Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Summary

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

Abstract

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2’-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

Introduction

Human milk oligosaccharides (HMOs) are lactose-derived oligosaccharides, usually comprising three to eight sugar monomers. They have a lactose (Gal-β1,4-Glc) reducing end and are further elongated by glycosidic links (β-1,3- or β-1,6-) to glucose (Glc), galactose (Gal), or N-acetylglucosamine (GlcNAc). In addition, fucose (Fuc, α-1,2- or α-1,3-) or sialic acid (Sia or NeuAc, α-2,3- or α-2,6-) residues are often added¹.

Current analysis of oligosaccharides and other carbohydrates is limited in throughput and scope by the need for chromatographic/mass spectrometric (MS) technology^2,3,4,5,6,7, which can take roughly an hour per sample, not to mention the necessity for expensive equipment, specialized columns and derivatizing agents, and expertise on the operation of this equipment⁸. Oligosaccharide linkages are particularly difficult to determine, requiring advanced MS^9,10 or nuclear magnetic resonance (NMR) techniques¹¹. Rapid optimization of synthesis of these oligosaccharides is thus limited by the throughput of this slow analytical step.

In this study, we demonstrate linkage-specific detection of fucosylated trisaccharide lactose-based HMOs, focusing on 2’-fucosyllactose (2’-FL) that is the most abundant HMO in human milk, using a genetically encoded Escherichia coli whole cell biosensor with a limit of detection at 4 mg/L. An important feature of this biosensor is its ability to distinguish between isomeric trisaccharides. The design principle is based on the expression of specific fucosidases in E. coli that liberate lactose from HMOs, the presence of which is detected by the lac operon, which in turn generates a fluorescent signal. We achieve this by building a two-plasmid system, one harboring the linkage-specific fucosidase and the other a fluorescent reporter protein. This biosensor platform is suitable for high-throughput screening by flow cytometry or micro-plate reader. We also demonstrate the utilization of the biosensor in quantifying 2’-FL produced by an engineered strain¹². Within this study, we also present three strategies on selective removal of lactose that can result in false positive signal from the biosensor, given that the engineered producer strain is grown on lactose.

Taken together, the genetically encoded biosensors allow us to detect and quantify HMOs in a linkage-specific manner, which is difficult even with chromatographic, MS, or NMR techniques. Due to its high throughput and ease of use, this method should have widespread applications in the metabolic engineering and synthesis of HMOs.

Protocol

1. Cell culture and induction conditions

NOTE: In the following experiments, three strains are used: E. coli BL21 (DE3) with an empty vector, E. coli BL21 (DE3) with plasmids pAfcA¹⁴ and pET28:green fluorescent protein (GFP), and E. coli BL21 (DE3) with plasmids pAfcB¹⁴ and pET28:GFP. All strains are grown in Luria-Bertani broth (LB) or minimal media with appropriate antibiotics. Prepare stock solutions of 1,000x kanamycin (50 mg/mL) and carbenicillin (100 mg/mL) in deionized (DI) water.

1. Media preparation
   1. Prepare LB media by adding 25 g of LB powder stock to 1 L of DI water. Autoclave the solution at 121 °C for 20 min to sterilize. Wait until the sterilized LB media temperature is below 60 °C before addition of 1 µL of antibiotic stock for every 1 mL of LB media.
   2. To prepare LB plates, add 15 g agar to the LB media before sterilization. Wait until the sterilized LB agar temperature is below 60 °C before addition of antibiotic.
   3. 1.1.3 For the biosensing cells, prepare media or plates with dual antibiotics, kanamycin and carbenicillin.
2. Carbohydrate inducers
   1. Prepare stock solutions of the carbohydrate inducers at molar equivalents to 20 g/L of lactose: 29 g/L of 2’-FL and 3-FL.
      NOTE: Each 200 µL of cells will require 20 µL of 2’-FL or 3-FL.
   2. Induce 200 µL of cells with 2.0 g/L final concentration of lactose or 2.9 g/L final concentration of 2’-FL/3-FL. Incubate at 37 °C with continuous shaking and let cultures grow overnight.
3. Cell culture
   1. The day before the experiment seed 5 mL of LB medium supplemented with kanamycin and carbenicillin from a fresh bacterial colony, using sterile technique.
   2. Incubate all cultures at 37 °C with agitation and grow the cultures overnight.
   3. Transfer 50 µL of overnight culture into 5 mL of fresh LB/M9 media with kanamycin and carbenicillin. Incubate at 37 °C with continuous shaking and grow until culture has reached optical density at 600 nm (OD₆₀₀) of 0.5–0.7. At this mid-log phase, the cells can be induced.

2. Measurement of fluorescence and limits of detection

1. Preparation of cells for flow cytometry
   1. Transfer the overnight cultures to 1.5 mL tubes and centrifuge at 12,500 x g for 1 min.
   2. Discard supernatant and re-suspend the pellet in 500 µL of 1x phosphate-buffered saline (PBS; 6 mM Na₂HPO₄, 1.8 mM NaH₂PO₄, 145 mM NaCl in DI water, pH 7.2) to prepare a single cell suspension and transfer the single cell suspension to 5 mL flow cytometry tubes.
   3. Keep the cells in the dark at 4 °C until running the samples on a flow cytometer. For best results, analyze the cells as soon as possible.
   4. Select 488 nm laser to excite GFP. Collect fluorescein isothiocyanate (FITC) fluorescence levels (20,000–40,000 events, gated for forward and side scatter to avoid aggregated cells and debris) with a 525/50 nm bandpass filter.
2. Calibrating the biosensor
   1. To make a calibration curve, make 8–10 dilutions of the standard 2’-FL in the range 0–2,500 mg/L.
   2. Culture the 2’-FL biosensing cells (containing pAfcA and pET28:GFP) and induce them with the standard dilutions, as described previously in section 1.3. Carry out three biological replicates for each dilution.

3. Detection and quantification of 2’-FL in a producer strain

1. Cultivation of producer strain
   1. For making minimal media, prepare separate solutions of 1 M K₂HPO₄, FeSO₄·7H₂O, sodium citrate, and 1 M thiamine-HCl. Sterilize the solutions using a 0.22 µm filter. Mix M9 media broth powder with 1 L of DI water. Autoclave the M9 solution at 121 °C for 15 min. Cool the solution down to 50 °C. Add MgSO₄, CaCl₂, K₂HPO₄, FeSO₄·7H₂O, sodium citrate, and thiamine to final concentrations of 2 mM, 0.1 mM, 12 g/L, 11 mg/L, 75 mg/L, and 7.5 µg/L, respectively.
   2. Start a culture of 2’-FL producing strain, e.g., JM109 gwBC-F2, in 5 mL of LB media and let it grow overnight at 37 °C, as described in Baumgartner et al.¹².
   3. Subculture at 1% as described earlier in step 1.3.3 in 250 mL of the minimal media prepared in step 3.1.1. Add glycerol to a final concentration of 10 g/L and incubate the culture at 37 °C.
   4. When the culture reaches an OD₆₀₀ of 0.5–0.7, add isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and lactose to a final concentration of 2 g/L.
   5. Incubate at 37 °C with continuous shaking and let cultures grow for 24 h.
2. Extraction of 2’-FL
   1. Transfer the overnight cultures to sterile 50 mL tubes and centrifuge at 900 x g for 15 min.
   2. 3.2.2 Discard supernatant and re-suspend the pellet in DI water. Repeat the wash step three times to remove residual lactose and finally re-suspend in 5 mL of DI water.
   3. To lyse the cell suspension, use a sonicator at 30% power, in 30 s pulses. Keep the cells on ice at all times.
   4. Centrifuge the lysed cells for 25 min at 2,000 x g. Remove the supernatant, pass it through a sterile (e.g., 0.22 µm) filter and store it at 4 °C until analysis.
3. Quantification with biosensor
   1. Inoculate a culture of 2’-FL biosensing cells and at mid-log phase, induce with cell lysate equivalent to estimated concentrations of 2’-FL used to make the calibration curve as described in section 2.2. Perform the calibration in the same milieu as the sample to be analyzed (e.g., cell lysate) by adding dilutions of 2’-FL to control (i.e., non-producer) filtered cell lysate before inducing biosensor cells.
   2. Run the samples on a flow cytometer. Generate a dose-response curve by plotting the fluorescence output against the oligosaccharide concentration. Compare the response of the standards to the cell lysate.

4. Selective removal of lactose

NOTE: The biosensing cells are sensitive to lactose and it is desirable for the biosensor to selectively detect HMOs over lactose. One strategy would include washing of cells as described in section 3.2. Three other strategies are described below.

1. Treatment with commercial purified β-galactosidase
   1. Dissolve lyophilized β-galactosidase to 1,000 (FCC lactase) units/mL, in 1 mM MgCl₂ or use commercial β-galactosidase in solution.
   2. To determine the optimum concentration of enzyme needed, grow an inoculum of 2’-FL biosensing cells and induce with 2 g/L lactose and 2.9 g/L 2’-FL at mid log phase.
   3. To 100 µL cultures, add variable amounts of β-galactosidase, up to 12 units, and let the cultures grow overnight at 37 °C with continuous shaking.
   4. Measure the fluorescence as described in section 2.1 and calculate the optimum units of enzyme needed by determining the minimum enzyme concentration to achieve desired attenuation of signal.
   5. To 2’-FL producing cells grown for 24 h, add optimum units of β-galactosidase and incubate overnight at 37 °C. Proceed with the protocol for determining the 2’-FL titer as described in section 3.3.
2. Pre-treatment with LacZ+ strain
   1. Start a 25 mL culture of 2’-FL producing strain and once induced at mid-log phase, incubate the culture at 37 °C for 24 h.
   2. Inoculate an E. coli BL21 (DE3) culture in LB, grow it to mid-log phase at 37 °C and add 50 mL of the culture (2:1) to the 24 h culture.
   3. Incubate at 37 °C for 3 h. Proceed with the protocol for determining the 2’-FL titer as described in section 3.3.
3. Lactose precipitation with ethanol
   NOTE: This section is adapted from Matsuki et al.¹².
   1. Start a 25 mL culture of 2’-FL producing strain and once induced at mid-log phase, incubate at 37 °C for 24 h.
   2. Add two volumes of 100% ethanol to the culture, shake well, and incubate at 37 °C for 4 h.
   3. Centrifuge the suspension at 900 x g for 15 min. Collect the supernatant and incubate overnight at 4 °C, which precipitates the lactose.
   4. Remove the precipitated lactose by passing the solution through a filter paper (11 µm). Collect the filtrate that is the cell lysate containing the 2’-FL, which can be quantified by following section 3.3.

Results

We designed a whole cell biosensor specific to 2’-FL that can be used in conjunction with the biotechnological production of the oligosaccharide. This relies on the specific enzymatic cleavage of modifying terminal sugars generating lactose and thereby activation of the lac operon, leading to expression of a reporter fluorescent protein under a lactose inducible promoter, in proportion to the quantity of 2’-FL. To demonstrate its linkage specificity, 3-fucosyllactose (3-FL), an i...

Discussion

We present a high-throughput strategy for the linkage-specific detection of fucosylated human milk oligosaccharides. This was accomplished by building whole cell biosensors by genetically engineering E. coli which upon induction with specific glycans respond with a fluorescent signal. The protocol also details on how the biosensor can be used to detect and quantify HMOs in a metabolically engineered bacterial strain.

Our protocol offers advantages over alternative methods due to its h...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2’-Fucosyllactose	Carbosynth	41263-94-9
3-Fucosyllactose	Carbosynth	41312-47-4
Agar	Fisher Scientific	BP9744500
Calcium Chloride, Dihydrate	Fisher Scientific	C79-500
Carbenicillin	Fisher Scientific	BP26481
Dextrose (D-Glucose), Anhydrous	Fisher Scientific	D16-1
Flow Cytometer	BD		FACSCanto Plus RUO
HPLC	Agilent Technologies		1100 Series HPLC system
HPLC Column	Luna		C18 reversed phase column
Kanamycin	Fisher Scientific	11815024
LB Broth, Miller	Fisher Scientific	12-795-027
Lactose	Fisher Scientific	64044-51-5
M9, Minimimal Salts, 5x	Sigma-Aldrich	M6030
Magnesium Sulfate, Anhydrous	Fisher Scientific	M65-500
MS	Agilent Technologies		Mass Selective Trap SL detector
Sodium chloride	Sigma-Aldrich	7647-14-5
Sodium phosphate dibasic	Sigma-Aldrich	7558-79-4
Sodium phosphate monobasic	Sigma-Aldrich	13472-35-0