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

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 st....

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