1.  Excess of sugar intake

Carbohydrates are the primary energy source that organisms use to sustain metabolism. Humans can digest and absorb saccharides(including glucose, maltose, fructose, sucrose)and starch,(which can be broken down into maltose, glucose)，etc. However, the excess intake of sugars nowadays due to the greater availability of foods and sweets [1] could not only hinder people from pursuing their fitness goals, but might also lead to numerous noncommunicable diseases (NCDs). The proportion of annual deaths caused by NCDs compared to total deaths is shown in Figure 1.

Figure 1. Causes of annual death [2–4].

 

It has been observed that the increased consumption of sugars, especially from sugar-sweetened beverages (SSBs), can lead to the elevation of blood lipid [5] and glucose [6] concentration (dyslipidemia and hyperglycemia). People with high sugar intake may become obese [7], which could give rise to multiple NCDs [8]. They may suffer from reduced insulin sensitivity [9] and even type 2 diabetes mellitus [10] in a dose-dependent manner [11]. Fats could accumulate, and adipose tissue may increase [12]. Due to increased intake of sugar, if excess lipid deposits in the coronary arteries, coronary heart disease (CHD) [13,14] may occur due to hyperglycemia and insulin resistance [15,16]; if too much fat builds up in the liver, this could incur non-alcoholic fatty liver disease (NAFLD) [12,17]. Also, stroke might develop [18] from fat deposits in brain vessels.

CHD and stroke have a high probability of leading to immediate death. At the same time, less potent conditions or diseases may also burden healthcare systems and individual households considerably. The disability-adjusted life years (DALYs) of other diseases or conditions are shown in Table 1. Regarding financial burdens, the average total costs per year to a patient or household in lower or middle-income countries of cardiovascular diseases and diabetes from 1st January 2000 to 7th May 2020, for example, were $6055.99 and $1017.05 in 2018 USD, respectively [19].

Table 1. The disability-adjusted life years (DALYs) of other diseases or conditions related to excessive sugar intake.

2.  Deficiency of dietary fibers

 Another issue that has drawn our attention is that people with a suboptimal diet are prone to dietary fiber deficiency. Several studies have unraveled the association between a diet low in fiber and the increased burdens of diseases such as diabetes mellitus, stroke, colon and rectum cancer (CRC), and ischemic heart disease (IHD) [24,25]. The age-standardized rate of DALY (per 100000 population) attributable to a diet low in fiber in 2019 was 186.89 [26].

1.  The reason for the design

 We devised a way to simultaneously ameliorate excessive sugar intake and dietary fiber deficiency via synthetic biology: converting sugars to fibers with enzymes produced by engineered strains.

 The first enzyme we produce is dextransucrase, coded by the gene EC.2.4.1.5, which originated in Leuconostoc citreum [27]. Dextransucrase transfers a D-glucosyl residue from sucrose to a glucan chain by an alpha (1→6) linkage. The reaction is shown in Figure 2. Hence, sucrose, which is normally broken down into glucose and fructose, does not produce glucose. Fructose and fiber dextran are produced instead, and glucose absorption declines.

Figure 2. The formation of dextran with dextransucrase from sucrose [28].

 

Similarly, the other enzyme, inulosucrase, catalyzes the reaction from sucrose to glucose and the fiber inulin by the transfer of the fructose group, as shown in Figure 3. The gene coding for the enzyme is EC.2.4.1.9 from Lactobacillus johnsonii [29].

Figure 3. The formation of inulin with inulosucrase from sucrose [30].

 

Both dextran and inulin are soluble dietary fibers (SDFs) 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 [31–33]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [34].

Either of the enzymes converts sucrose into 50% monosaccharides and 50% SDFs. However, combining the two enzymes in an ideal ratio can further reduce the proportion of monosaccharides.

 

We went through several previous studies on the properties and applications of these two enzymes, although not all of them were pertinent to our project.

For example, Yi A-R et al. (2009) found that transformed E. coli containing the dextransucrase (LcDS) gene from Leuconostoc citreum HJ-P4 induced by 0.1 mM IPTG at 15℃ produced over 330 times more enzymes than at 37℃. Nadir Naveed Siddiqui et al. (2013) spotted a 6.75-fold increase in dextransucrase activity when mutagenesis occurred in Leuconostoc mesenteroides KIBGE IB-22 due to UV irradiation compared to the wild type [35]. Nisha and Wamik Azmi (2019) found that dextransucrase had the best activity at pH 5.5 and 37℃ for 30 min in both the free as well as immobilized state, in which the standardized calcium-alginate immobilized enzymes retained 99.5% relative activity [36]. The review by Jürgen Seibel et al. (2010) noted that the initial reaction rates follow Michaelis–Menten kinetics up to 200 mM sucrose as the single substrate. However, the enzyme could be inhibited when the substrate concentration (730 mM for sucrose) was too high. Raising the concentration of acceptors (dextran) could alter Vmax, Km, and KI and, therefore, reverse the inhibition by the substrate [37]. Chen Z. (2023) summarised the mechanisms of the reaction of glucansucrases, the α-retention double displacement involving the three catalytic residues (nucleophiles, acid/base catalysts, and transition state stabilizers). The α1-β2 glycosidic bond of sucrose was first broken by the attack of the nucleophile, forming a β-D glucosyl-enzyme intermediate, which was stabilized. The fructose moiety was protonated by an acid/base catalyst to form fructose. If the glucosyl moiety is transferred to dextran, a receptor reaction could elongate the chain [38].

As for inulosucrase, Olivares-Illana V et al. (2003) revealed its unusual structure-bearing features of both glucosyltransferases (C- and N-terminal region) and fucosyltransferase (catalytic domain). Sequence comparison indicated it was a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase (a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355) by a fructosyltransferase [39]. L.K. Ozimek et al. (2005) discovered the critical structural role of Ca2+ ions in bacterial fructosyltransferase enzymes (including inulosucrase) belonging to glycoside hydrolase family 68 (GH68) as a metal cofactor [40]. Anwar MA et al. (2008) found similar non-Michaelian behaviors in inulosucrase from L. johnsonii, which might be attributable to the oligosaccharides, the initial products of the reaction, as better acceptors than the growing dextran chain [41].

The mainstream method to restrain sugar while maintaining sweetness is using sugar substitutes which are low in calories to replace sucrose and syrup sweeteners. However, the use of sugar substitutes is disputed. It was validated by some research that the elevated intake of sugar substitutes is associated with increased risks of obesity, insulin resistance, glucose intolerance, thrombosis and cancer [42,43]. Sugar substituted could also change the host gut microbiome, leading to decreased satiety and altered glucose homeostasis [44]. The use of sugar substitutes seems to be counterproductive.

Hence, for the same purpose, instead of altering the ingredients of the food or drink, it could be more sensible to cut sugar absorption. Novozymes developed a β-galactosidase solution called Saphera® Fiber that converts lactose into dairy-based fiber during production [45]. Better Juice uses immobilized microorganisms that contain active enzymes to convert sugars to non-digestible fibers or sugars (sucrose to dietary fiber, glucose to gluconic acid, and fructose to sorbitol) [46]. Wyss Institute collaborated with Kraft Heinz to develop their micro-encapsulated enzymes. The plant-originated enzyme converts sugar to fiber. This enzyme was engineered to only be activated by raised pH (when entering the intestines). A special aspect of it which makes it more versatile is that it could be incorporated into food recipes [47].Inspired by those predecessors, we decided to target sucrose only and maximize the effect of converting it into fibers using two enzymes in capsules.

As shown in the technology roadmap (Figure 4), we obtained the sequence information of the genes coding for dextransucrase and inulosucrase from NCBI and subsequently constructed the recombinant plasmids and transformed them into E. coli for heterologous expression through genetic engineering and the verification of enzyme functions.

SDS-PAGE was utilized to identify successful protein expression. Further, the activity of the recombinant enzymes was verified using thin-layer chromatography and quantified by establishing a digestion model. Our desired plasmids are illustrated in Figure 5.

Figure 4. The technology roadmap.

Figure 5. Plasmid maps of pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9.

 

We design to produce capsules that release their contents, dextransucrase, and inulosucrase, in the intestines. Administrated orally, these enzymes could catalyze the conversion from sucrose to soluble dietary fibers, dextran, and inulin. This design kills two birds with one stone. On the one hand, it reduces the absorption of sugars, hence the chance of getting obese or contracting NCDs such as hyperglycemia, dyslipidemia, diabetes, CHD, NAFLD, and stroke, without diminishing the sweetness of food and drink. On the other hand, people may benefit from the capsules as the functioning of the guts is fostered by SDFs since the diversity of gut microbiota is enhanced.

The high acidity of gastric acid will denature the two protein-based enzymes, so the enzymes must be protected from the loss of activity due to low pH before arriving at their sites of action. We adopted the microencapsulation technique for better targeting and smoother content release, where the enzymes are coated in small spheres. The reaction makes the shells of sodium alginate and calcium chloride as calcium ions replace the sodium ions to form high-strength, elasticity, and thermal stability gels resistant to pepsin and hydrochloric acid in the stomach. The enzymes are also added to the reaction mixture to be encapsulated [48].

 

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