Background & Inspiration

Breast milk oligosaccharides (HMO) are very important to the health of infants, among which 3'- sialyllactose (3'-SL) has attracted much attention because of its various benefits in promoting brain development and enhancing immunity. With the global demand for high-quality infant formula increasing, the market demand for 3'-SL is expected to rise sharply.Breast milk oligosaccharide (HMO) is the third richest solid component in breast milk after lactose and lipid in mature breast milk, and its content is about 5 to 15 g/L[1].

In recent years, it has been widely concerned because of its impact on human health and its application in high-quality infant formulas. At present, more than 200 HMOs with different structures have been identified, including fucosylation, non-fucosylation and sialylation[2]. Among them, sialylated HMO accounts for 12-14%, and 3'- sialyllactose (3'-SL) is its representative monomer. Its structure is Neu5Ac-α2,3Gal-β1,4Glc, which is composed of N- acetylneuraminic acid and lactose units[3].

Studies have shown that 3'-SL has prebiotic activity, which can play a role by inhibiting pathogen adhesion, preventing necrotizing enterocolitis, enhancing immune function and promoting brain development[4]. The European Union has approved 3'-SL sodium salt as a new resource of infant formula (EU 2023/1582), and it has been added to Nestle BEBA formula at a dosage of 1-5 mg/100 mL[1]. The global HMO market has grown rapidly from $200 million in 2022 and is expected to reach $780 million in 2028 [4]. As the main component of HMO, the application demand of 3'-SL will continue to be released with market expansion.

In view of its diversified and valuable applications, the demand for 3'-SL will continue to increase. However, the traditional extraction and chemical synthesis methods are inefficient and cannot meet the rapidly growing market demand. Therefore, sustainable biotechnology methods have attracted more and more research attention.

It is worth noting that fermentation medium may account for 30% of the total production cost of commercial chemicals, which urges people to find economic alternatives[5]. soy whey is a by-product of soybean food production process, which produces as much as 70 million tons in China every year[6].

However, more than 80% of carbohydrates, 20% of protein and 20% of oil in soybeans will be lost to soy whey, resulting in soy whey containing nearly 1.5% of carbohydrate, 0.1-0.8% of protein and about 0.4% of trace elements[7]. Therefore, soy whey is a nutrient-rich culture condition, which is likely to be used as a culture medium for our biosynthesis. However, most of the soy whey is discharged into sewage, which not only causes serious environmental pollution, but also leads to the waste of its nutrients.

Our research is based on the above background. First, using soy whey as the culture medium for our biosynthesis can not only save costs, but also control water pollution. Second, we synthesized 3'-SL with high value by microorganisms on the premise of using soy whey as culture medium, which has high economic benefits and market value.

Target

Based on synthetic biology principles, this project aims to construct an E. coli that uses low-cost soy whey as a fermentation medium and leverages the natural galactose in soy whey to achieve an auto-induction expression system. The experiment is mainly divided into the following three parts. First of all, at present, E.coli can't use carbohydrates (such as sucrose and stachyose) in soy whey as carbon sources, which makes it difficult to use this rich food industry by-product as fermentation medium. Therefore, we first need to genetically modify E.coli so that it can survive normally in soy whey. Secondly, Inducible expression system depends on T7-lac promoter (T7 promoter with lac longitudinal), which is one of the most commonly used expression systems in microbial cell factories, and IPTG is the most effective inducer to trigger gene expression. However, the potential cytotoxicity and high cost of IPTG hinder the economic feasibility of this system. Therefore, we want to construct a self-induced protein expression system. Based on this system, bacteria can directly use galactose in the culture medium as an inducer, thus spontaneously inducing the expression of downstream proteins. Finally, we need to transfer the synthesis module of 3'-SL into plasmid, so that bacteria can synthesize 3'-SL protein in soy whey.

Design

Preliminary preparation and safety training: Conduct laboratory safety training to ensure all operators master standard operating procedures; learn basic experimental skills such as plasmid construction, PCR, electrophoresis, protein expression, and identification.

Based on the background, our experiment is divided into three modules.

1. Module construction for carbohydrate utilization in soy whey: Target non-fermentable sugars (stachyose, raffinose) in soy whey; construct a carbohydrate utilization module by introducing key genes (e.g., SACC, AGA). Aga and Sacc can convert carbon sources that E.coli can't use into available carbon sources (such as glucose, fructose and galactose) [8].

2. Construction of galactose-based auto-induction module:

In our experiment, galactose is not only an inducer of downstream protein expression, but also an important nutrient for cell growth. Therefore, how to improve the inducible ability of downstream proteins as much as possible under the premise of maintaining cell growth is the key to improving protein yield. Therefore, we constructed three PgalP variants in the promoter stage: PgalP-a, PgalP-b and PgalP-c, and introduced the green fluorescent protein EGFP downstream of the promoter to characterize the induction intensity of galactose on the promoter. Through the preliminary analysis of fluorescence intensity, we can know which promoter has the strongest ability to induce downstream protein expression under galactose induction. After screening the optimal promoter, we replaced EGFP gene with galP gene, so that galactose could be transported as much as possible to induce the expression of downstream proteins, and further improve the expression level of proteins.

3. Assembly of 3'-SL synthesis module into auto-induction system:

CMP-Neu5Ac Synthase (CSS) and α2,3- sialyltransferase (α2,3-SiaT) are the key enzymes for 3'-SL synthesis. CSS catalyzes the formation of CMP-Neu5Ac, and free N- acetylneuraminic acid (Neu5Ac) combines with CTP (cytidine triphosphate) under the action of CSS to form CMP-Neu5Ac. As an activated form of sialic acid, CMP-Neu5Ac is the donor of subsequent sialylation reaction. α2,3-SiaT is responsible for transferring Neu5Ac in CMP-Neu5Ac to galactose residue of acceptor lactose to form 3'-SL.

Goal

Our experiment is expected to use metabolic engineering and synthetic biology strategies to construct an Escherichia coli which can use cheap soy whey as culture medium and galactose in soy whey to realize self-induced expression system for efficient and economical production of 3'-SL. On the one hand, our strain can survive 3'-SL efficiently; on the other hand, we can also treat wastewater pollution and improve the environment.

References

[1] HU M, ZHANG T 2023. Expectations for employing Escherichia coli BL21 (DE3) in the synthesis of human milk oligosaccharides. Journal of agricultural and food chemistry [J], 71: 6211-6212.

[2] LEE S Y, STUCKEY D C 2022. Separation and biosynthesis of value-added compounds from food-processing wastewater: Towards sustainable wastewater resource recovery. Journal of Cleaner Production [J], 357: 131975.

[3] ZHANG J, ZHU Y, ZHANG W, et al. 2022. Efficient production of a functional human milk oligosaccharide 3′-sialyllactose in genetically engineered Escherichia coli. Acs synthetic biology [J], 11: 2837-2845.

[4] ZHU Y, ZHANG J, ZHANG W, et al. 2023. Recent progress on health effects and biosynthesis of two key sialylated human milk oligosaccharides, 3′-sialyllactose and 6′-sialyllactose. Biotechnology advances [J], 62: 108058.

[5] WANG Z, DAI Y, AZI F, et al. 2024. Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system. Bioresource technology [J], 393: 130145.

[6] LIANG J, XU N, NEDELE A-K, et al. 2023. Upcycling of Soy Whey with ischnoderma benzoinum toward production of bioflavors and mycoprotein. Journal of agricultural and food chemistry [J], 71: 9070-9079.

[7] LEE S Y, STUCKEY D C 2022. Separation and biosynthesis of value-added compounds from food-processing wastewater: Towards sustainable wastewater resource recovery. Journal of Cleaner Production [J], 357: 131975.

[8] WANG Z, DAI Y, AZI F, et al. 2024. Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system. Bioresource technology [J], 393: 130145

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