Biosynthetic routes for producing various fucosyl-oligosaccharides

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Biosynthetic routes for producing various fucosyl-oligosaccharides Eun Ju Yun, JINGJING LIU, Jaewon Lee, Suryang Kwak, Sora Yu, Kyoung Heon Kim, and Yong-Su Jin ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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ACS Synthetic Biology

Revised for ACS Synthetic Biology

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Biosynthetic Routes for Producing Various Fucosyl-oligosaccharides

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Eun Ju Yun,†,‡ Jing-Jing Liu,‡ Jae Won Lee,‡ Suryang Kwak,‡ Sora Yu,†

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Kyoung Heon Kim,*,† and Youg-Su Jin*,‡

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†Department

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Republic of Korea

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‡Carl

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Champaign, Urbana, IL 61801, United States

of Biotechnology, Graduate School, Korea University, Seoul 02841,

R. Woese Institute for Genomic Biology, University of Illinois at Urbana-

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*Corresponding authors.

17

E-mail address: [email protected] (Y.-S. Jin)

18

E-mail address: [email protected] (K. H. Kim)

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ABSTRACT

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Fucosyl-oligosaccharides (FOSs) play physiologically important roles as prebiotics,

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neuronal growth factors, and inhibitors of enteropathogens. However, challenges in

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designed synthesis and mass production of FOSs hamper their industrial applications.

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Here, we report flexible biosynthetic routes to produce various FOSs, including

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unnatural ones, through in vitro enzymatic reactions of various sugar acceptors, such

32

as glucose, cellobiose, and agarobiose, and GDP-L-fucose as the fucose donor by

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using α1,2-fucosyltransferase (FucT2). Also, the whole-cell conversion for fucosylation

34

of various sugar acceptors by overexpressing the genes associated with GDP-L-

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fucose production and FucT2 gene in Escherichia coli was demonstrated by producing

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17.74 g/L of 2′-fucosylgalactose (2′-FG). Prebiotic effects of 2′-FG were verified based

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on selective fermentability of 2′-FG by probiotic bifidobacteria. These biosynthetic

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routes can be used to engineer industrial microorganisms for more economical, more

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flexible, and safer production of FOSs than chemical synthesis of FOSs.

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KEYWORDS: Fucosyltransferase, fucosyl-oligosaccharides, 2′-fucosylgalactose,

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enzymatic synthesis, prebiotics, chemotherapeutics

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Fucosylation, a type of glycosylation, is one of the most common oligosaccharide

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modifications, wherein fucose residue is added to N-glycans, O-glycans, or

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glycolipids.1

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fucosyltransferase—is implicated in the pathological conditions involving immune

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response induced by inflammation, cancer, and signal transduction.1-3 Recently,

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fucosyl-oligosaccharides (FOSs) have gained much attention owing to their prebiotic

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and chemotherapeutic activities.4,5

In

a

biological

system,

fucosylation—catalyzed

2 ACS Paragon Plus Environment

by

enzyme

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FOSs, mainly found in human milk oligosaccharides (HMOs), have been

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reported to prevent infant diarrhea and to inhibit colonization of enteric pathogens.6,7

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More specifically, FOSs act as receptor homologs by inhibiting adhesion of diarrheal

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pathogens such as Escherichia coli, Vibrio cholerae, and Salmonella fyris to intestinal

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epithelial cells.8 In particular, 2′-fucosyllactose (2′-FL), one of the most abundant

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HMOs found in human breast milk, is a well-known prebiotic oligosaccharide that

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modulates the composition of the infant gut microbiota, mainly by selectively

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stimulating growth of bifidobacterial.9,10 Meanwhile, a structurally similar disaccharide,

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2′-fucosylgalactose (2'-FG) moiety has been reported to give a crucial structure to

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glycoproteins for interacting with lectin receptors in the brain.4,11,12 Specifically, the

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formation of 2'-FG termini resulting from α1,2-fucosylation of terminal galactose

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residues of brain glycoproteins is implicated to modulate neuronal communication or

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to enable neuronal plasticity related to long-term potentiation and memory storage.11,12

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Interestingly, not only the 2'-FG termini of glycoproteins but also 2'-FG carbohydrate

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itself also can promote neuronal growth that was verified by treating embryonic

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hippocampal neurons with a 2'-FG-biotin probe.4 Moreover, 2′-FL was also reported to

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enhance hippocampal long-term potentiation in rats.13

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Despite these beneficial effects of FOSs, effective synthesis methods for

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mass production of FOSs have not yet been established. Chemical synthesis methods

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have been devised for synthesizing FOSs, including 2′-FL14 and 2′-FG4. However,

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these methods require the use of toxic chemicals, such as methanol, pyridine, or

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toluene, at high temperatures and generate structurally similar byproducts that are

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difficult to separate.4,14 Therefore, to produce structurally correct FOSs, enzymatic

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synthesis of FOSs might be more favorable than chemical synthesis, but by using

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enzymatic methods, only 2′-FL has been synthesized so far.9,15-17 3 ACS Paragon Plus Environment


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In this study, we aimed to develop flexible biosynthetic platforms producing

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various FOSs, including unnatural FOSs. To accomplish this, in vitro enzymatic

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reactions of various sugar acceptors and a GDP-L-fucose as a fucose donor, using an

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α1,2-fucosyltransferase, FucT2, originating from Helicobacter pylori, were performed.

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The reaction products obtained from the in vitro enzymatic reactions were analyzed

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using hybrid ion trap/time-of-flight mass spectrometry coupled with liquid

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chromatography (LC/MS−IT−TOF). Then, E. coli was engineered to produce not only

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2′-FG but other FOSs by introducing the genes associated with de novo pathway to

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accumulate GDP-L-fucose intracellularly and fucT2 into E. coli. Moreover, to

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investigate the potential prebiotic effect of 2′-FG, selective fermentability of 2′-FG by

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bifidobacteria was tested. To our knowledge, this is the first report of biosynthetic

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routes for producing not only natural but also unnatural FOSs and of an engineered

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microbial platform that enables mass production of 2′-FG. These biosynthetic routes

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can be applied to metabolic engineering of industrial microorganisms for producing

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various FOSs more economically and environmental-friendly.

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RESULTS AND DISCUSSION

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In Vitro Enzymatic Reactions of Various Sugar Acceptors by α1,2-

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Fucosyltransferase

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Prior to in vitro enzymatic reactions of various sugar acceptors by α1,2-

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fucosyltransferase, FucT2, in vitro control experiments using crude enzymes extracted

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from E. coli BL21(DE3) strain harboring an empty vector and lactose as a sugar

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acceptor was performed. The results obtained from the in vitro experiments showed

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that when crude enzymes extracted from E. coli BL21(DE3) strain harboring an empty 4 ACS Paragon Plus Environment

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vector was incubated with lactose and GDP-L-fucose, 2′-FL was not produced

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(Supporting Information, Figure S1A). However, when crude enzymes extracted from

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E. coli BL21(DE3) strain expressing fucT2 gene was incubated with lactose and GDP-

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L-fucose,

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from the in vitro enzymatic reaction was identified by gas chromatography/time-of-

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flight mass spectrometry (GC/TOF MS) analysis (Supporting Information, Figure

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S1C,D). These results showed that 2′-FL production is due to the activity of α1,2-

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fucosylation of lactose by FucT2 originating from H. pylori as already verified in

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previous studies.16-19

2′-FL was produced (Supporting Information, Figure S1B). 2′-FL produced

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To produce various FOSs, in vitro enzymatic reactions of GDP-L-fucose and

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various sugar acceptors were performed using α1,2-fucosyltransferase, FucT2 (Figure

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1A). Five different monosaccharides and disaccharides, namely glucose, galactose,

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lactose, cellobiose, and agarobiose were used as sugar acceptors (Figure 1B). Among

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various sugar acceptors tested in this study, in particular, cellobiose and agarobiose,

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the most abundant disaccharide units comprising plant lignocellulose and red

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macroalgae (Rhodophyta), respectively20,21,have never been used as sugar acceptors

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for α1,2-fucosyltransferase reaction.22-24

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Agarobiose, a disaccharide unit of agarose consisting of D-galactose and

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3,6-anhydro-L-galactose, is not commercially available. Therefore, agarobiose

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produced in-house by acid hydrolysis of agarose using phosphoric acid (Supporting

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Information, Figure S2) was used. Specifically, 10% (w/w) agarose was treated with

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2% (w/v) phosphoric acid at 90 °C, and the optimal reaction time for producing

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agarobiose was 180 min (Supporting Information, Figure S2).

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Identification of Enzymatic Reaction Products by LC/MS−IT−TOF Analysis 5 ACS Paragon Plus Environment


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Enzymatic reactions of GDP-L-fucose and five sugar acceptors were

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performed using FucT2, and the reaction products were analyzed using

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LC/MS−IT−TOF (Figure 2). After the enzymatic reaction of galactose and GDP-L-

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fucose by FucT2, two peaks, corresponding to the exact masses of lithium adduct ions

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of 2′-FG and dimeric 2′-FG, were detected at m/z values of 333 and 659, respectively

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(Figure 2A). After the enzymatic reaction of glucose and GDP-L-fucose by FucT2, a

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peak corresponding to the exact mass of lithium adduct of 2′-fucosylglucose was

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detected at m/z 333 (Figure 2B). Three disaccharides acceptors (lactose, cellobiose,

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and agarobiose) were also examined for the biosynthesis of fucosyl-disaccharides via

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the enzymatic reaction of GDP-L-fucose using FucT2. When lactose and cellobiose

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were used in the enzymatic reaction of GDP-L-fucose using FucT2, a peak

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corresponding to the exact masses of lithium adducts of 2′-FL and 2′-fucosylcellobiose

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was detected at m/z of 495 (Figure 2C, D). Also, when agarobiose was added to this

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enzymatic reaction, two major peaks corresponding to the exact masses of lithium

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adduct ions of 2′-fucosylagarobiose and its hydrated form were detected at m/z values

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of 477 and 495, respectively (Figure 2E). 2′-Fucosylagarobiose was found to exist

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predominantly in the hydrated form because of 3,6-anhydro-L-galactose (AHG) at the

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reducing end of agarobiose, because AHG is readily hydrated under aqueous

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conditions.25 Notably, 2′-fucosylcellobiose and 2′-fucosylagarobiose have not been

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synthesized using either chemical or enzymatic method so far. Therefore, this is the

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first

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fucosylagarobiose via enzymatic routes.

report

demonstrating

the

production

of

2′-fucosylcellobiose

and

2′-

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To further identify the FOSs produced by the enzymatic reactions, the FOSs

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were analyzed in two stages using tandem MS (Figure 3). For instance, the precursor

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ion of 2′-FG detected at m/z 333 was found to be fragmented into fucose and galactose 6 ACS Paragon Plus Environment

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ions, detected at m/z values of 169 and 187, respectively (Figure 3A). Similarly, the

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precursor ion of 2′-fucosylglucose, detected at m/z value of 333, was also found to be

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fragmented into fucose and glucose, detected at m/z values of 169 and 187,

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respectively (Figure 3B). In the case of FOSs produced using disaccharides, because

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of the two glycosidic linkages within the molecules, the precursors of 2′-FL, 2′-

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fucosylagarobiose, and 2′-fucosylcellobiose ions can be fragmented in two different

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ways. In the case of 2′-FL, two fragment ions of fucose and lactose, detected at m/z

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values of 169 and 349, respectively, were generated from the cleavage of the α1,2-

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glycosidic linkage of 2′-FL (Figure 3C). Additionally, the fragment ions of

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fucosylgalactose and glucose at m/z 331 and 187 were identified from the cleavage of

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the β1,4-glycosidic linkage of 2′-FL (Figure 3C). Similarly, the fragment ions from the

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cleavages of 2′-fucosylagarobiose and 2′-fucosylcellobiose molecules were identified

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by tandem MS to confirm the production of 2′-fucosylagarobiose and 2′-

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fucosylcellobiose, respectively, by FucT2 (Figure 3D, E).

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Production of 2′-FG by Metabolically Engineered E. coli

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Among various FOSs synthesized in this study, 2′-FG is the most promising

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one, because it can promote neuronal growth, specifically by interacting with lectin

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receptors in the brain.4 Moreover, 2′-FG was suggested to possess prebiotic effect,

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based on the fact that fucosylated HMOs, such as 2′-FL, lacto-N-fucopentaose, and

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lacto-N-difucohexaose, contain a 2′-FG unit.17 In this study, for the potential of mass

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production of 2′-FG, we constructed an engineered microbial platform using E. coli

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BL21(DE3) without the capability to galactose utilization (Figure 4A).26 The incapability

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of E. coli BL21(DE3) to use galactose as a carbon source allowed the use of galactose

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as an acceptor for synthesizing 2′-FG from the enzymatic reaction of FucT2 with GDP7 ACS Paragon Plus Environment


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L-fucose

(Figure 4A). The incapability of E. coli BL21(DE3) to use galactose as a

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carbon source is due to the knockout of its 16 genes involved in the galactose catabolic

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pathway from galM to ybhJ.26,27 To accumulate GDP-L-fucose intracellularly, the

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endogenous de novo pathway for producing GDP-L-fucose was augmented by

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overexpressing

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guanylyltransferase (ManC), GDP-mannose 4,6-dehydratase (Gmd), and GDP-L-

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fucose synthase (WcaG) (Figure 4A). Glycerol was supplied as a carbon source

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instead of glucose to allow the transport of galactose without catabolite repression

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(Figure 4A).

phosphomannomutase

(ManB),

mannose

1-phosphate

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2′-FG was produced at a titer of 1.79 g/L with a yield of 1.52 g 2′-FG/g

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galactose from a fed-batch fermentation of an engineered E. coli BL21(DE3)

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pmBCGWF in a shake flask, which is equivalent to 84% of the theoretical maximum

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yield (Figure 4B). From a high cell density fed-batch fermentation of the engineered E.

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coli in a bioreactor, the 2′-FG titer increased to 17.74 g/L (Figure 4C), but the yield of

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2′-FG significantly decreased to 0.55 g 2′-FG/g galactose (Figure 4C). The lower 2′-

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FG yield of the high cell density fed-batch fermentation in a bioreactor was probably

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because of an inefficient export system of 2′-FG in the E. coli cells, similar to the case

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of 2′-FL production in E. coli

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(Supporting Information, Figure S3). The final titer of intracellular 2′-FG was 3.17 g/L,

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which accounted for 15% of total 2′-FG produced by the engineered E. coli (Supporting

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Information, Figure S3). Moreover, galactitol, a reduced form of galactose, and acetate

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were produced as byproducts at 4.91 g/L (with a yield of 0.15 g galactitol/g galactose)

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and 5.21 g/L, respectively (Supporting Information, Figure S3).

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and higher accumulation of galactitol and acetate

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Identification of α1,2-Glycosidic Linkage of 2′-FG 8 ACS Paragon Plus Environment

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To confirm the α1,2-glycosidic linkage in 2′-FG in the culture of the

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engineered E. coli BL21(DE3), the culture supernatant was purified by gel-permeation

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chromatography using a G-10 column (Figure 5A and B), and the purified 2′-FG was

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incubated with the crude extract of E. coli expressing α1,2-fucosidase that specifically

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recognizes and acts on the α1,2-glycosidic linkage.29 As a result, the generation of

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fucose and galactose from the hydrolysis of α1,2-glycosidic linkage

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fucosidase were confirmed (Figure 5C). On the contrary, in the control reactions

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performed by using the crude enzymes extracted from E. coli BL21(DE3) harboring

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the empty vector and the purified 2′-FG, fucose and galactose were not detected as

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reaction products (Figure 5D).

by α1,2-

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In vitro Prebiotic Activity of 2′-FG

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Before investigating a potential prebiotic activity of 2′-FG, the stability of 2′-

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FG in a simulated gastric fluid in the absence of pepsin needed to be verified first. This

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was because oral administration of 2′-FG might lead to its degradation in stomach

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before reaching to intestines (Supporting Information, Figure S4). However, 2′-FG was

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not degraded for 3 h in the simulated gastric fluid at 37 °C, indicating that 2′-FG might

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stably pass through the stomach and reach the gut (Supporting Information, Figure

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S4).

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Next, to investigate the in vitro prebiotic activity of 2′-FG, two bifidobacteria,

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namely Bifidobacterium longum ssp. infantis ATCC 15697 and B. bifidum DSM 20082,

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which are predominant species of the infant bifidobacterial community in the infant gut,

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were cultured on 2′-FG as a carbon source (Figure 6). When the two probiotic strains

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of bifidobacteria were cultured with 2′-FG, the cell growth of B. infantis was faster than

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that of B. bifidum (Figure 6A). The fermentation profiles of B. infantis and B. bifidum in 9 ACS Paragon Plus Environment


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the presence of 2′-FG revealed that the two strains used different metabolic pathways

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for utilizing 2′-FG (Figure 6B–E). 2′-FG was transported into the cytosol of B. infantis,

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and intracellular 2′-FG was hydrolyzed into fucose and galactose by intracellular α1,2-

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fucosidases belonging to GH29 and GH95 (Figure 6B).30 As a result of 2′-FG

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fermentation by B. infantis, acetate, lactate, and 1,2-propanediol were produced, in

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particular, 1,2-propanediol production was a result of fucose fermentation (Figure

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6C).31

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On the contrary, B. bifidum hydrolyzed 2′-FG into fucose and galactose

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extracellularly using secreted α1,2-fucosidases,10,32 and galactose was consumed

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(Figure 6D). Because of the extracellular hydrolysis of 2′-FG and selective utilization

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of galactose, fucose accumulated extracellularly during the fermentation of 2′-FG by

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B. bifidum (Figure 6D). Through the galactose fermentation by B. bifidum, acetate and

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lactate were produced, but 1,2-propanediol was not produced (Figure 6E), indicating

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that fucose is not metabolized by B. bifidum.33

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To confirm the selective fermentability of 2′-FG by bifidobacteria, the cell

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growth of two pathogenic bacteria, namely E. coli O1:K1:H7 and Salmonella enterica

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serotype Typhimurium, was examined using 2′-FG as a carbon source (Figure 7).

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Unlike the two bifidobacteria, B. infantis and B. bifidum (Figure 6), E. coli O1:K1:H7

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and S. enterica Typhimurium could not metabolize 2′-FG (Figure 7).

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Our results suggest that 2′-FG can be used as a new prebiotic for

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bifidobacteria, i.e., bifidus factor, owing to its stability in simulated gastric fluid, and

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selective fermentability by bifidobacteria. In addition to the growth-promoting activity

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of bifidobacteria, 2′-FG is expected to exhibit protective activities against pathogens,

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such as Campylobacter jejuni,7 S. enterica serotype Typhimurium,34 enterotoxigenic

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E. coli,35 H. pylori,36 and norovirus,6 as 2′-FG can perturb the binding of these 10 ACS Paragon Plus Environment

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pathogens to their adhesion sites in the gut mucosa. Furthermore, the neuronal growth

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stimulation by 2′-FG has applications in understanding and treatment of

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neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases.4

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CONCLUSIONS

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In summary, we demonstrated biosynthetic routes for producing various

257

FOSs, including unnatural FOSs which are not found among HMOs, by in vitro

258

enzymatic reactions using α1,2-fucosyltransferase with unprecedented sugar

259

acceptors for fucosylations. In particular, to produce 2′-FG, which is known to play a

260

biologically crucial role as a neuronal growth factor, we developed a microbial platform

261

using engineered E. coli as a host. Fed-batch fermentation of an engineered E. coli

262

yielded 17.74 g/L of 2′-FG. Furthermore, for the first time, the prebiotic effect of 2′-FG

263

was investigated. Two distinct bifidobacteria, B. infantis and B. bifidum utilized 2′-FG

264

as a carbon source whereas the pathogenic bacteria, E. coli O1:K1:H7 and S. enterica,

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were not capable of utilizing 2′-FG. Our results can be applied to synthesize high

266

value-added FOSs by providing the new design principles for metabolic engineering

267

of industrial microorganisms.

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METHODS

270 271

Strains and Plasmids

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The strains and plasmids used in this study are summarized in Table 1. E. coli DH5α

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and BL21(DE3) (NEB, Ipswich, MA) were used for plasmid construction and as a host

274

for 2′-FG production, respectively. To construct pFucT2 plasmid , fucT2 gene,

275

originating from H. pylori, encoding α1,2-fucosyltransferase was cloned into a pET21a 11 ACS Paragon Plus Environment


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vector.17 To construct plasmid pmBCGWF, two DNA fragments (manB-manC-gmd-

277

wcaG and fucT2) were amplified from pmBCGW and pfucT2, respectively.17 The two

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PCR products were ligated together by in vitro homologous recombination using a

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CloneEZ PCR cloning kit (GenScript, Piscataway, NJ). To construct pFC plasmid, the

280

gene AO826_11705, originating from Xanthomonas manihotis, encoding α1,2-

281

fucosidase was cloned into a pETduet-1 vector. The recombinant plasmid harboring

282

fucT2 or AO826_11705 was transformed into E. coli BL21(DE3) for recombinant

283

protein expression.

284 285

Expression of FucT2 and α1,2-Fucosidase in E. coli BL21(DE3)

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To express recombinant FucT2 and α1,2-fucosidase, the recombinant E. coli cells

287

were grown at 37 °C and 250 rpm in Luria-Bertani (LB) broth (BD, San Jose, CA)

288

containing 100 µg/mL of ampicillin (Sigma-Aldrich, St. Louis, MO) until the mid-

289

exponential phase of growth. The recombinant proteins were then expressed with 0.1

290

mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Roche, Basel, Switzerland) at 16 °C

291

and 250 rpm for 16 h. Following induction, cells were harvested by centrifugation at

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5,488 x g for 30 min at 4 °C, and the cell pellets were suspended in 20 mM Tris-HCl

293

buffer (pH 7.4). The cell suspensions were disrupted by a ultrasonic homogenizer

294

(Branson Digital Sonifier 450, Branson, Danbury, CT), and the supernatants

295

containing soluble recombinant proteins were obtained by centrifugation at 24,371 x g

296

for 30 min at 4 °C. To concentrate soluble crude proteins and to remove intracellular

297

metabolites and media components from crude proteins, the supernatants were

298

filtered by using an Amicon Ultra-15 10K centrifugal filter (EMD Millipore, Billerica, MA).

299 300

Production of Agarobiose by Acid Hydrolysis of Agarose 12 ACS Paragon Plus Environment

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To produce agarobiose, a disaccharide unit of agarose consisting of D-galactose and

302

3,6-anhydro-L-galactose with a β1,4-glycosidic linkage, acid hydrolysis of agarose was

303

used since α1,3-glycosidic bonds of agarose are preferentially hydrolyzed by acid

304

hydrolysis, resulting in the formation of agarooligosaccharides, including agarobiose.37

305

For acid hydrolysis, 10% (w/w) agarose was treated with 2% (w/v) phosphoric acid at

306

90 °C. Time course analyses of the reaction products using high-performance liquid

307

chromatography (HPLC) were performed to determine the optimal reaction time for

308

agarobiose production.

309 310

In Vitro Enzymatic Reactions of Various Sugar Acceptors by FucT2

311

The enzymatic reactions of various sugar acceptors by FucT2 were performed as

312

follows: the reaction mixtures containing 2 mM GDP-L-fucose, 5 mg/mL of the crude

313

enzyme, 1 mM dithiothreitol, and 5 mM of one of the following sugar acceptors,

314

galactose, glucose, lactose, agarobiose, or cellobiose, in 50 mM Tris-HCl (pH 7.0)

315

buffer were incubated at 30 °C and 600 rpm for 12 h. A total volume of the reaction

316

mixture was 200 µL. The enzymatic reactions were stopped by heat inactivation of

317

FucT2 at 95 °C for 5 min.

318 319

Analyses of Reaction Products using LC/MS–IT–TOF and HPLC

320

To analyze various FOSs produced from the enzymatic reaction mentioned above,

321

LC/MS analysis was performed using an LC/MS–IT–TOF system (Shimadzu, Kyoto,

322

Japan) equipped with a Hypercarb Porous Graphitic Carbon LC Column (100 mm ×

323

2.1 mm packed with a 3 µm particle size; Thermo Fisher Scientific, Waltham, USA).38

324

Electrospray ionization was operated in a positive ion mode. The mobile phase

325

comprised solution A (25 µM lithium chloride [LiCl] in water) and B (acetonitrile). The 13 ACS Paragon Plus Environment


326

gradient elution was from 0% to 80% in 41 min at a flow rate of 0.2 mL/min, while the

327

injection volume was 20 µL. The temperatures of LC column and autosampler were

328

maintained at 70 °C and 10 °C, respectively. Source-dependent parameters were set

329

as follows: nebulizing gas flow, 1.5 L/min; interface voltage, 4.5 kV; detector voltage,

330

1.65 kV; a curved desolvation line (CDL) temperature, 200 °C; and heat block

331

temperature, 200 °C. The mass scan range was set from 100 to 700 m/z. LabSolutions

332

LCMS software (version 3.8, Shimadzu) was used for the analysis of LC/MS−IT−TOF

333

data. To obtain tandem MS data from the precursor ions, the collision-induced

334

dissociation (CID) parameters, CID energy, collision gas parameter, and frequency,

335

were set at 50%, 50%, and 45 kHz, respectively. Raw LC/MS−IT−TOF data were

336

processed using LabSolutions LCMS software (version 3.8; Shimadzu).

337

For HPLC analysis, an HPLC system (1200 Series, Agilent Technologies,

338

Waldbronn, Germany) equipped with an H+ (8%) column (Rezex ROA-Organic Acid;

339

Phenomenex, Torrance, CA) and a refractive index detector (RI) were used. The flow

340

rate of the mobile phase, 0.005 N H2SO4, was set at 0.6 mL/min, and the column and

341

RI detector temperatures were set at 50 °C.

342 343

GC/TOF MS Analysis

344

For the GC/TOF MS analysis, 30 µL of reaction product was dried and derivatized with

345

10 μL of 40 mg/mL methoxyamine hydrochloride in pyridine (Sigma-Aldrich) at 200

346

rpm for 90 min at 30 °C for methoxyamination of carbonyl groups. Then,

347

trimethylsilylation of hydroxyl groups was performed by adding 45 μL of N-methyl-N-

348

(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich) at 200 rpm for 30 min at 37 °C. The

349

GC/TOF MS analysis was performed using an Agilent 7890A GC (Agilent

350

Technologies, Wilmington, DE) that was coupled to a Pegasus HT TOF MS (LECO, 14 ACS Paragon Plus Environment

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351

St. Joseph, MI) equipped with an RTX-5Sil MS column (30 m × 0.25 mm, 0.25 μm film

352

thickness; Restek, Bellefonte, PA) with an additional 10-m integrated guard column.

353

Oven temperature was initially set to 50 °C for 1 min, it was increased to 330 °C at 20

354

°C/min, and then held for 5 min. Mass spectra were acquired in a scan range of 85-

355

500 m/z at an acquisition rate of 10 spectra/s. The ionization mode was subjected to

356

electron impact at 70 eV, and the ion source temperature was set to 250 °C. The mass

357

spectra acquired were preprocessed using a LECO ChromaTOF software (version

358

2.32, LECO).

359 360

2′-FG Production by an Engineered E. coli BL21(DE3)

361

For 2′-FG production, E. coli BL21(DE3) pmBCGWF (Table 1) was used as a

362

recombinant strain. The strain was precultured in LB broth containing 50 µg/mL of

363

kanamycin to maintain pmBCGWF plasmid overnight at 37 °C and 200 rpm. The

364

precultured cells were harvested by centrifugation at 5,000 rpm for 30 min at 4 °C, and

365

the cell pellets were then inoculated into 20 mL of LB broth containing 50 µg/mL of

366

kanamycin in a 125-mL Erlenmeyer flask. An initial cell concentration measured by

367

optical density at 600 nm (OD600) was adjusted to 0.1, and the cells were incubated at

368

37 °C and 200 rpm. When the OD600 reached 1.0 (after 6 h), 5 g/L glycerol, a carbon

369

source for the engineered E. coli, 2 g/L galactose, an acceptor for synthesizing 2′-FG,

370

and 0.1 mM IPTG, an inducer for production of recombinant proteins, were added to

371

induce 2′-FG production. The temperature of incubation was switched to 25 °C after

372

the induction. To provide an additional carbon source, 4 g/L of glycerol was supplied

373

when depleted. The concentrations of extracellular metabolites, especially glycerol,

374

galactose, and 2′-FG were monitored by HPLC analysis. The OD600 was measured

375

using a spectrophotometer (BioMate 3S; Thermo Scientific, Waltham, MA). Then, the 15 ACS Paragon Plus Environment


376

value of OD600 was converted into dry cell weight (DCW) by using a conversion factor,

377

0.36, since it was reported that OD600 was correlated to DCW with a ratio DCW / OD600

378

= 0.36.15

379

A fed-batch fermentation for 2′-FG production was carried out in a 3-L bioreactor

380

containing 1 L of defined medium [13.5 g/L KH2PO4, 4.0 g/L (NH4)2HPO4, 1.7 g/L citric

381

acid, 1.4 g/L MgSO4·7H2O, 10 mL/L trace element solution (10 g/L Fe(III) citrate, 2.25

382

g/L ZnSO4·7H2O, 1.0 g/L CuSO4·5H2O, 0.35 g/L MnSO4·H2O, 0.23 g/L

383

Na2B4O7·10H2O, 0.11 g/L (NH4)6Mo7O24, 2.0 g/L CaCl2·2H2O)] containing 20 g/L

384

glycerol and 50 µg/mL kanamycin at 25 °C. After depletion of 20 g/L glycerol added

385

initially, a feeding solution comprising of 800 g/L glycerol and 20 g/L MgSO4·7H2O was

386

fed limitedly to avoid acetate generation. When OD600 reached 60, 0.1 mM IPTG and

387

6 g/L galactose were added to the bioreactor. During the fermentation, pH of the

388

medium was maintained at 6.8. The agitation speed was increased to 1000 rpm to

389

prevent the deficiency of dissolved oxygen; the air flow rate was also increased during

390

the fermentation.

391 392

Purification of 2′-FG by Gel-permeation Chromatography

393

For purification of 2′-FG by gel-permeation chromatography, a column (I.D. 6 × 100

394

cm) packed with Sephadex G-10 resin (Sigma Aldrich) was equilibrated with water.

395

The concentrated culture supernatant containing 2′-FG obtained from fed-batch

396

fermentation using a bioreactor was directly applied onto top of the column, and then

397

eluted using water as a mobile phase. Fractions of 1.5 mL each were eluted, and

398

fractions verified by HPLC to contain 2′-FG only were collected.

399 400

Identification of α1,2-Glycosidic Linkage of 2′-FG by α1,2-Fucosidase Reaction 16 ACS Paragon Plus Environment

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401

To confirm the α1,2-glycosidic bond of 2′-FG, an enzymatic reaction of α1,2-

402

fucosidase was performed on the purified 2′-FG. The reaction mixture containing 1.5

403

mg/mL of 2′-FG and 1 mg/mL of the crude enzyme obtained from E. coli BL21(DE3)

404

harboring α1,2-fucosidase gene in 50 mM sodium phosphate buffer (pH 7.0) was

405

incubated at 30 °C and 500 rpm for 12 h by using a thermomixer (Eppendorf, Hamburg,

406

Germany). As a control, the crude enzyme obtained from E. coli BL21(DE3) harboring

407

the empty vector was used. The reaction products were analyzed using HPLC.

408 409

Stability Test of 2′-FG on Simulated Gastric Fluid

410

To test the stability of 2′-FG on the simulated gastric fluid, the reaction mixture

411

containing 4 g/L 2′-FG with the simulated gastric fluid comprising 0.2% (w/v) sodium

412

chloride in 0.7% (v/v) hydrochloric acid was incubated at 37 °C until 3 h. Degradation

413

of 2′-FG was monitored by HPLC analysis.

414 415

Growth Test of Probiotic and Pathogenic Bacteria Under 2′-FG Carbon Source

416

To investigate the prebiotic effect of 2′-FG, two distinct bifidobacteria, namely

417

Bifidobacterium longum ssp. infantis ATCC 15697 and B. bifidum DSM 20082 were

418

cultured on synthetic De Man, Rogosa and Sharpe (sMRS; Sigma-Aldrich) broth

419

supplemented with 4 g/L 2′-FG as a carbon source. The sMRS broth was composed

420

of 10 g/L peptone, 5 g/L yeast extract, 2 g/L K2HPO4 anhydrous, 5 g/L sodium acetate

421

anhydrous, 2 g/L NH4 citrate tribasic, 0.2 g/L MgSO4·7H2O, 0.05 g/L MnSO4, 1 mL/L

422

Tween 80, and 0.5 g/L cysteine 39. The cells of B. infantis and B. bifidum were cultured

423

in an anaerobic chamber with an anaerobic atmosphere comprising 85% N2, 10% H2,

424

and 5% CO2 (Airgas, Radnor, PA) at 37 °C. During the fermentation, cell growth was



425

monitored by measuring optical density at 595 nm. Moreover, 2′-FG consumption and

426

acetate, lactate, and 1,2-propanediol production were monitored using HPLC.

427

To test the fermentability of 2′-FG on pathogenic bacteria, two pathogens,

428

namely E. coli O1:K1:H7 and Salmonella enterica serovar Typhimurium, were cultured

429

in the modified M9 broth with 0.25% (w/v) yeast nitrogen base, and 4 g/L glucose,

430

lactose, or 2′-FG was supplemented as a sole carbon source. The cells of E. coli

431

O1:K1:H7 and S. enterica were inoculated into 200 µL of the modified M9 media. The

432

cells were incubated at 37 °C with maximum shaking speed, and cell growth was

433

measured using a BioscreenC (Bioscreen, Helsinki, Finland).

434 435

ASSOCIATED CONTENT

436

Supporting Information

437

Supporting Figures S1–S4 generated from the results of in vitro control experiment,

438

acid hydrolysis of agarose, final concentrations of the fermentation products, and

439

stability of 2′-FG in the presence of a simulated gastric fluid.

440 441

AUTHOR INFORMATION

442

Corresponding Authors

443

E-mail address: [email protected] (Y.-S. Jin)

444

E-mail address: [email protected] (K. H. Kim)

445 446

Author Contributions

447

YSJ and KHK conceived the project. JL and SK constructed the plasmids and

448

engineered the strain. EJY, JJL, and JL designed the experiments. JJL, JL, and EJY


Page 18 of 37

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449

performed the fermentation. EJY analyzed data. EJY, SY, KHK, and YSJ wrote the

450

manuscript. All authors read and approved the final manuscript.

451 452

Notes

453

The authors declare no conflict of interest

454 455

ACKNOWLEDGMENTS

456

KHK acknowledges grant supports from the National Research Foundation of Korea

457

(NRF-2017R1A2B2005628 and NRF-2018R1A4A1022589) and the facility support at

458

the Korea University Food Safety Hall for the Institute of Biomedical Science and Food

459

Safety.

460 461

Abbreviations

462

FOSs, fucosyl-oligosaccharides; 2′-FG, 2'-fucosylgalactose; HMOs, human milk

463

oligosaccharides; 2′-FL, 2'-fucosyllactose; GDP-L-fucose, guanosine 5'-diphospho-β-

464

L-fucose; HPLC, high-performance liquid chromatography; LC/MS−IT−TOF, hybrid ion

465

trap/time-of-flight mass spectrometry coupled with liquid chromatography; AHG, 3,6-

466

anhydro-L-galactose.

467 468

References

469

(1) Miyoshi, E., Moriwaki, K., and Nakagawa, T. (2008) Biological function of

470

fucosylation in cancer biology. J. Biochem. 143, 725-729.

471

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472

Goltzman, D. (1992) Structural requirements for the growth factor activity of the amino-

473

terminal domain of urokinase. J. Biol. Chem. 267, 14151-14156. 19 ACS Paragon Plus Environment


474

(3) Pickard, J. M., Maurice, C. F., Kinnebrew, M. A., Abt, M. C., Schenten, D.,

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Golovkina, T. V., Bogatyrev, S. R., Ismagilov, R. F., Pamer, E. G., Turnbaugh, P. J.,

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and Chervonsky, A. V. (2014) Rapid fucosylation of intestinal epithelium sustains host-

477

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(4) Kalovidouris, S. A., Gama, C. I., Lee, L. W., and Hsieh-Wilson, L. C. (2005) A Role

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for fucose α(1−2) galactose carbohydrates in neuronal growth. J. Ame. Chem. Soc.

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127, 1340-1341.

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(5) Yu, Z.-T., Chen, C., Kling, D. E., Liu, B., McCoy, J. M., Merighi, M., Heidtman, M.,

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and Newburg, D. S. (2013) The principal fucosylated oligosaccharides of human milk

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exhibit prebiotic properties on cultured infant microbiota. Glycobiology. 23, 169-177.

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(6) Newburg, D. S., Ruiz-Palacios, G. M., and Morrow, A. L. (2005) Human milk

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glycans protect infants against enteric pathogens. Annu. Rev. Nutr. 25, 37-58.

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(7) Morrow, A. L., Ruiz-Palacios, G. M., Altaye, M., Jiang, X., Lourdes Guerrero, M.,

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Meinzen-Derr, J. K., Farkas, T., Chaturvedi, P., Pickering, L. K., and Newburg, D. S.

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in breast-fed infants. J. Pediatr. 145, 297-303.

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(8) Coppa, G. V., Zampini, L., Galeazzi, T., Facinelli, B., Ferrante, L., Capretti, R., and

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Orazio, G. (2006) Human milk oligosaccharides inhibit the adhesion to Caco-2 cells of

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diarrheal pathogens: Escherichia coli, Vibrio cholerae, and Salmonella fyris. Pediatr.

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Res. 59, 377-382.

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synthesis of 2′-fucosyllactose. Microb. Cell Fact. 12, 40.

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(10) Matsuki, T., Yahagi, K., Mori, H., Matsumoto, H., Hara, T., Tajima, S., Ogawa, E.,

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key genetic factor for fucosyllactose utilization affects infant gut microbiota

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rat hippocampus. NeuroReport. 9, 813-817.

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(12) Murrey, H. E., Ficarro, S. B., Krishnamurthy, C., Domino, S. E., Peters, E. C., and

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Hsieh-Wilson, L. C. (2009) Identification of the plasticity-relevant fucose-α(1-2)-

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galactose proteome from the mouse olfactory bulb. Biochemistry. 48, 7261-7270.

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(13) Krug, M., Wagner, M., Staak, S., and Smalla, K. H. (1994) Fucose and fucose-

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containing sugar epitopes enhance hippocampal long-term potentiation in the freely

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(14) Dékany, G., Agoston, K., Bajza, I., Boutet, J., BØJSTRUP, M., Fanefjord, M.,

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PÉREZ, I. F., Hederos, M., Horvath, F., and KOVÁCS-PÉNZES, P. Synthesis of 2'-O-

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fucosyllactose. US Patent WO2010115935A1; 2010.

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(15) Chin, Y.-W., Kim, J.-Y., Lee, W.-H., and Seo, J.-H. (2015) Enhanced production

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of 2′-fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation of

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lactose metabolism and fucosyltransferase. J. Biotechnol. 210, 107-115.

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(16) Chin, Y.-W., Seo, N., Kim, J.-H., and Seo, J.-H. (2016) Metabolic engineering of

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Escherichia coli to produce 2′-fucosyllactose via salvage pathway of guanosine 5′-

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diphosphate (GDP)-L-fucose. Biotechnol. Bioeng. 113, 2443-2452.

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(17) Lee, W.-H., Pathanibul, P., Quarterman, J., Jo, J.-H., Han, N. S., Miller, M. J., Jin,

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Y.-S., and Seo, J.-H. (2012) Whole cell biosynthesis of a functional oligosaccharide,

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2′-fucosyllactose, using engineered Escherichia coli. Microb. Cell Fact. 11, 48.

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(18) Liu, J. J., Kwak, S., Pathanibul, P., Lee, J. W., Yu, S., Yun, E. J., Lim, H., Kim, K.

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H., and Jin, Y. S. (2018) Biosynthesis of a functional human milk oligosaccharide, 2'21 ACS Paragon Plus Environment


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fucosyllactose, and L-fucose using engineered Saccharomyces cerevisiae. ACS

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Synth. Biol. 7, 2529-2536.

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(19) Yu, S., Liu, J.-J., Yun, E. J., Kwak, S., Kim, K. H., and Jin, Y.-S. (2018) Production

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of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered

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Saccharomyces cerevisiae. Microb. Cell Fact. 17, 101.

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(20) Yun, E. J., Choi, I.-G., and Kim, K. H. (2015) Red macroalgae as a sustainable

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resource for bio-based products. Trends Biotechnol. 33, 247-249.

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(21) Kim, S. R., Ha, S.-J., Wei, N., Oh, E. J., and Jin, Y.-S. (2012) Simultaneous co-

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fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol.

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Trends Biotechnol. 30, 274-282.

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(22) Engels, L., and Elling, L. (2014) WbgL: a novel bacterial α1,2-fucosyltransferase

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for the synthesis of 2'-fucosyllactose. Glycobiology. 24, 170-178.

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(23) Li, M., Liu, X. W., Shao, J., Shen, J., Jia, Q., Yi, W., Song, J. K., Woodward, R.,

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Chow, C. S., and Wang, P. G. (2008) Characterization of a novel α1,2-

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fucosyltransferase of Escherichia coli O128:B12 and functional investigation of its

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common motif. Biochemistry. 47, 378-387.

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(24) Wang, G., Boulton, P. G., Chan, N. W., Palcic, M. M., and Taylor, D. E. (1999)

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Novel Helicobacter pylori α1,2-fucosyltransferase, a key enzyme in the synthesis of

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Lewis antigens. Microbiology. 145, 3245-3253.

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3,6-anhydro-L-galactose dehydrogenase indicates its essentiality in the agar

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catabolism of a marine bacterium. Process Biochem. 64, 130-135.

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ancestors and relatives of Escherichia coli B, and the derivation of B strains REL606

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Understanding the differences between genome sequences of Escherichia coli B

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strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes.

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(28) Huang, D., Yang, K., Liu, J., Xu, Y., Wang, Y., Wang, R., Liu, B., and Feng, L.

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(2017) Metabolic engineering of Escherichia coli for the production of 2′-fucosyllactose

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and 3-fucosyllactose through modular pathway enhancement. Metab. Eng. 41, 23-38.

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(29) Wong-Madden, S. T., and Landry, D. (1995) Purification and characterization of

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novel glycosidases from the bacterial genus Xanthomonas. Glycobiology. 5, 19-28.

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(30) Sela, D. A., Garrido, D., Lerno, L., Wu, S., Tan, K., Eom, H. J., Joachimiak, A.,

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Lebrilla, C. B., and Mills, D. A. (2012) Bifidobacterium longum subsp. infantis ATCC

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15697 α-fucosidases are active on fucosylated human milk oligosaccharides. Appl.

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Environ. Microbiol. 78, 795-803.

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(31) Sela, D. A., Chapman, J., Adeuya, A., Kim, J. H., Chen, F., Whitehead, T. R.,

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Lapidus, A., Rokhsar, D. S., Lebrilla, C. B., German, J. B., Price, N. P., Richardson,

564

P. M., and Mills, D. A. (2008) The genome sequence of Bifidobacterium longum subsp.

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infantis reveals adaptations for milk utilization within the infant microbiome. Proc. Natl.

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Acad. Sci. U. S. A. 105, 18964-18969.

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(32) Ashida, H., Miyake, A., Kiyohara, M., Wada, J., Yoshida, E., Kumagai, H.,

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Katayama, T., and Yamamoto, K. (2009) Two distinct α-L-fucosidases from

569

Bifidobacterium bifidum are essential for the utilization of fucosylated milk

570

oligosaccharides and glycoconjugates. Glycobiology. 19, 1010-1017.

571

(33) Bunesova, V., Lacroix, C., and Schwab, C. (2016) Fucosyllactose and L-fucose

572

utilization of infant Bifidobacterium longum and Bifidobacterium kashiwanohense.

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BMC Microbiol. 16, 248. 23 ACS Paragon Plus Environment


574

(34) Chessa, D., Winter, M. G., Jakomin, M., and Baumler, A. J. (2009) Salmonella

575

enterica serotype Typhimurium Std fimbriae bind terminal a(1,2)fucose residues in the

576

cecal mucosa. Mol. Microbiol. 71, 864-875.

577

(35) Newburg, D. S., Pickering, L. K., McCluer, R. H., and Cleary, T. G. (1990)

578

Fucosylated oligosaccharides of human milk protect suckling mice from heat-stabile

579

enterotoxin of Escherichia coli. J. Infect. Dis. 162, 1075-1080.

580

(36) Magalhaes, A., and Reis, C. A. (2010) Helicobacter pylori adhesion to gastric

581

epithelial cells is mediated by glycan receptors. Braz. J. Med. Biol. Res. 43, 611-618.

582

(37) Yang, B., Yu, G., Zhao, X., Jiao, G., Ren, S., and Chai, W. (2009) Mechanism of

583

mild acid hydrolysis of galactan polysaccharides with highly ordered disaccharide

584

repeats leading to a complete series of exclusively odd-numbered oligosaccharides.

585

FEBS J. 276, 2125-2137.

586

(38) Kim, J. H., Yun, E., Yu, S., Kim, K., and Kang, N. (2017) Different levels of skin

587

whitening activity among 3,6-anhydro-L-galactose, agarooligosaccharides, and

588

neoagarooligosaccharides. Mar. Drugs. 15, 321.

589

(39) Barrangou, R., Altermann, E., Hutkins, R., Cano, R., and Klaenhammer, T. R.

590

(2003) Functional and comparative genomic analyses of an operon involved in

591

fructooligosaccharide utilization by Lactobacillus acidophilus. Proc. Natl. Acad. Sci. U.

592

S. A. 100, 8957-8962.

593 594

Figure captions

595 596

Figure 1. (A) Biosynthetic route for producing 2′-FG using an α1,2-fucosyltransferase

597

originating from H. pylori, FucT2, with galactose as an acceptor and GDP-L-fucose as


Page 24 of 37

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598

a glycosylation donor. (B) Sugar acceptors used in this study for the biosynthesis of

599

various fucosyl-oligosaccharides.

600 601

Figure 2. Mass spectra of (A) 2′-FG, (B) 2′-fucosylglucose, (C) 2′-fucosyllactose, (D)

602

2′-fucosylcellobiose, and (E) 2′-fucosylagarobiose, which were obtained from in vitro

603

enzymatic reactions of galactose, glucose, lactose, cellobiose, and agarobiose as

604

acceptors and GDP-L-fucose as a donor by an α1,2-fucosyltransferase, FucT2,

605

respectively. Mass spectra were acquired by LC/MS−IT−TOF analysis.

606 607

Figure 3. Tandem mass spectra of (A) 2′-FG and (B) 2′-fucosylglucose, (C) 2′-

608

fucosyllactose, (D) 2′-fucosylcellobiose, and (E) 2′-fucosylagarobiose, which were

609

obtained from in vitro enzymatic reactions of galactose, glucose, lactose, cellobiose,

610

and agarobiose as acceptors and GDP-L-fucose as a donor by an α1,2-

611

fucosyltransferase,

612

LC/MS−IT−TOF analysis.

FucT2,

respectively.

Mass

spectra

were

acquired

by

613 614

Figure 4. (A) Metabolic design to produce 2′-FG in E. coli BL21(DE3). In order to

615

accumulate intracellular GDP-L-fucose, a de novo pathway for producing GDP-L-

616

fucose was amplified by overexpressing ManB, ManC, Gmd, and WcaG. H. pylori

617

fucT2 coding for α1,2-fucosyltransferase was introduced into the engineered E. coli

618

accumulating GDP-fucose. Galactose and glycerol were supplied as an acceptor and

619

as a carbon source, respectively. Production of 2′-FG by an engineered Escherichia

620

coli BL21(DE3) from fed-batch fermentations in (B) a shake flask and (C) a bioreactor.

621

During the fermentations, concentrations of dry cell weight (DCW), glycerol, galactose,



622

and 2′-FG were monitored. For the shake flask fermentation, data are presented as

623

mean value and standard deviations of two independent biological replicates.

624 625

Figure 5. Purification of 2'-FG and identification of α1,2-glycosidic linkage in 2'-FG.

626

(A–B) Purification of 2′-FG from the supernatant of the culture of engineered E. coli

627

BL21(DE3). HPLC chromatograms of (A) culture supernatant and (B) fraction

628

containing solely 2′-FG obtained from gel permeation chromatography of culture

629

supernatant using a G-10 column. (C–D) Identification of α1,2-glycosidic linkage in 2′-

630

FG by in vitro enzymatic reaction of α1,2-fucosidase. HPLC chromatograms of the

631

reaction products obtained from in vitro enzymatic reactions with crude enzymes of

632

(C) E. coli BL21(DE3) harboring α1,2-fucosidase gene or (D) an empty vector (control).

633

For the in vitro enzymatic reactions, the reaction mixture containing 1.5 mg/mL of 2′-

634

FG and 1 mg/mL of crude enzymes from E. coli BL21(DE3) harboring α1,2-fucosidase

635

gene or an empty vector (control) in 50 mM sodium phosphate buffer (pH 7.0) was

636

incubated at 30 °C and 500 rpm for 12 h.

637 638

Figure 6. Prebiotic effect of 2′-FG. (A) Cell growth of Bifidobacterium longum ssp.

639

infantis ATCC 15697 (B. infantis) and B. bifidum DSM 20082 (B. bifidum). (B and C)

640

Fermentation profiles of B. infantis and (D and E) B. bifidum. Two distinct bifidobacteria,

641

B. infantis and B. bifidum, were cultured in the MRS broth supplemented with 4 g/L of

642

2′-FG as a carbon source at 37 °C under anaerobic conditions. During fermentation,

643

the cell density (optical density at 595 nm) and the concentrations of 2′-FG, galactose,

644

fucose, acetate, lactate, and 1,2-propanediol were monitored. All experiments were

645

performed in duplicate.

646 26 ACS Paragon Plus Environment

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647

Figure 7. Growth tests of two pathogenic bacteria using 2′-FG as a carbon source. (A)

648

E. coli O1:K1:H7 and (B) S. enterica serotype Typhimurium were cultured on the

649

modified M9 medium supplemented 4 g/L of glucose, lactose, or 2′-FG as a sole

650

carbon source at 37 °C for 60 h. All experiments were performed in duplicate.

651 652 653 654 655 656 657 658 659 660 661 662 663

Fig. 1

664



665 666 667 668 669 670 671 672 673 674

Fig. 2


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676 677 678 679 680

Fig. 3



682 683 684 685 686

Fig. 4


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688 689 690 691 692 693 694 695 696 697 698

Fig. 5



700 701 702 703 704 705 706 707 708 709 710 711

Fig. 6


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713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728

Fig. 7



730 731 732 733 734 735 736 737 738 739 740 741 742 743


Page 34 of 37

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Table 1. Strains and plasmids used in this study. Strain/Plasmid

Description

Reference/source

Strain F−, ϕ80d, lacZΔM15, endA1, recA1, hsdR17(rK-mK-), supE44, E. coli DH5α

Invitrogen thi-1, gyrA96, relA1, Δ(lacZYA-argF)U169

E. coli BL21(DE3)

F−, ompT, hsdSB(rB-mB-), gal, dcm rne131 (DE3)

Invitrogen

E. coli BL21(DE3) pET21a

BL21(DE3) harboring pET21a plasmid

This study

E. coli BL21(DE3) pFucT2

BL21(DE3) harboring pFucT2 plasmid

This study

E. coli BL21(DE3) pmBCGWF

BL21(DE3) harboring pmBCGWF plasmid

This study

E. coli BL21(DE3) pETduet-1

BL21(DE3) harboring pETduet-1 plasmid

This study

E. coli BL21(DE3) pFC

BL21(DE3) harboring pFC plasmid

This study

pET21a

T7-lac promoter, pBR322 replicon, AmpR

Novagen

pETduet-1

Two T7 promoters with two MCS, pBR322 replicon, AmpR

Novagen

pCOLAduet-1

Two T7 promoters with two MCS, ColA replicon, KanR

Novagen

pFucT2

Derived from pET21a, PT7- fucT2-TT7, AmpR

This study

Plasmid


ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

pmBCGWF

Derived from pCOLAduet-1, PT7-manB, manC-PT7-gmd, wcaG-

Page 36 of 37

This study

PT7-fucT2-TT7, KanR pFC

Derived from pETduet-1, PT7-α-L-fucosidase-PT7-MCS2-TT7, AmpR


This study

Page 37 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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Biosynthetic routes for producing various fucosyl-oligosaccharides

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