In our project, we developed a capsule containing two catalytic enzymes, dextransucrase, and inulosucrase, that are coded by genes EC.2.4.1.5 and EC.2.4.1.9 to catalyze the conversion from sucrose to two types of soluble dietary fibers (SDFs) dextran and inulin respectively as shown in Figure 1. Therefore, individuals may enjoy their diets without change in flavor while reducing the human body's effective absorption of sugars in food and increasing fiber intake. With no change to the sugar content of the food, this initiative intends to promote bowel movement, regulate gut bacteria, and lower the quantity of sugar absorbed by the human body into the bloodstream.

1. New basic part: BBa_K5093000(EC 2.4.1.5)

Profile

Name: EC 2.4.1.5

Base Pairs: 4440 bp

Origin: Leuconostoc citreum [1], synthesised

Properties: EC 2.4.1.5 codes for the enzyme dextransucrase, which transfers a D-glucosyl group from sucrose, a disaccharide, to a growing dextran chain, a fiber, by an alpha (1→6) linkage. The reaction is illustrated in Figure 2.

Figure 1. The gene map of EC 2.4.1.5

 

This new essential part, EC 2.4.1.5, originated in Leuconostoc citreum [1] and is 4440 bp long, as shown in Figure 1. The gene samples we used are artificially synthesized by the biotech company Gene Script. EC 2.4.1.5 codes for the enzyme dextransucrase, which transfers a D-glucosyl group from sucrose, a disaccharide, to a growing dextran chain, a fiber, by an alpha (1→6) linkage. The reaction is illustrated in Figure 2.

 

Figure 2. The formation of dextran with dextransucrase from sucrose [2].

 

Use of Dextransucrase

Sucrose is usually broken down into two monosaccharides, glucose and fructose, which are absorbed and metabolized. However, the protein that the gene codes for converts this disaccharide into 50% fructose and 50% dextran. This is the primary approach to dextran synthesis.

Dextran is a versatile substance. As a soluble dietary fiber, it can be efficiently fermented by the gut microbiota. High diversity of the gut microbiota has been proven to reduce the incidence of many chronic diseases, including inflammatory bowel disease, colorectal cancer, obesity, and T2DM [3]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [4]. Its medical uses include its antithrombotic and anticoagulant activities and its role in maintaining blood osmolarity to replace expensive plasma proteins. Its excellent biological compatibility and degradability have made it a promising drug administration vector. Dextran is also a food additive with great stabilizing, thickening, and emulsifying abilities. In cosmetics, dextran and its derivatives also function as lubricants, humectants, and thickeners in skin and hair care products. Dextran could also facilitate imaging technologies and the manufacture of paper.

The majority of attention on dextransucrase is associated with the functions of dextran. However, as our project aims to synthesize dextran in the human gut, its effect on its substrate, sucrase, is also vital. It reduces the absorption of glucose by adding them to dextran chains, and hence the chance of getting obese or contracting non-communicable diseases such as hyperglycemia, dyslipidemia, diabetes, coronary heart disease, non-alcoholic fatty liver disease, and stroke [5–18], without diminishing the sweetness of food and drink.

 

Obtaining, Amplifying, and Identifying the Gene

The biotechnology company Gene Script synthesizes the DNA molecules containing our desired gene. Then, they are cut with restriction endonucleases NdeI and XhoI and amplified using polymerase chain reaction (PCR). The gene's length is 4400 bp. The bands representing EC 2.4.1.5 successfully appear at their corresponding positions in the gel, as shown in Figure 3, indicating that the cutting and amplifying are successful.

Figure 3. The results of gel electrophoresis of EC 2.4.1.5.

2. New basic part: BBa_K5093001(EC 2.4.1.9)

Profile

Name: EC 2.4.1.9

Base Pairs: 2400 bp

Origin: Lactobacillus johnsonii [1], synthesized

Properties: EC 2.4.1.9 codes for the enzyme inulosucrase, which transfers a fructose group from the disaccharide sucrose to a growing inulin chain, a fiber, to produce glucose and inulin. The reaction is illustrated in Figure 5.

Figure 4. The gene map of EC 2.4.1.9.

 

Figure 5. The formation of inulin with inulosucrase from sucrose [2].

 

Use and Biology

Sucrose is usually broken down into two monosaccharides, glucose and fructose, which are absorbed and metabolized. However, the protein that the gene codes for converts this disaccharide into 50% glucose and 50% dextran.

Inulin is a soluble dietary fiber (SDF) and has been approved by the Food and Drug Administration to facilitate the nutritional values of foods. The gut microbiota can efficiently ferment it. High diversity of the gut microbiota has been proven to reduce the incidence of many chronic diseases, including inflammatory bowel disease, colorectal cancer, obesity, and T2DM [3–5]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [6]. Research has also shown inulin calcium-ion-absorption-facilitating and antioxidant properties [7].

Inulin also has wide applications in pharmacy. Because it cannot be digested or fermented in the initial portion of the human alimentary canal and quickly reaches the distal portion of the colon, inulin can be utilized as a drug carrier, especially for colon diseases. Furthermore, its structure and properties enable it to be a stabilizing agent and cryoprotectant [7].

Most of the attention on inulosucrase is due to the functions of its product, inulin. However, as our project aims to synthesize inulin in the human gut, its effect on its substrate, sucrase, is also vital. It reduces the absorption of fructose by adding it to inulin chains, and hence the chance of getting obese or contracting non-communicable diseases such as hyperglycemia, dyslipidemia, diabetes, coronary heart disease, non-alcoholic fatty liver disease, and stroke [8–21], without diminishing the sweetness of food and drink.

 

Obtaining, Amplifying, and Identifying the Gene

The biotechnology company GeneScript synthesizes the DNA molecules containing our desired gene. Then, they are cut with restriction endonucleases NdeI and XhoI and amplified using polymerase chain reaction (PCR). The gene's length is 2400 bp. The bands representing EC 2.4.1.9 successfully appear at their corresponding positions in the gel, as shown in Figure 6, indicating that the cutting and amplifying are successful.

Figure 6. The results of gel electrophoresis of EC 2.4.1.9.

 

3. New composite part: BBa_K5093002(pET28a-EC.2.4.1.5)

Construction Design:

This engineered plasmid comprises a pET28a backbone (BBa_K3521004) and the gene EC 2.4.1.5 (BBa_K5093000), as shown in Figure 7.

Figure 7. Plasmid maps of pET28a-EC.2.4.1.5

 

Essential parts in the plasmid

EC 2.4.1.5 codes for the enzyme dextransucrase, which transfers a D-glucosyl group from sucrose, a disaccharide, to a growing dextran chain, a fiber, by an alpha (1→6) linkage. The reaction is illustrated in Figure 8.

In our project, this enzyme reduces the absorption of glucose by catalyzing this reaction using sucrose as its substrate, hence lowering the chance of getting obese or contracting non-communicable diseases such as hyperglycemia, dyslipidemia, diabetes, coronary heart disease, non-alcoholic fatty liver disease, and stroke [2–15], without diminishing the sweetness of food and drink.

Its product, dextran, is a soluble dietary fiber that the gut microbiota can efficiently ferment. The high diversity of the gut microbiota has been proven to reduce the incidence of many chronic diseases, including inflammatory bowel disease, colorectal cancer, obesity, and T2DM [16]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [17].

Figure 8. The formation of dextran with dextransucrase from sucrose [18].

 

         T7 Promoter (BBa_K3521002) and T7 Terminator (BBa_K3521002)

The T7 promoter and terminator originated in the T7 bacteriophage. The T7 promoter has a high affinity to its specific polymerase, the T7 RNA polymerase, which has a high transcription rate. When Isopropyl β-D-1-thiogalactopyranoside (IPTG), a lactose-like substance that bacteria cannot metabolize, is added, it binds to the Lac repressor on the operator to cause a conformation change, so the repressor is detached from the operator. T7 RNA polymerase can bind to the T7 promoter to initiate a fast transcription. This is very beneficial in the heterologous expression because the induction by IPTG can endure a high protein production rate. Figure 9 is an illustration of the T7 promoter and terminator.

Figure 9. An illustration of the T7 promoter and terminator.

 

         Plasmid backbone: pET28a (BBa_K3521004)

The pET28a plasmid has other desirable features that make it an ideal vector besides the T7 promoter and terminator it possesses. It has multiple restriction sites for the insertion of a new gene. Meanwhile, it has a sequence coding for a 6×histidine tag between the T7 promoter and terminator, so the protein produced can be purified via nickel column affinity chromatography. Another pronounced feature is the kanamycin resistance gene (KanR). When the target protein is made, this gene on the same plasmid is also expressed so that the bacteria containing the plasmid can survive under kanamycin. This essentially facilitates the preliminary selection of successfully transformed bacteria on kanamycin-containing media because the ones that fail to take in the plasmids are killed and unable to reproduce into clones.

1. Obtaining, Amplifying, and Identifying the Gene

The biotechnology company GeneScript synthesizes the DNA molecules containing our desired gene. Then, they are cut with restriction endonucleases NdeI and XhoI and amplified using polymerase chain reaction (PCR). The gene's length is 4400 bp. The bands representing EC 2.4.1.5 successfully appear at their corresponding positions in the gel, as shown in Figure 10, indicating that the cutting and amplifying are successful.

Figure 10. The results of gel electrophoresis of EC 2.4.1.5.

2. Plasmid Construction and Transformation

NdeI and XhoI are also used to cut pET28a to make complementary sticky ends. EC 2.4.1.5 is connected to the linear plasmids with T4 ligase. Then, the heat shock conversion of the recombinant plasmids is applied to competent E. coli DH5α. We then incubated them overnight at 37°C after streak inoculating them on LB solid medium plates that included appropriate antibiotics (LB-kana), as shown in Figure 11B. Afterward, we picked three colonies from each petri dish and extracted their plasmids. PCR and gel electrophoresis (shown in Figure 11A) were run to confirm the extracted plasmids were the ones we required. EC 2.4.1.5 (4440 bp long) appears at its corresponding position. Then, the pET28a-EC 2.4.1.5 plasmids are sent to Azenta Life Sciences for sequencing, whose results further proved our success in constructing the plasmid (Figure 12).

Figure 11. A: Gel electrophoresis to confirm pET28a-EC 2.4.1.5 plasmids. B: pET28a-EC 2.4.1.5 containing strain clones.

 

Figure 12. The DNA sequencing diagram for pET28a-EC 2.4.1.5.

3. Protein Expression

The verified plasmids were transformed into E.coli BL21(DE3), incubated on LB solid medium plates (Kana+), and cultured at 37°C overnight. Four colonies were selected for PCR amplification to confirm success in the second transformation (Figure 13) and then transferred into 1L fresh LB (Kana+) culture medium for the scale-up cultivation. IPTG (0.2 mM) was used to induce the expression of genes EC.2.4.1.5 with OD600 around 0.6-0.8 and cultured at 16℃ for 20h. The proteins were then extracted from the supernatant of the E.coli BL21(DE3) after ultrasonic cell disruption and centrifugation. The size of dextransucrase is 167 kDa, as confirmed in the SDS-PAGE in Figure 14.

Figure 13. DNA gel electrophoresis of EC 2.4.1.5 (A) and clones of E.coli BL21(DE3) containing the gene (B).

 

4. Protein Purification

Nickel affinity chromatography effectively purifies dextransucrase because the protein contains a 6×his tag. We got a more precise result with little interference by non-specifically bound proteins. Figure 14 shows only one band with a molecular weight of 167 kDa. This demonstrates that dextransucrase is successfully expressed and purified.

Figure 14. SDS-PAGE of dextransucrase from EC 2.4.1.5-containing E.coli BL21(DE3)

 

4. New composite part: BBa_K5093003 (pET28a-EC.2.4.1.9)

Construction Design

This engineered plasmid comprises a pET28a backbone (BBa_K3521004) and the gene EC 2.4.1.9 (BBa_K5093001).

Figure 15. Plasmid maps of pET28a-EC.2.4.1.9.

EC 2.4.1.9 codes for the enzyme inulosucrase, which transfers a fructose group from the disaccharide sucrose to a growing inulin chain, a fiber, to produce glucose and inulin. The reaction is illustrated in Figure 17.

In our project, this enzyme reduces the absorption of glucose by catalyzing this reaction using sucrose as its substrate, hence lowering the chance of getting obese or contracting non-communicable diseases such as hyperglycemia, dyslipidemia, diabetes, coronary heart disease, non-alcoholic fatty liver disease, and stroke [2–15], without diminishing the sweetness of food and drink.

Its product, inulin, is a soluble dietary fiber that the gut microbiota can efficiently ferment. The high diversity of the gut microbiota has been proven to reduce the incidence of many chronic diseases, including inflammatory bowel disease, colorectal cancer, obesity, and T2DM [16–18]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [19].

Figure 16. The gene map of EC 2.4.1.9.

 

Figure 17. The formation of inulin with inulosucrase from sucrose [20].

 

         T7 Promoter (BBa_K3521002) and T7 Terminator (BBa_K3521002)

The T7 promoter and terminator originated in the T7 bacteriophage. The T7 promoter has a high affinity to its specific polymerase, the T7 RNA polymerase, which has a high transcription rate. When Isopropyl β-D-1-thiogalactopyranoside (IPTG), a lactose-like substance that bacteria cannot metabolize, is added, it binds to the Lac repressor on the operator to cause a conformation change, so the repressor is detached from the operator. T7 RNA polymerase can bind to the T7 promoter to initiate a fast transcription. This is very beneficial in the heterologous expression because the induction by IPTG can endure a high protein production rate. Figure 18 is an illustration of the T7 promoter and terminator.

Figure 18. An illustration of the T7 promoter and terminator.

 

         Plasmid backbone: pET28a (BBa_K3521004)

The pET28a plasmid has other desirable features that make it an ideal vector besides the T7 promoter and terminator it possesses. It has multiple restriction sites for the insertion of a new gene. Meanwhile, it has a sequence coding for a 6×histidine tag between the T7 promoter and terminator, so the protein produced can be purified via nickel column affinity chromatography. Another pronounced feature is the kanamycin resistance gene (KanR). When the target protein is made, this gene on the same plasmid is also expressed so that the bacteria containing the plasmid can survive under kanamycin. This essentially facilitates the preliminary selection of successfully transformed bacteria on kanamycin-containing media because the ones that fail to take in the plasmids are killed and unable to reproduce into clones.

1. Obtaining, Amplifying, and Identifying the Gene

The biotechnology company GeneScript synthesizes the DNA molecules containing our desired gene. Then, they are cut with restriction endonucleases NdeI and XhoI and amplified using polymerase chain reaction (PCR). The gene's length is 2400 bp. The bands representing EC 2.4.1.9 successfully appear at their corresponding positions in the gel, as shown in Figure 19, indicating that the cutting and amplifying are successful.

Figure 19. The results of gel electrophoresis of EC 2.4.1.9.

2. Plasmid Construction and Transformation

NdeI and XhoI are also used to cut pET28a to make complementary sticky ends. EC 2.4.1.9 is connected to the linear plasmids with T4 ligase. Then, the heat shock conversion of the recombinant plasmids is applied to competent E. coli DH5α. We then incubated them overnight at 37°C after streak inoculating them on LB solid medium plates that included appropriate antibiotics (LB-kana), as shown in Figure 20B. Afterward, we picked three colonies from each petri dish and extracted their plasmids. PCR and gel electrophoresis (shown in Figure 20A) were run to confirm the extracted plasmids were the ones we required. EC 2.4.1.9 (2400 bp long) appears at its corresponding position. Then, the pET28a-EC 2.4.1.9 plasmids are sent to Azenta Life Sciences for sequencing, whose results further proved our success in constructing the plasmid (Figure 21).

Figure 20. A: Gel electrophoresis to confirm pET28a-EC 2.4.1.9 plasmids. B: pET28a-EC 2.4.1.9 containing strain clones.

 

Figure 21. The DNA sequencing diagram for pET28a-EC 2.4.1.9.

 

3. Protein Expression

The verified plasmids were transformed into E.coli BL21(DE3), incubated on LB solid medium plates (Kana+), and cultured at 37°C overnight. Four colonies were selected for PCR amplification to confirm success in the second transformation (Figure 22) and then transferred into 1L fresh LB (Kana+) culture medium for the scale-up cultivation. IPTG (0.2 mM) was used to induce the expression of genes EC.2.4.1.9 with OD600 around 0.6-0.8 and cultured at 16℃ for 20h. The proteins were then extracted from the supernatant of the E.coli BL21(DE3) after ultrasonic cell disruption and centrifugation. The size of inulosucrase is 88 kDa, as confirmed in the SDS-PAGE in Figure 23.

Figure 22. DNA gel electrophoresis of EC 2.4.1.9 (A) and clones of E.coli BL21(DE3) containing the gene (B).

 

4. Protein Purification

Nickel affinity chromatography effectively purifies inulosucrase because the protein contains a 6×his tag. We got a more precise result with little interference by non-specifically bound proteins. Figure 8 shows only one band with a molecular weight of 88 kDa. This demonstrates that inulosucrase is successfully expressed and purified.

Figure 23. SDS-PAGE of inulosucrase from EC 2.4.1.9-containing E.coli BL21(DE3)

Our enzyme mixture capsules are designed to restrain sugar absorption and increase fiber content without affecting food flavor. It can help people maintain health and good figures while relishing delicious foods for healthy people and those afflicted by high-sugar-related non-communicable diseases or fiber deficiency.

Throughout our product's research and development stages, we respected academic ethics and adhered to accuracy and precision when experimenting and analyzing our data. We strictly followed moral and commercial standards by conducting market research, education programs, and product promotion. Our ultimate goal is to help more people live healthier lives and contribute to the sustainable development of human society.

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