Name: analysis of fucosylated human milk trisaccharides in biotechnological context using genetically encoded biosensors
Uploaded: 2019-04-13T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors at JoVE.com

This work was supported by Iowa State University Startup Funds. F.E. was partially funded by the NSF Trinect Fellowship and Manley Hoppe Professorship. &#173;&#173;T.J.M. was partially supported by the Karen and Denny Vaughn Faculty Fellowship. The authors thank the Iowa State University Flow Cytometry Facility and the W.M. Keck Metabolomics Research Laboratory for assistance with fluorescence and LC-MS studies.

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 carbenici...

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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...

Human milk oligosaccharides (HMOs) are lactose-derived oligosaccharides, usually comprising three to eight sugar monomers. They have a lactose (Gal-&#946;1,4-Glc) reducing end and are further elongated by glycosidic links (&#946;-1,3- or &#946;-1,6-) to glucose (Glc), galactose (Gal), or N-acetylglucosamine (GlcNAc). In addition, fucose (Fuc, &#945;-1,2- or &#945;-1,3-) or sialic acid (Sia or NeuAc, &#945;-2,3- or &#945;-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) technology2,3,4,5,6,7, 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 MS9,10 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&#8217;-fucosyllactose (2&#8217;-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&#8217;-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.

<table border="0" cellpadding="0" cellspacing="0" style="width:993px;" width="992">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">2&#8217;-Fucosyllactose&#160;</td>
			<td style="width:247px;">Carbosynth&#160;</td>
			<td style="width:204px;">41263-94-9</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-Fucosyllactose&#160;</td>
			<td>Carbosynth&#160;</td>
			<td>41312-47-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Fisher Scientific</td>
			<td>BP9744500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride, Dihydrate</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbenicillin&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP26481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose (D-Glucose), Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer</td>
			<td>BD</td>
			<td></td>
			<td>FACSCanto Plus RUO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>1100 Series HPLC system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC Column</td>
			<td>Luna</td>
			<td></td>
			<td>C18 reversed phase column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>11815024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB Broth, Miller&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-795-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactose</td>
			<td>Fisher Scientific</td>
			<td>64044-51-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M9, Minimimal Salts, 5x</td>
			<td>Sigma-Aldrich</td>
			<td>M6030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate, Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>Mass Selective Trap SL detector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>13472-35-0</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of fucosylated human milk trisaccharides in biotechnological context using genetically encoded biosensors

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&#8217;-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.

We designed a whole cell biosensor specific to 2&#8217;-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&#8217;-FL. To demonstrate its linkage specificity, 3-fucosyllactose ...

Watch this Scientific Journal Video about Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors at JoVE.com

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

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.

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

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