing a green and sustainable solution for the development of next-generation prebiotic functional sugar substitutes.^[1-3]

Cycle 1

Design 1

Two prokaryotic expression systems were designed to express α-agarase AgaE (BBa_2552W5YK) from Thalassomonas sp. LD5 and endoinulinase Inulinase (BBa_25KTPH1H) from Aspergillus arachidicola (Fig. 1). To improve the expression efficiency of these two enzymes in E. coli, their native coding sequences were optimized for codon usage—optimization strategies included adapting to E. coli codon preference, reducing rare codons, and enhancing the stability of mRNA secondary structure. The optimized genes were synthesized by Nanjing GenScript Biotechnology Co., Ltd., which also provided the cloning plasmids. Using the laboratory-preserved pET-22b(+) vector as the expression backbone, recombinant plasmids AgaE_pET-22b(+) and Inulinase_pET-22b(+) were constructed separately.

The coding sequences of AgaE and Inulinase were amplified from the cloning plasmids using specific primers, while the pET-22b(+) vector was extracted and linearized. The gene fragments and vector were double-digested with EcoRI and XhoⅠ, then ligated using T4 DNA ligase to successfully construct the two recombinant expression plasmids. Subsequently, the constructed plasmids were transformed into the cloning host strain E. coli TOP10 and the expression host strain E. coli Rosetta, respectively, enabling the production of AgaE and Inulinase using E. coli (Fig. 2).

After induced expression, α-agarase AgaE from Thalassomonas sp. LD5 specifically cleaves the α-1,3-glycosidic bond in agarose (this bond links D-galactose and 3,6-anhydro-α-L-galactose residues), ultimately generating AOS. In contrast, endoinulinase Inulinase from Aspergillus arachidicola hydrolyzes the internal β-2,1-glycosidic bonds in inulin molecules, producing FOS (Fig. 3).

Build 1

1. Plasmid Construction Results

(1) Construction of AgaE Recombinant Plasmid

The AgaE gene fragment was amplified via PCR and identified by agarose gel electrophoresis (Fig. 4A). A clear target band was observed, with a size consistent with expectations (approximately 2.8 kb). The specific band was then purified using a gel extraction kit. The purified AgaE fragment and the empty pET-22b(+) vector were separately subjected to double digestion with EcoRI/XhoⅠ. Results in Figs. 4B and 4E showed that the digestion reactions were completed successfully, yielding linearized fragments of the expected sizes (the digested AgaE fragment was approximately 2.8 kb, and the digested pET-22b(+) vector was approximately 5.4 kb).

The digested and purified AgaE fragment was ligated with the vector fragment using T4 DNA ligase, and the ligation product was transformed into E. coli TOP10 competent cells. Positive clones were screened via colony PCR (Figs. 5A and 5B), and results showed that clones 2, 3, 5, and 6 amplified bands consistent with the size of the target gene. The plasmid AgaE_pET-22b(+) was extracted from positive clones and sequenced. Sequence alignment with the reference sequence confirmed no base mutations or frame shifts, indicating the successful construction of the AgaE recombinant plasmid.

(2) Construction of Inulinase Recombinant Plasmid

Following the same plasmid construction protocol as for AgaE, the Inulinase gene fragment was amplified via PCR. Agarose gel electrophoresis (Fig. 4C) showed a clear target band (approximately 1.5 kb), consistent with expectations. After double digestion (Fig. 4D), linearized Inulinase fragments and vector fragments were obtained. After transforming the ligation product into E. coli TOP10, colony PCR verification (Figs. 5C and 5D) showed that all positive clones amplified the target band. Sequencing results further confirmed no mutations in the Inulinase_pET-22b(+) plasmid, indicating successful construction.

(A) PCR amplification of the AgaE gene fragment. M: DNA marker; 1-3: AgaE PCR products. (B) Enzymatic digestion of the AgaE PCR product. M: DNA marker; 1: Digestion product of the gel-purified AgaE PCR product. (C) PCR amplification of the Inulinase gene fragment. M: DNA marker; 1-2: Inulinase PCR products. (B) Enzymatic digestion of the Inulinas PCR product. M: DNA marker; 1-2: Digestion product of the gel-purified Inulinase PCR product. (E) Enzymatic digestion of the pET-22b (+) vector. M: DNA marker; 1-2: Digestion product of the pET-22b (+) plasmid; 4: Negative control (undigested pET-22b (+) plasmid).

(A) Transformation of AgaE_pET-22b (+) into E. coli TOP10. (B) PCR verification of the cloned strains. M: DNA marker. (C) Transformation of Inulinase_pET-22b (+) into E. coli TOP10. (D) PCR verification of the cloned strains. M: DNA marker.

Test 1

1. Expression Results Of AgaE And Inulinase

(1) AgaE Protein Expression

E. coli Rosetta harboring AgaE_pET-22b(+) was cultured on a large scale in a 200 mL system. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.025 mM, and induction was carried out at 20°C for 18 h (this induction concentration was optimized via pre-experiments to balance expression level and solubility). After ultrasonic disruption of the bacterial cells, SDS-PAGE analysis (Fig. 6A) confirmed that, compared with the uninduced group (Lane 1), the induced group (Lanes 2-3) showed a specific band at approximately 97 KD—consistent with the theoretical molecular weight of AgaE. The target protein existed in both soluble form (Lane 3) and inclusion body form (Lane 4), with quantities sufficient for subsequent enzyme activity detection and AOS preparation. The protein concentration of the crude enzyme solution was determined to be 2.09 mg/mL using the Bradford method, with a total protein yield of 37.62 mg.

(2) Inulinase Protein Expression

E. coli Rosetta harboring Inulinase_pET-22b(+) was cultured on a large scale in a 200 mL system. IPTG was added to a final concentration of 0.5 mM, and induction was performed at 20°C for 18 h. SDS-PAGE results (Fig. 6B) showed that the induced group had a specific band at approximately 55 KD—consistent with the theoretical molecular weight of Inulinase—while no such band was observed in the uninduced group. The target protein existed in both soluble form (Lane 2) and inclusion body form (Lane 3), with the soluble fraction accounting for approximately 55%. The protein concentration of the crude enzyme solution was 3.59 mg/mL as determined by the Bradford method, with a total protein yield of 64.62 mg, which was suitable for subsequent FOS synthesis experiments.

(A) Expression of AgaE_pET-22b(+) protein. M: Protein marker. (B) Expression of Inulinase protein. M: Protein marker.

2. Enzyme Activity Detection Results

To quantitatively evaluate the activities of AgaE and Inulinase, the 3,5-dinitrosalicylic acid (DNS) colorimetric method was used in this study. The principle is as follows: under alkaline conditions, DNS reacts with reducing sugars to form a reddish-brown product with characteristic absorption at 540 nm. The absorbance value is positively correlated with the content of reducing sugars. Since both the hydrolysis products of AgaE (AOS) and Inulinase (FOS) have reducing ends, this method is suitable for the accurate determination of the activities of both enzymes.

(1) AgaE Enzyme Activity Detection

Experimental procedure: 3 mL of crude AgaE enzyme solution (experimental group) or heat-inactivated crude enzyme solution (control group) was mixed with 2 mL of 2% agarose solution, and the reaction was incubated in a constant temperature water bath at 35°C for 30 min (35°C was determined via pre-experiments as the optimal reaction temperature for AgaE). After the reaction, the enzyme was inactivated by heat treatment at 95°C for 10 min. A 200 µL aliquot of the reaction solution was taken, mixed with an equal volume of DNS reagent, and heated at 95°C for 5 min. After cooling to room temperature, the color development was observed and the absorbance at 540 nm was measured.

Results (Fig. 7A) showed that the color of the AgaE experimental group was significantly darker than that of the control group, with an absorbance value 3.2 times higher than that of the control group. This indicated a significant increase in the production of reducing sugars (AOS), confirming that AgaE can effectively degrade agarose and exhibits the expected biocatalytic activity.

(2) Inulinase Enzyme Activity Detection

Experimental procedure: 3 mL of crude Inulinase enzyme solution (experimental group) or heat-inactivated crude enzyme solution (control group) was mixed with 2 mL of 2% inulin solution, and the reaction was incubated in a constant temperature water bath at 55°C for 30 min (55°C is the optimal reaction temperature for Inulinase). The subsequent reaction termination and color development steps were the same as those for AgaE enzyme activity detection.

Results (Fig. 7B) showed that the color of the Inulinase experimental group was significantly darker than that of the control group, with an absorbance value 4.5 times higher than that of the control group. This indicated a large amount of reducing sugars (FOS) produced, confirming that Inulinase can efficiently catalyze the hydrolysis of inulin and that its enzyme activity meets the experimental requirements.

(A) AgaE enzyme activity assay using the DNS method (left: experimental group; right: control group). (B) Inulinase enzyme activity assay using the DNS method (left: experimental group; right: control group).

3. Product Analysis

(1) AOS Product Identification (HPLC Method)

High-performance liquid chromatography (HPLC) was used to analyze the products of agarose hydrolysis by AgaE. A mixed standard of agarobiose (A2), agarotetraose (A4), and agarohexaose (A6) was used as a reference, with retention times of 10.26 min, 18.97 min, and 36.81 min, respectively.

Results (Fig. 8A) showed that the HPLC chromatogram of the AgaE hydrolysis product had significant characteristic peaks at 18.97 min and 36.81 min, which were completely consistent with the retention times of the A4 and A6 standards. No characteristic peak of A2 was observed, indicating that AgaE has product specificity and preferentially produces AOS with a degree of polymerization of 4-6. The control group (heat-inactivated enzyme group) had no such characteristic peaks, confirming that AgaE can directionally generate high-purity A4 and A6.

(2) FOS Product Identification (HPLC Method)

The same HPLC conditions were used to analyze the products of inulin hydrolysis by Inulinase. A mixed standard of 1-kestose (GF2) and nystose (GF3) was used as a reference, with retention times of 23.49 min and 34.88 min, respectively.

Results (Fig. 8B) showed that the HPLC chromatogram of the Inulinase hydrolysis product had obvious new peaks at 23.49 min and 34.88 min, which matched the retention times of the GF2 and GF3 standards. The control group had no characteristic peaks, indicating that Inulinase can specifically produce GF2- and GF3-type FOS, with product purity sufficient for subsequent prebiotic activity verification.

4. Product Activity Verification

Prebiotics are substances that can be selectively utilized by host microorganisms to confer health benefits. In this experiment, the prebiotic activities of FOS and AOS were verified by evaluating their promoting effects on the in vitro growth of Lactobacillus acidophilus and Lactobacillus paracasei LPC100 (two common beneficial intestinal bacteria). Under anaerobic conditions, L. acidophilus and L. paracasei LPC100 can metabolize specific oligosaccharides as carbon sources. The degree of their proliferation was quantified by measuring the absorbance (OD₆₀₀) of the culture medium at a wavelength of 600 nm—an increase in absorbance directly reflects an increase in bacterial biomass, thereby enabling the evaluation of the prebiotic potential of FOS and AOS.

Single colonies were isolated from L. acidophilus tablets and yogurt drinks using the spread plate method. Typical single colonies were picked and inoculated into liquid MRS medium, followed by anaerobic culture at 37°C for 24 h to obtain seed cultures in the logarithmic growth phase. Fresh MRS medium was used, and treatments were divided into the following groups (with 3 replicates per group):

1) Control group: 6 mL MRS medium + 700 μL PBS buffer + 100 μL seed culture;

2) FOS group: 6 mL MRS medium + 600 μL PBS buffer + 100 μL FOS solution (5.41 mg/mL) + 100 μL seed culture;

3) AOS group: 6 mL MRS medium + 100 μL PBS buffer + 600 μL AOS solution (0.81 mg/mL) + 100 μL seed culture;

4) FOS+AOS group: 6 mL MRS medium + 50 μL FOS solution + 300 μL AOS solution + 350 μL PBS buffer + 100 μL seed culture.

All groups were cultured in a shaker at 37°C for 24 h and 48 h, respectively. After culture, 200 μL of the culture medium was transferred to a 96-well plate, and the OD₆₀₀ value was measured using a microplate reader. Uninoculated MRS medium was used as a blank control for zero adjustment.

Data showed that compared with the control group, the OD₆₀₀ values of the FOS group, AOS group, and FOS+AOS group were significantly higher after 24 h and 48 h of culture. This indicated that both FOS and AOS can effectively promote the proliferation of L. acidophilus and L. paracasei LPC100, demonstrating significant prebiotic activity.

Learn 1

This study developed two green preparation strategies for novel functional sugar substitutes based on prokaryotic expression systems: 1) Codon optimization was performed on the AgaE gene from Thalassomonas sp. LD5 and the Inulinase gene from Aspergillus arachidicola, and two recombinant expression plasmids (AgaE_pET-22b(+) and Inulinase_pET-22b(+)) were successfully constructed; 2) Soluble expression of both enzymes was achieved in E. coli Rosetta, with a total protein yield of 37.62 mg for AgaE and 64.62 mg for Inulinase—both meeting the requirements of subsequent experiments; 3) Enzyme activity detection confirmed that AgaE can effectively degrade agarose and Inulinase can efficiently catalyze the hydrolysis of inulin; 4) HPLC identification showed that AgaE directionally produces A4/A6-type AOS and Inulinase specifically produces GF2/GF3-type FOS, with high product purity; 5) Prebiotic activity verification indicated that both AOS and FOS can significantly promote the proliferation of L. acidophilus and L. paracasei LPC100, exhibiting the core characteristics of functional sugar substitutes.

This study provides a new approach for the preparation of prebiotic sugar substitutes using natural polysaccharides (agarose, inulin) as raw materials, and in particular offers a feasible enzymatic solution for the industrial production of AOS. Future work will focus on two directions: first, further optimizing the soluble expression ratio and enzyme activity efficiency of AgaE to reduce the production cost of AOS; second, systematically evaluating the biosafety of AOS and FOS, and determining their safe and effective doses as food additives to promote the translation of functional sugar substitutes from laboratory research to practical applications.

Cycle 2

The second-phase work focuses on the preparation of food-grade functional sugar substitute enzymes (agarase AgaE and inulinase Inulinase), with the core objective of establishing a "full-chain safe and controllable" production system. The specific plan is as follows: Priority will be given to screening strains that meet the GRAS (Generally Recognized as Safe) standard as hosts, such as Lactobacillus plantarum, Lactococcus lactis (lactic acid bacteria), and Kluyveromyces marxianus (yeast). Whole-genome sequencing will be conducted to verify the absence of pathogenic genes and antibiotic resistance genes in these strains. For the construction of food-grade expression plasmids, pNZ8148, pLP252, or pKM1 will be used as the basic backbones. Nutrient-deficient complementary markers (e.g., thyA, URA3) will replace traditional antibiotic resistance markers. Meanwhile, the gene sequences of AgaE and Inulinase will be optimized based on the codon preference of the host strains, and food-grade signal peptides (such as SPusp45 and α-factor signal peptide) will be added to achieve extracellular secretory expression of the enzymes. In subsequent steps, conditions including nisin induction concentration and carbon source ratio will be optimized to improve enzyme yield (with the target of not lower than the yield of the prokaryotic system, and a 20% or more increase in yield for yeast hosts). Simultaneously, the optimal temperature, pH, and product specificity of the enzymes will be verified—ensuring that AgaE directionally produces A4/A6-type AOS (agarooligosaccharides) and Inulinase specifically produces GF2/GF3-type FOS (fructo-oligosaccharides), with a product purity of ≥90%. Finally, the safety of the enzyme preparations will be validated through qPCR (quantitative real-time PCR) for detecting host DNA residues, acute toxicity tests, and allergenicity assessments. Additionally, the adaptability of the enzymes to food processing scenarios will be tested to address the safety risks associated with traditional prokaryotic expression systems.

Due to time constraints, the second-phase work has not yet been initiated; however, its technical route and implementation plan have been systematically designed. In the future, experimental parameters will be further refined (e.g., specific genotyping verification methods for host strains, response surface optimization design for induction conditions). Priority will be given to resolving issues such as vector compatibility and gene insertion efficiency in the construction of food-grade plasmids, and the experimental implementation will be advanced step by step. This will provide continuous technical support for the food-grade industrial application of AOS and FOS.

References

[1] Li, J., Wang, L., & Zhang, M. (2023). Research progress on enzymatic preparation technology and application of fructo-oligosaccharides. Food and Fermentation Industries, 49(12), 312-320.

[2] Xu, Y., Hu, Y., Li, M., et al. (2024). Enzymatically prepared neoagarooligosaccharides improve gut health and function through promoting the production of spermidine by Faecalibacterium in chickens. Science of The Total Environment, 889, 164621.

[3] Xu, L., Liu, L., Zhang, H., et al. (2022). Sustainable bioproduction of natural sugar substitutes: Strategies and challenges. Trends in Food Science & Technology, 129, 512-527.