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.

Figure 1 Schematic diagram of experimental design

1.Construction and verification of carbohydrate utilization module

1.1 Construction of pCOLAtrc(lost O)-sacc-aga

At present, most of the E.coli can't use carbohydrates (such as Sucrose, stachyose and raffinose) in soy whey as carbon source, which makes it difficult to use this rich food industry by-product as fermentation medium. In order to enable E.coli to make normal use of these nutrients, we introduced AGA and SACC into E.coli. AGA is α-galactosidase and SACC is extracellular sucrase. AGA and SACC can convert carbon sources that E.coli can't use into available carbon sources (such as glucose, fructose and galactose). After introducing them into bacteria, we initially obtained the strains that could be successfully constructed by antibiotic screening and further verified them by DNA sequencing, and the sequencing results showed that the construction of our strains was successful.

Figure 2. Constructing carbohydrate utilization module.

1.2 SDS-PAGE was used to verify the protein expression level

After confirming the success of the strain construction, we want to further verify whether these two genes can express the protein normally. We have carried out experimental tests in soy whey medium. The experimental group used IPTG to induce protein expression. The experimental results showed that the strain could grow normally and express Aga and Sacc proteins under the induction of IPTG in soy whey medium. After introducing this module, the strain can grow normally in soy whey and express protein normally under the induction of IPTG. Since the self-induction module of galactose has not been constructed, the protein expression was less when galactose is used as inducer. Therefore, the next step is to construct a self-induction module of galactose in bacteria.

Figure 3 SDS-PAGE of carbohydrate utilization module

1.3. Determination of strain growth after introducing carbohydrate utilization module

After confirming that the protein can be successfully induced to express, we want to know whether these two proteins can function normally. That is, whether Escherichia coli can grow normally in soy whey after Aga and Sacc are introduced. Therefore, We tested the growth of strains with carbohydrate utilization module and without carbohydrate utilization module in soy whey. The experimental results show that the strain can grow normally in soy whey after introducing protein, but the growth of the strain without introducing protein is obviously inhibited. This result shows that the strain can grow normally in soy whey after containing carbohydrate utilization module.

Figure 4. Growth ability of strains under different culture conditions.

2. Construction and verification of galactose self-induction module

2.1. Construction of pCOM4-PgalP-a-EGFP, pCOM4-PgalP-b-EGFP, pCOM4-PgalP-c-EGFP

After constructing the carbohydrate utilization module, we have solved the normal growth problem of bacteria in soy whey. Next, we want bacteria to express the protein we need. 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. Through antibiotic screening (Figure 5A), colony PCR verification(Figure 5B), and DNA sequencing(Figure 5C), we confirmed that the construction of the strain was successful.

Figure 5. Construction and Verification of Promoter Screening Module.

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

2.2. SDS-PAGE was used to verify the protein expression level

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. Furthermore, we want to determine the expression level of EGFP, so as to screen the promoter with the highest induced expression intensity.

Figure 6 SDS-PAGE of galactose self-induction module.

2.3. The promoter with the strongest induction ability was screened by 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 7. Fluorescence intensity determination.

2.4. Construction of pCOM4-PgalP-GalP

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 8A), colony PCR verification (Figure 8B), and DNA sequencing(Figure 8C), we confirmed that the plasmid construction was correct.

Figure 8. Construction of pCOM4-PgalP-GalP. A. Antibiotic screening plate.

B. Agarose gel electrophoresis verification. C. The results of DNA sequencing verification.

2.5. SDS-PAGE was used to verify the protein expression level

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 9), we can find that Galp protein can be expressed normally under the induction of IPTG and galactose.

Figure 9. The result of SDS-PAGE.

3.Construction and verification of 3'-SL synthesis module

3.1. Construction of pET-CSS-SaiT

After confirming that bacteria can survive in soy whey and can spontaneously induce protein production by using nutrients in soy whey, the next step is to obtain the synthesis module of 3'-SL.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. We introduced these two enzymes into E.coli, enabling E.coli to synthesize 3'-SL. First, we obtained the strains that could be successfully constructed by antibiotic screening(Figure 10A), and then we further confirmed that the construction of this strain was successful by colony PCR (Figure 10B) and DNA sequencing (Figure 10C).

Figure 10. Construction and verification of 3'-SL synthesis module. A. Antibiotic screening plate. B. Agarose gel electrophoresis verification. C. The results of DNA sequencing verification.

3.2. SDS-PAGE was used to verify the protein expression level

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 11), we can find that two proteins can be expressed normally under the induction of IPTG and galactose.

Figure 11. The result of SDS-PAGE.

4. Construction and verification of three plasmid co-transformed strains

4.1. The Construction of Co-transformed Plasmids

After all the three modules are constructed, we need to co-transform the plasmids of the three modules into the same bacteria to make the bacteria have complete functions. After the three plasmids were transformed into bacteria, we also made a preliminary screening through antibiotics to obtain the strains that could be successfully constructed (Figure 12A). Then we further verified it by colony PCR(Figure 12B) and DNA sequencing (Figure 12C), and the experimental results showed that our co-transformed strain was constructed correctly.

Figure 12. Construction and verification of three plasmid co-transformed strains. A. Antibiotic screening plate. B. Agarose gel electrophoresis verification. C. The results of DNA sequencing verification.

4.2. Determination of strain growth curve after co-transformation

After transferring the plasmid of three modules, we first want to determine whether the co-transformed bacteria can survive normally in soy whey. Therefore, we cultured the co-transformed bacteria in LB and soy whey respectively and determined the growth curve. Through the determination of growth curve, we found that bacteria can grow normally in Soy whey after plasmid co-transformation, and the growth ability is completely consistent with that of bacteria cultured in LB medium. Next, we need to determine the ability of the strain to produce 3'-SL in soy whey.

Figure 13. Determination of strain growth curve after co-transformation.

4.3. The synthetic amount of 3'-SL was determined by HPLC

After confirming that the strain can survive normally in soy whey, we need to measure the yield of 3'-SL in the next step. After streaking and activating the fermentation strain, a single colony was picked and inoculated into 5 mL of LB liquid medium (with 5 μL Chl and Amp) and then cultured at 37°C and 200 r/min for 8-12 hours. The culture was transferred to 100 mL of soy whey at an initial OD₆₀₀ of 0.1 and cultured at 37°C and 200 r/min for approximately 8 hours. The culture was centrifuged at 4°C and 5000 rpm for 10 minutes. The bacterial cells were resuspended in 24 mL of Tris-HCl buffer (50 mmol/L, pH 7.0, containing 250 mg Neu5Ac or 500 mg Neu5AC). A substrate solution with a concentration of 10 g/L (1 mL of 250 g/L lactose) was added to start the reaction. The results of HPLC showed that when adding 250 mg/L of Neu5Ac, E.coli could produce more than 80 mg/L of 3'-SL (Figure 14A) in 16 hours, and the conversion rate of 3'-SL was over 30% (Figure 14). When adding 500 mg/L of Neu5Ac, E.coli can produce more than 80mg/L of 3'-SL (Figure 14C) in 24 hours, and the conversion rate of 3'-SL is over 40% (Figure 14D).

Figure 14. Determination of synthetic yield of 3'-SL by HPLC.

A.3'-SL yield (Neu5AC 250 mg) B. 3'-SL conversion rate (Neu5AC 250 mg)

C.3'-SL yield (Neu5AC 500 mg) D. 3'-SL conversion rate (Neu5AC 500 mg)

5. Future plans

Based on our experimental 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 yellow serous water, which is of great significance in protecting the ecological environment.

There are also some areas that need to be optimized in our experiment. At present, the conversion rate of 3'-SL is about 40%. We can further optimize the promoter or other biological elements in the future, so as to further improve the conversion rate and increase the protein yield. Secondly, we 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. Finally, if our products need to be put into industrial production in the future, we need to optimize the fermentation process, and further research is needed to maintain the stability of strains and ensure the stable supply and quality consistency of raw materials of soy whey. And we also need to optimize the product separation and purification process to develop an efficient and low-cost product separation and purification process suitable for industrial production.

6. 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