Excessive sugar intake poses risks to people's health. The World Health Organization (WHO) recommends reducing the intake of free sugars to less than 10% of total energy intake at different life stages to mitigate risks such as unhealthy weight gain and dental caries[1]. Free sugars encompass all sugars added to foods and beverages, as well as those naturally present in honey, syrups, fruit juices, and fruit juice concentrates. Additionally, the WHO considers further reducing free sugar intake to below 5% of total energy intake a reasonable long-term health policy goal. However, adhering to this threshold remains challenging, as many convenience foods, condiments, and beverages contain high levels of added sugars[2]. The discovery and proper use of sweeteners help reduce sugar content in food. However, as research progresses, an increasing number of sweeteners are being applied, but while people focus on "sugar control," they often overlook other health impacts. For example, some sweeteners may decompose or change color under high temperatures and can undergo the Maillard reaction when in the presence of amino acids[3]. These potential risks are often ignored. Therefore, it is particularly important to choose safer and more stable sweeteners.

Currently, there are many sweeteners on the market, each with its own advantages and drawbacks. Erythritol, a natural sweetener, has unique physicochemical properties and does not present the aforementioned risks[4]. It also boasts low caloric value, high stability, and does not cause digestive discomfort[4-5]. Especially as "sugar control" trends target younger age groups, many sweeteners have the potential to cause tooth decay[5]. However, erythritol is particularly effective in preventing cavities. Unlike sucrose, erythritol does not undergo fermentation by oral bacteria to produce acid, which is why it does not contribute to tooth decay. Therefore, erythritol is a healthier option as a sweetener[6]. Sugar substitutes (sweeteners) are a type of food additive that can provide sweetness with lower or no calories. They are widely used in foods, beverages, and medicines. Common sugar substitutes include erythritol, aspartame, sucralose, stevia, and xylitol. There are notable differences between these substitutes[7].

Erythritol is a natural sugar alcohol, with a sweetness level about 60%-70% of that of sucrose. It has an extremely low calorie content, only 0.4 kcal/g, and is almost not metabolized by the body, so it has minimal impact on blood sugar and insulin levels[8]. Additionally, erythritol does not cause tooth decay, is well-tolerated, and is less likely to cause digestive discomfort. However, it has a relatively low sweetness, so it is often used in combination with other high-sweetness substitutes (like stevia). Excessive intake may cause mild digestive issues, such as bloating[8]. Aspartame is much sweeter, about 200 times sweeter than sucrose, and has very low calories, making it popular in sugar-free beverages and low-calorie foods. However, it is unstable at high temperatures, so it is not suitable for baking. Some people may also be sensitive to it, and although the FDA considers it safe, some studies have raised concerns about its long-term safety[9].. Sucralose is even sweeter, about 600 times sweeter than sucrose, and has zero calories. It is stable and suitable for high-temperature processing. However, some studies suggest it might affect the gut microbiota, and its long-term safety is still under debatey[10]. Stevia is a natural sweetener with a high sweetness level, around 200-300 times that of sucrose, and has zero calories. It does not affect blood sugar levels. However, some stevia products may have a bitter or metallic taste, so it is often blended with other sweeteners to improve flavor[11]. Xylitol has a sweetness similar to sucrose, with a lower calorie content (2.4 kcal/g), and is tooth-friendly, making it commonly used in gum and oral care products. However, excessive consumption can cause digestive issues, and it is toxic to dogs[12]. The advantage of glucose as a carbon source is that it does not require the use of additional ATP and can rapidly produce GA-3-P by glycolysis, which enters the PPP pathway directly with high metabolic efficiency. The disadvantage is that the cost is high, and large-scale production of dextrose is limited by economic constraints. And glucose as the main food raw material resource constraints, will cause resource allocation controversy.

The advantage of glycerol as a carbon source is that it is less expensive, and a price of one-third or one-half that of glucose can reduce production costs. Moreover, glycerol is widely available and in stable supply. The disadvantage is that the metabolic pathway is complicated, and it needs to be converted to DHAP by GUT1 and GUT2, which consumes 1 molecule of ATP and may affect the cellular distribution. And the actual glycerol yield fluctuates greatly, and the result may be lower than the theoretical value.[13] Erythritol is produced through fermentation, utilizing glucose or glycerol as substrates, and is synthesized via the pentose phosphate pathway (PPP) in yeast. One of its key advantages is that it is a natural and environmentally friendly sweetener, as its production does not rely on chemical synthesis. Additionally, erythritol is highly tolerable by the human body, with few by-products produced during its fermentation. However, its production comes with some disadvantages, such as the need for high-purity carbon sources, which contributes to higher production costs, as well as significant energy consumption during the fermentation process.[14][15]Mogroside is produced through a plant extraction method, where the mogroside-rich fruit is crushed, and the active compounds are extracted using water or alcohol. The extract is then purified through column chromatography. One of the main advantages of mogroside is that it is derived from natural ingredients, which leads to high consumer acceptance. However, the production process has certain drawbacks, such as low purity in the initial extract, which requires multiple rounds of purification. Additionally, its production is dependent on raw materials, and the long cultivation period of Luo Han Guo results in an unstable supply of the material.[16][17]Steviol glycosides are produced through plant extraction and enzyme modification processes. One of the key advantages of steviol glycosides is their high sweetness with low energy consumption, making them an attractive alternative to sugar. However, there are some disadvantages, such as an undesirable taste and a bitter aftertaste, which can affect the overall sensory experience. Additionally, the availability of raw materials for steviol glycosides is limited, which poses a challenge for consistent production.[18][19].The comparison of heat, advantages and defects of various sugar substitutes is shown in Table 1.The production of erythritol mostly relies on a single pathway, usually using glucose as a substrate and synthesizing erythritol through the fermentation of specific microorganisms. However, this single - pathway approach has some limitations.

Table 1. The comparison of heat, advantages and defects of various sugar substitutes.

Erythritol stands out for its safety, tolerance, and health benefits, particularly for people with diabetes or those focused on healthy living. However, due to its lower sweetness, it is typically combined with other sweeteners. Erythritol (C₄H₁₀O₄) is a natural low-calorie sugar substitute. It appears as a white crystalline powder with a sweetness level of about 60%-70% of sucrose and has only 0.2 kcal/g[8]. It is highly soluble in water, slightly soluble in ethanol, and naturally found in fruits (such as grapes and pears) and fermented foods (like soy sauce and wine)[8]. It can also be industrially produced through glucose fermentation. Erythritol’s key features include low calorie content, minimal metabolism in the body (90% is excreted via urine, and 10% through the digestive system), blood sugar friendliness (suitable for diabetics), good tolerance (rarely causing digestive issues), and high stability (suitable for baking and cooking)[21].

In recent years, erythritol has rapidly grown in the global market as a natural, low-calorie sweetener, driven by the trend toward healthier eating and the rising prevalence of diabetes. According to a report by Grand View Research, the global erythritol market was valued at approximately $250 million in 2022, with an estimated compound annual growth rate (CAGR) of 8.5% from 2023 to 2030, reaching a market size of $520 million by 2030. The Asia-Pacific region, especially China and India, is the main production and consumption market for erythritol[22]. Erythritol is widely used in the food and beverage industry, including in sugar-free drinks, baked goods, candies, dairy products, and health supplements[23].

Production methods for erythritol include chemical synthesis, microbial fermentation, and bioextraction.Chemical synthesis is a multi-step process where starch is first oxidized via periodate oxidation to produce dialdehyde starch, which is then hydrogenated and cleaved to yield erythritol along with other derivatives. This method requires harsh reaction conditions, including high pressure (20 MPa) and elevated temperatures (125–200°C). Due to its high production costs, energy-intensive nature, and inferior product quality, chemical synthesis has not been widely adopted in industry[24]. In addition, certain lactic acid bacteria can also produce erythritol.The primary substrate for large-scale erythritol production is glucose. The separation and purification of erythritol are critical steps in its manufacturing process. Recovering erythritol from the fermentation broth typically involves its separation from microbial cells, followed by preferred methods such as ion-exchange chromatography and crystallization. The erythritol fractions obtained from chromatographic separation may also undergo activated carbon treatment for further purification.However, compared to the fermentation production of other sugar alcohols, microbial fermentation of erythritol still faces challenges such as low yield and suboptimal conversion rates. Therefore, further research into optimizing the bioproduction of erythritol remains highly important[25].

By the passage of “Advances in efficient biosynthesis of erythritol by metabolic engineering of Yarrowia lipolytica”, we can see that when glucose is used as a carbon source to convert erythritol, it has a conversion rate of 67.7%. When glycerol is used as a carbon source the conversion rate is 66.3%[25]. Currently, glycerol and glucose are the main substrates for the synthesis of erythritol. The advantages and disadvantages are as follows:  In regions that are not starch - producing areas, using sucrose as a raw material for erythritol production has a significantly greater competitive advantage than using glucose.[26] When glucose is used as the carbon source, the yield of erythritol is relatively high, but there are more by - products. When glycerol is used as the substrate, although the yield is slightly lower, there are fewer by - products, resulting in a higher ratio of erythritol to by - products.
Moreover, the production of erythritol using glucose and glycerol as substrates not only incurs higher costs but also goes against the concept of green and sustainable development in China [27]. We can utilize the ability of dual - substrate utilization, compare the impacts of the two substrates on product production along with detailed data, and specifically analyze the substrate that the strain prefers and is more suitable for the metabolic production of erythritol, in order to achieve more efficient production and higher - quality products [28]. Expanding the scope of substrate utilization can not only reduce the generation of by - products and enable the resource utilization of the by - products generated during erythritol production[29]. With the reduction of by-products, a series of experiments will also be reduced, saving experimental consumables. It can also broaden the utilization of low - value renewable resources, representing a green and safe production method.

Therefore, we enhanced and integrated the erythritol biosynthesis pathway in bacteria through molecular biology techniques, we not only utilize glucose as a substrate but also incorporate the glycerol metabolic pathway. This approach reduces by-product formation, expands the utilization of low-cost substrates (glycerol), and redirects metabolic flux toward erythritol production. Specifically, we engineered two distinct biosynthetic pathways for erythritol synthesis in Escherichia coli (Figure 1).

Pathway 1 - Metabolic pathway of erythritol synthesis with glucose as substrate[30]：

Step 1: Glucose Phosphorylation

The enzyme hexokinase catalyzes the phosphorylation of glucose at the C6 position, forming glucose-6-phosphate(G-6-P).

Step 2: Isomerization to Fructose 6-phosphate

Glucose-6-phosphate is isomerized to fructose-6-phosphate(F-6-P).

Step 3: Phosphoketolase Cleavage

Fructose-6-phosphate is cleaved by phosphoketolase (PK) to produce acetyl-phosphate and erythrose-4-phosphate (Erythrose-4-p).

Step 4: Reduction to Erythritol-4-phosphate

Erythrose-4-phosphate is converted to erythritol-4-phosphate by erythritol-4-phosphate dehydrogenase(EPDH).

Step 5: Dephosphorylation to Erythritol

The phosphate group is removed from erythritol-4-phosphate by phosphatase(PTase), yielding the final product erythritol.

Pathway 2-Metabolic pathway for the synthesis of erythritol with glycerol as substrate[31]：

Step 1: Glycerol Phosphorylation

Glycerol is converted to glycerol 3-phosphate by glycerol kinase (GUT1), consuming 1 molecule of ATP.

Step 2: Dehydrogenation to Dihydroxyacetone Phosphate

Glycerol 3-phosphate is oxidized to dihydroxyacetone phosphate (DHAP) by glycerol-3-phosphate dehydrogenase (GUT2), with the concomitant reduction of 1 NAD⁺ to NADH.

Step 3: The enzyme triosephosphate isomerase (TPI1) catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate ( Glyceraldehyde-3-P). Part of GA-3-P flows into the tricarboxylic acid cycle ( TCA cycle) through pyruvate, providing raw materials, energy and cofactors for various life activities of cells. Another part of GA-3-P produced fructose-6-phosphate (F-6-P) under gluconeogenesis and entered the pentose phosphate pathway ( PPP ). The gluconeogenesis pathway includes the intermediates F-6-P and GA-3-P produced by the PPP pathway, which flow to the synthesis of erythritol in pathway 1.

Figure 1.Synthesis pathway of erythritol

(Note： PK: phosphoketolase；EPDH: erythritol-4-phosphate dehydrogenase; PTase: phosphatase; GUT1: Encoding glycerol kinase; GUT2: Encoding glycerol-3-phosphate dehydrogenase; TPI1: Encoding triosephosphate isomerase)

This project addresses this situation by strengthening and integrating the production pathway of erythritol in bacteria through molecular biology techniques. Additionally, to broaden the utilization of glycerol, the metabolic pathway of glycerol has been added and directed towards erythritol production. Glycerol, a low-cost and abundant renewable resource, has attracted significant attention from researchers for its use as a substrate in erythritol fermentation production in recent years. Thus, we selected three genes for erythritol production using glucose as a substrate: (Phosphoketolase gene PK, 4-phosphate-erythritol dehydrogenase gene EPDH, and phosphatase gene PTase). Additionally, we constructed a glycerol utilization pathway containing three genes: (Glycerol kinase GUT1, glycerol-3-phosphate dehydrogenase GUT2, and triosephosphate isomerase TPI1). After amplifying these genes using PCR, they were connected to the co-expression vectors Pet- Duet and PCDF-Duet through homologous recombination, resulting in plasmids PCDF-Duet-PK-EPDH-PTase and Pet- Duet- GUT1- GUT2—TPL1. Then we transformed the constructed plasmid into E.coli for monoclonal verification and sequencing. Meanwhile, the three-dimensional structure of the protein was predicted using SWISS-MODEL and AlphaFold2. The protein was purified and identified by SDS-PAGE and Western blot (WB).

Finally, after co-transformation with the dual plasmids, fermentation was carried out. We set up different ratios of glycerol and glucose as substrates for fermentation cultures: glucose-only complete medium, glucose:glycerol at 2:1, glucose:glycerol at 1:2, glucose:glycerol at 1:1, and glycerol-only complete medium. We measured the growth curves to examine whether the metabolic pathways affected bacterial growth. In addition, we determined erythritol content using HPLC and calculated the conversion rate (Figure. 2)

Figure 2. Technical Roadmap

The form of our product is erythritol. There are two innovations in our product. Firstly, our product adds the glycerol metabolic pathway, this way, cheap and low-purity glycerol can be used as the main carbon source. Besides fewer by-products are produced in the production process, so this is a green and safe production method. Secondly, the product has a high conversion rate and a high yield.

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