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 [ 1 ]. However, the traditional extraction and chemical synthesis methods are inefficient, costly and may cause pollution, so it is difficult to meet the market demand [ 2 ]. The purpose of this project is to construct an Escherichia coli chassis which can use cheap soy whey as culture medium and galactose in soy whey to realize self-induced expression system, so as to produce 3'-SL efficiently and economically. This study not only provides a new solution for the industrial production of 3'-SL but also provides a useful reference for the biological manufacture of other high value-added chemicals, which have remarkable economic benefits and environmental protection significance.

Our design consists of three main cycles:

Cycle 1: Construction and verification of carbohydrate utilization module. (pCOLAtrc(lost O)-sacC-aga, BBa_25TFWJDX)

Cycle 2: Construction and verification of galactose self-induction module.

pCOM4-PgalP-a-EGFP, BBa_2572ULRB

pCOM4-PgalP-b-EGFP, BBa_25NRQBKP

pCOM4-PgalP-c-EGFP, BBa_25Q32CXO

pCOM4-PgalP-GalP, BBa_25M3YRM6

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

pET-CSS-SaiT, BBa_251TW3T1

Cycle 1: Construction and verification of carbohydrate utilization module.

Design:

The carbon sources in soy whey are mainly sucrose, stachyose and raffinose. In order to utilize these carbohydrates, it is necessary for the strain to express extracellular sucrase(Sacc) and α -galactosidase(Aga), so as to hydrolyze the carbon sources that bacteria cannot use (sucrose, stachyose and raffinose) into carbon sources that bacteria can use (glucose, fructose and galactose). Therefore, we first need to introduce Sacc and Aga into E.coli, so that E.coli can survive in soy whey.

 

Figure 1 The plasmid map of pCOLAtrc(lost O)-scaC-aga

Build:

Agaa 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. It was found that the DNA fragments were consistent with expectations (AGA (2193bp) and SACC (1509bp), the sequencing results showed that the construction of our strains was successful.

Figure 2. Constructing carbohydrate utilization module.

Test:

1. SDS-PAGE of carbohydrate utilization module

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.

Figure 3 SDS-PAGE of carbohydrate utilization module.

 

2. Strain growth determination

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.

Cycle 2: Construction and verification of galactose self-induction module.

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

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

Build:

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 6A). Subsequently, we selected monoclonal colonies from the plate and verified them by PCR(Figure 6B). After the PCR verification was successful, we further confirmed that the strain construction was successful through DNA sequencing(Figure 6C).

Figure 6 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 7A), colony PCR verification (Figure 7B), and DNA sequencing (Figure 7C), we confirmed that the plasmid construction was correct.

Figure 7 Construction of pCOM4-PgalP-GalP. A. Antibiotic screening plate. B. Agarose gel electrophoresis verification. C. The results of DNA sequencing verification.

Test:

1. The expression level of EGFP was verified by SDS-PAGE.

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 8 SDS-PAGE of galactose self-induction module.

2.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 9 Fluorescence intensity determination.

3.The expression level of Galactose transporter was verified by SDS-PAGE.

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

Figure 10. The result of SDS-PAGE.

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

Design:

 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.

Figure 11 The plasmid map of pET-CSS-SaiT

Build:

First of all, 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 12A). Subsequently, we selected monoclonal colonies from the plate and verified them by PCR (Figure 12B). After the PCR verification was successful, we further confirmed that the strain construction was successful through DNA sequencing (Figure 12C).

Figure 12. Construction and verification of 3'-SL synthesis module. A. Antibiotic screening plate. B. Agarose gel electrophoresis verification. C. The results of DNA sequencing verification.

Test:

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

Figure 13. The result of SDS-PAGE.

Construction and verification of three plasmid co-transformed strains

Build:

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 14A). Then we further verified it by colony PCR (Figure 14B) and DNA sequencing (Figure 14C), and the experimental results showed that our co-transformed strain was constructed correctly.

Figure 14. 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.

Test:

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 15. Determination of strain growth curve after co-transformation.

After confirming the correct construction of the strain, 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 16A) in 16 hours, and the conversion rate of 3'-SL was over 30% (Figure 16B). When adding 500 mg/L of Neu5Ac, E.coli can produce more than 80mg/L of 3'-SL (Figure 16C) in 24 hours, and the conversion rate of 3'-SL is over 40% (Figure 16D).

Figure 16. 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)

Learn:

In conclusion, 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. 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 construction 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.

References

---

[1] PALUR D S, PRESSLEY S R, ATSUMI S 2023. Microbial production of human milk oligosaccharides. Molecules [J], 28.

[2] SCHELCH S, ZHONG C, PETSCHACHER B, et al. 2020. Bacterial sialyltransferases and their use in biocatalytic cascades for sialo-oligosaccharide production. Biotechnology Advances [J], 44: 107613.