Overview

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.

During our experiment, we added some new parts for iGEM part and new information to an existing part (Table 1).

Table 1. Part contributions

Part contribution

1    Add new basic part

1.1        SacC，BBa_25V1IYRD

Name: SacC

Base Pairs: 1509 bp

Origin: AAU37516.1 from Mannheimia succiniciproducens

Usage and Biology

SacC is an extracellular enzyme that primarily catalyzes the hydrolysis of large fructan polymers (such as Levan and Inulin) into fructose molecules[9].

1.2        Aga，BBa_25MWK240

Name: Aga

Base Pairs: 2193 bp

Origin: Bacillus coagulans MA-13, WP_133537615

Usage and Biology

Alpha-galactosidase is a glycoside hydrolase that specifically catalyzes the hydrolysis of α-galactosidic bonds. It cleaves the terminal α-1,6-linked galactose residues from oligosaccharides and polysaccharides, yielding galactose and the corresponding substrate[10].

1.3        PgalP-a，BBa_25K4V860

Name: PgalP-a

Base Pairs: 179 bp

Origin: E.coli

Usage and Biology

Mutants of PgalP were engineered to improve the initial expression level of GalP, thereby enhancing the intracellular availability of galactose[11].

1.4        PgalP-b，BBa_255LQDVV

Name: PgalP-b

Base Pairs: 84 bp

Origin: E.coli

Usage and Biology

Mutants of PgalP were engineered to improve the initial expression level of GalP, thereby enhancing the intracellular availability of galactose [11].

1.5        PgalP-c，BBa_25ZG3I79

Name: PgalP-c

Base Pairs: 38 bp

Origin: E.coli

Usage and Biology

Mutants of PgalP were engineered to improve the initial expression level of GalP, thereby enhancing the intracellular availability of galactose [11].

1.6        galP，BBa_254XGI7O

Name: galP

Base Pairs: 1395 bp

Origin: Escherichia coli str. K-12 substr. MG1655;NC_000913.3 (3088284..3089678)

Usage and Biology

Mutants of PgalP were engineered to improve the initial expression level of GalP, thereby enhancing the intracellular availability of galactose [11].

1.7        CSS，BBa_250SNH60

Name: CSS

Base Pairs: 666 bp

Origin: WP_218994138.1; Campylobacter jejuni

Usage and Biology

CSS catalyzes the formation of CMP-Neu5Ac. Under the action of CSS, free N-acetylneuraminic acid (Neu5Ac) combines with cytidine triphosphate (CTP) to form CMP-Neu5Ac, simultaneously releasing pyrophosphate (PPi) [11].

1.8        α2,3-SiaT，BBa_25JU2UK2

Name: α2,3-SiaT

Origin: P. multocida Pm70;AAK02272.1

Usage and Biology

α2,3-SiaT is responsible for transferring the Neu5Ac moiety from CMP-Neu5Ac to the galactose residue of lactose, forming an α2,3-glycosidic bond to produce 3'-SL [11].

Figure 1 Target gene

2     Add new Composite Part

Name: pCOM4-PgalP-a-EGFP、pCOM4-PgalP-b-EGFP、pCOM4-PgalP-c-EGFP、pCOM4-PgalP-GalP

Construction Design：

At present, our bacteria need to use exogenous IPTG for protein induction, IPTG has potential cytotoxicity and high cost, so we wonder if we can build a module. After the module is introduced, bacteria can spontaneously induce protein expression by using galactose in soy whey. On the one hand, this can reduce the cost, on the other hand, it can avoid introducing chemical components that may have side effects. Galactose can not only be used as a nutrient for normal cell growth, but also as an inducer to induce protein expression, so how to induce protein expression as much as possible on the basis of maintaining bacterial growth is extremely critical. In order to improve the induced yield of protein with galactose as inducer, we constructed three promoters regulated by galactose, and constructed EGFP fluorescent protein downstream of the promoters. By observing the expression intensity of fluorescent protein, we can quickly obtain the induced yield of protein. Through screening, we can find the promoter with the strongest inducing ability when galactose is used as a protein inducer. After selecting the most suitable promoter, we further want to replace EGFP gene with galactose transporter. After adding galactose inducer, the promoter is regulated by galactose, which strongly induces the expression of galactose transporter downstream. Galactose transporter transports galactose to 3'-SL module and activates the synthesis of 3'-SL.

Figure 2 The plasmid maps. A. pCOM4-PgalP-a-EGFP.B. pCOM4-PgalP-b-EGFP.

C. pCOM4-PgalP-c-EGFP.D. pCOM4-PgalP-GalP.

 

 

 

Experimental Approach

Plasmid transformation and verification

In order to obtain E.coli with different promoters, we obtained DNA fragments of different promoters by PCR and connected them with plasmid vectors by homologous recombination. After obtaining the complete plasmid, we transformed the plasmid into E.coli, and carried out preliminary screening by antibiotics (Figure 3A). Subsequently, we selected monoclonal colonies from the plate and verified them by PCR (Figure 3B). After the PCR verification was successful, we further confirmed that the strain construction was successful through DNA sequencing (Figure 3C).

Figure 3 Construction and Verification of Promoter Screening Module.

A.    Antibiotic screening plate. B. Agarose gel electrophoresis verification. C. The results of DNA sequencing verification.

After selecting the most suitable promoter, we further want to replace EGFP gene with galactose transporter. After adding galactose inducer, the promoter is regulated by galactose, which strongly induces the expression of galactose transporter downstream. Galactose transporter transports galactose to 3'-SL module and activates the synthesis of 3'-SL. Therefore, we first replaced EGFP gene with galactose transporter GALP by molecular biology. Through antibiotic screening (Figure 4A), colony PCR verification (Figure 4B), and DNA sequencing (Figure 4C), we confirmed that the plasmid construction was correct.

Figure 4 Construction of pCOM4-PgalP-GalP. A. Antibiotic screening plate. B. Agarose gel electrophoresis verification. C. The results of DNA sequencing verification.

Protein expression

After confirming the success of the strain construction, we want to further verify whether these genes can express the protein normally. Three strains were cultured in LB medium, and IPTG and galactose were used as inducers to induce protein expression, and the protein expression level was verified by SDS-PAGE. Through the experimental results, we can find that EGFP protein can be expressed normally under the culture conditions with IPTG and galactose as inducers.

Figure 5 SDS-PAGE of galactose self-induction module.

Measurement of EGFP fluorescence intensity

By measuring the fluorescence intensity, we quickly measured the induction intensity of the promoter under the condition of galactose induction. Through the experimental results, we can find that the fluorescence intensity of the blank control group and the control group has hardly changed with the increase of time. However, with the increase of time, the fluorescence intensity of EGFP increased with different strains containing different promoters. Among them, pGalp-a promoter has the strongest ability to induce protein expression, so we chose pGalp-a promoter for the follow-up experiment.

Figure 6 Fluorescence intensity determination.

Protein expression

After the successful construction of the strain, we also verified the protein expression level of the strain by SDS-PAGE. Through the experimental results (Figure 7), we can find that Galp protein can be expressed normally under the induction of IPTG and galactose.

Figure 7. The result of SDS-PAGE.

Other Contributions

This study provides an important reference for other iGEM teams. Other teams can carry out further exploration based on our research results. We constructed a strain, which can survive in soy whey and can spontaneously induce and express 3'-SL protein by using soy whey. First of all, our experiment broke through the bottleneck that E. coli has a defect in the utilization of carbon source in soy whey, which enables us to cultivate E. coli with cheap soy whey. Secondly, we constructed an IPTG-independent self-induced expression system to solve the toxicity and cost problems caused by using chemical inducers in protein induction. Finally, our experiment can not only synthesize high-value 3'-SL but also control the water pollution caused by soy whey, which is of great significance in protecting the ecological environment. Other teams can apply the constructed strain to other products. By replacing the 3'-SL synthesis module, we can realize the efficient production of other high value-added products.

Reference

[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

[9] Kannan R, Mukundan G, Aït-Abdelkader N, Augier-Magro V, Baratti J, Gunasekaran P. Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis. Arch Microbiol. 1995 Mar;163(3):195-204. doi: 10.1007/BF00305353. PMID: 7778976.

[10] Bhatia S, Singh A, Batra N, Singh J. Microbial production and biotechnological applications of α-galactosidase. Int J Biol Macromol. 2020 May 1;150:1294-1313. doi: 10.1016/j.ijbiomac.2019.10.140. Epub 2019 Nov 17. PMID: 31747573.

[11] Wang Z, Dai Y, Azi F, Wang Z, Xu W, Wang D, Dong M, Xia X. Engineering Escherichia coli for cost-effective production of medium-chain fatty acids from soy whey using an optimized galactose-based autoinduction system. Bioresour Technol. 2024 Feb;393:130145. doi: 10.1016/j.biortech.2023.130145. Epub 2023 Nov 30. PMID: 38042430.