Biosynthesis of a functional human milk oligosaccharide, 2

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Biosynthesis of a functional human milk oligosaccharide, 2#Fucosyllactose, and L-fucose, using engineered Saccharomyces cerevisiae JINGJING LIU, Suryang Kwak, Panchalee Pathanibul, Jaewon Lee, Sora Yu, Eun Ju Yun, Hayoon Lim, Kyoung Heon Kim, and Yong-Su Jin ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00134 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

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ACS Synthetic Biology

Submitted to ACS Synthetic Biology

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Biosynthesis of a functional human milk oligosaccharide, 2′-Fucosyllactose, and L-fucose,

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using engineered Saccharomyces cerevisiae

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Jing-Jing Liu a, Suryang Kwak a,b, Panchalee Pathanibul b, Jae Won Lee b, Sora Yu c, Eun Ju Yun a,c,

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Hayoon Lim a, Kyoung Heon Kim c, Yong-Su Jin a,b,*

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a

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Urbana, IL, USA

Carl R. Woose Institute for Genomic Biology, University of Illinois at Urbana-Champaign,

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b

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Urbana, IL, USA

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c

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Korea

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign,

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

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* Corresponding author: Yong-Su Jin ([email protected]).

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Mailing address: 1206 W. Gregory Drive, Carl R. Woese Institute for Genomic Biology, Urbana,

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IL 61801, USA

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2 ACS Paragon Plus Environment

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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2’-Fucosyllactose (2-FL), one of the most abundant human milk oligosaccharides (HMOs), has

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received much attention due to its health-promoting activities, such as stimulating the growth of

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beneficial gut microorganisms, inhibiting pathogen infection, and enhancing the host immune

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system. Consequently, large quantities of 2-FL are on demand for food applications as well as in

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depth investigation of its biological properties. Biosynthesis of 2-FL has been attempted primarily

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in Escherichia coli, which might not be the best option to produce food and cosmetic ingredients

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due to the presence of endotoxins on the cell surface. In this study, an alternative route to produce

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2-FL using a food-grade microorganism, Saccharomyces cerevisiae, has been devised.

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Specifically, heterologous genes, which are necessary to achieve the production of 2-FL from a

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mixture of glucose and lactose, were introduced into S. cerevisiae. When the lactose transporter

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(Lac12), de novo GDP-L-fucose pathway (consisting of GDP-D-mannose-4,6-dehydratase (Gmd)

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and GDP-4-keto-6-deoxymannose-3,5-epimerase- 4-reductase (WcaG)), and α1,2-

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fucosyltransferase (FucT2) were introduced, the resulting engineered strain (D452L-gwf)

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produced 0.51 g/L of 2-FL from a batch fermentation. In addition, 0.41 g/L of L-fucose was

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produced when α-L-fucosidase was additionally expressed in the 2-FL producing strain (D452L-

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gwf). To our knowledge, this is the first report of 2-FL and L-fucose production in engineered S.

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cerevisiae via the de novo pathway. This study provides the possibility of producing HMOs by a

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food grade microorganism S. cerevisiae, and paved the way for more HMO productions in the

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future.

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Keywords: Saccharomyces cerevisiae, de novo pathway, 2’-fucosyllactose, α-L-fucosidase, L-

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fucose. 3 ACS Paragon Plus Environment

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Human milk oligosaccharides (HMOs) are unique oligosaccharides which are found

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only in human milk 1. HMOs have been reported to confer many health benefits to the host

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including stimulating the growth of Bifidobacteria (prebiotic effect), serving as receptor

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analogs for pathogens, and modulating immune responses 2, 3. The most abundant type of HMO

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is a fucosylated (L-fucose containing) type constituting over 70% of total HMOs 4. In

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particular, 2’-fucosyllactose (2-FL) is the most prevalent fucosylated HMO accounting for

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about 30% of total HMOs 5. 2-FL has the most basic structure of fucosylated HMOs with one

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fucose unit connected to lactose on the galactose end. 2-FL has been demonstrated protective

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activities against several pathogenic microorganisms, such as Campylobacter jejuni,

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enteropathogenic Escherichia coli (EPEC), Salmonella enterica and Norovirus 6-9.

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In order to further evaluate putative biological functions of 2-FL, and enable the

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addition of 2-FL to various foods, economic and scalable production of 2-FL is necessary. 2-FL

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has been isolated from surplus human milk, and artificially synthesized via chemical or

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enzymatic reactions 10-13. Isolation of 2-FL from human milk requires complex purification

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steps which makes this technique impractical for large-scale production. Chemical synthesis of

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2-FL is challenging due to intricate reaction processes and the use of toxic reagents. To

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produce 2-FL via the enzymatic reaction route, the present shortcomings are high costs of

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GDP-L-fucose substrate and purification of enzyme α1,2-fucosyltransferase (FucT2) catalyzing

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the transfer of fucose from GDP-L-fucose to the acceptor, lactose 14.

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Whole cell synthesis of 2-FL by engineered microorganisms is an alternative strategy

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bypassing the abovementioned drawbacks of other synthetic methods. Whole cell synthesis of 2-

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FL has been attempted primarily in Escherichia coli 15-19 thus far, but no case of 2-FL

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biosynthesis in Saccharomyces cerevisiae has been reported. S. cerevisiae could be a preferred



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host to produce certain fucosylated HMOs owing to its rich intracellular pool of GDP-D-

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mannose 20. As GDP-D-mannose is a substrate of de novo pathway of GDP-L-fucose, which is a

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precursor of 2-FL biosynthesis, this gives an advantage for the bulk production of 2-FL by

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engineered yeast. Additionaly, as a GRAS (generally recognized as safe) microorganism,

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engineered S. cerevisiae capable of secreting HMOs might be utilized in food fermentation

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directly to generate HMOs in foods without the concerns regarding endotoxin carryover from

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engineered E. coli 21, 22. As such, 2-FL producing yeast would be favorable in multiple

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applications to provide additional health benefits and values to the products.

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To achieve 2-FL production in S. cerevisiae, ample supply of key precursors including

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lactose and GDP-L-fucose is necessary. Native S. cerevisiae cannot metabolize nor import

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lactose. Genetic modifications are necessary to enable S. cerevisiae to transport lactose in the

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culture medium into the cytosol. Lactose permease (Lac12) from Kluyveromyces lactis, or

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cellodextrin transporter (Cdt-1) from Neurospora crassa can be introduced into S. cerevisiae to

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allow the uptake of external lactose into the cytosol 23-25. GDP-L-fucose, as another precursor

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for 2-FL production, may be generated through an in vivo metabolic conversion from GDP-D-

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mannose (de novo pathway) or L-fucose (salvage pathway). The de novo pathway involves two

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enzymatic reactions of GDP-D-mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-

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deoxymannose-3,5-epimerase- 4-reductase (WcaG) 20, 26, 27. In the salvage pathway, L-fucose

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provided extracellularly and transported to the cytosol is converted into GDP-L-fucose by a

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bifunctional enzyme, L-fucose kinase/L-fucose-1-phosphate guanylyltransferase (FKP) 28.

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GDP-L-fucose production in S. cerevisiae can then be attained through the de novo pathway 20,

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component for 2-FL production in vivo is α1,2-fucosyltransferase (FucT2), which catalyzes the

or the salvage pathway if L-fucose is extracellularly supplemented 30. Finally, the last


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transfer of L-fucose from GDP-L-fucose to lactose 14. FucT2 from Helicobacter pylori has been

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studied extensively and shown to be functionally expressed in E. coli 31. In addition, α-L-

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fucosidase from Xanthomonas manihotis was reported to hydrolyze 2-FL into L-fucose and

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lactose 32. By introducing α-L-fucosidase into a 2-FL producing yeast, in vivo L-fucose

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production can be feasible.

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L-fucose, a precursor for biosynthesis of GDP-L-fucose in the salvage pathway, can be

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produced through chemical modifications of other hexose sugars 33, direct extraction from

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brown algae hydrolysates, and enzymatic hydrolysis of L-fucose-rich microbial

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exopolysaccharide (EPS) 33. However, economic and large-scale production of L-fucose is still

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limited and challenging. Thus, it might not be cost-effective to use L-fucose for the large-scale

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industrial production of 2-FL via the salvage pathway. Production of L-fucose by engineered S.

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cerevisiae could also be appealing as the demand of L-fucose is increasing in cosmetics, food

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products, pharmaceuticals, and biomedical applications. Although GDP-L-fucose production in S. cerevisiae is successful through both de novo

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and salvage pathways 20, 29, 30, 2-FL production in yeast has not been reported. In this study,

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three genetic perturbations to enable 2-FL synthesis in yeast have been introduced into S.

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cerevisiae (Fig. 1). First, a lactose transporter (Lac12) from K. lactis was integrated into S.

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cerevisiae D452-2. Second, Gmd and WcaG from E. coli K-12 were overexpressed in S.

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cerevisiae to empower in vivo GDP-L-fucose production through de novo pathway. Third,

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FucT2 from H. pylori was expressed in S. cerevisiae to transfer fucose unit from GDP-L-fucose

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to lactose. The production of 2-FL was observed in the resulting engineered yeast strain.

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Finally, L-fucose production was accomplished by additional expression of α-L-fucosidase

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from X. manihotis in the 2FL producing strain. In this study, we demonstrate the feasibility of



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producing 2-FL and L-fucose by engineered S. cerevisiae via de novo pathway.

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RESULTS AND DISCUSSION

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Lactose transport and GDP-L-fucose accumulation in engineered S. cerevisiae. As S.

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cerevisiae does not naturally assimilate lactose which is a precursor for 2-FL synthesis, the

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introduction of a heterologous lactose transporter is necessary to produce 2-FL in the cytosol

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of S. cerevisiae. Therefore, LAC12 coding for lactose permease from K. lactis, 34 was

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integrated into the genome of the D452-2 strain under the control of a constitutive promoter

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(pGPD). To evaluate the functional expression of LAC12 in S. cerevisiae, the intracellular

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lactose concentrations of the D452L strain and a parental strain (D452-2) were measured after

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incubating cells with 3 g/L of lactose for 6 h. The D452L strain expressing LAC12

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accumulated 0.11 g lactose/g cell intracellularly while the parental strain D452-2 showed no

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accumulation of intracellular lactose (Fig. 2a and Fig. 2b). It was reported that lactose could

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be toxic to the mutant K. lactis lacking β-galactosidase gene due to an excessive accumulation

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of lactose in the cytosol 35. We also tested lactose toxicity in our engineered strain D452L (Fig.

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S1 and S2). Consistent with a previous report, lactose toxicity towards glucose and galactose

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uptake was observed and the toxicity was lactose dose-dependent. Thus, low lactose

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concentration was chosen for 2-FL production. Nonetheless, lactose assimilation in the

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engineered S. cerevisiae (D452L) via heterologous expression of LAC12 was confirmed.

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The other precursor for 2-FL biosynthesis is GDP-L-fucose that serves as a fucosyl

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donor for the fucosylation of transported lactose. GDP-L-fucose can be synthesized from

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GDP-D-mannose which is already synthesized in yeast by introducing two enzymes: GDP-D-

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mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase


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(WcaG) from E. coli (Fig. 1). The products of E. coli Gmd/WcaG was previously

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characterized as GDP-L-fucose via NMR 36. Overexpression of Gmd and WcaG in the D452L

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strain resulted in the D52L-gw strain which can accumulate GDP-L-fucose intracellularly. The

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D452L-gw strain accumulated 1.56 mg GDP-L-fucose /g cell after incubating for 50 h in YPD

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(Fig. 2c and Fig. 2d). By overexpressing E. coli Gmd and WcaG, efficient production of GDP-

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L-fucose in S. cerevisiae was achieved.

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Production of 2-FL in engineered S. cerevisiae. As the D452L-gw strain harboring lactose

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permease, and GDP-L-fucose producing enzymes can assimilate lactose and produce GDP-L-

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fucose intracellularly, the last enzymatic reaction that was introduced for the production of 2-

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FL is α-1,2-fucosyltransferase (FucT2), which can transfer fucosyl group from GDP-L-fucose

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into lactose. FucT2 from H. pylori was introduced into the D452L-gw to construct the D452L-

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gwf strain expressing all of the necessary enzymes needed to produce 2-FL. When the D452L-

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gwf was cultured in YP medium with 40 g/L of glucose and 3 g/L of lactose (Fig. 3), 2-FL

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production was observed (Fig. 3c). All the glucose was consumed within 20 h (Fig. 3a) and

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the yeast cells continued to grow, utilizing ethanol as a carbon source after glucose depletion

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(Fig. 3a). The extracellular 2-FL concentration measured from the culture broth, reached 0.42

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g/L, and the total 2-FL concentration measured after cell lysis of cells was 0.56 g/L with a

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productivity of 0.0058 g/L/h, indicating that 25% of synthesized 2-FL was trapped inside the

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yeast cells (Fig. 3c and Fig. 3d). Until 96 h, 1.7 g/L of lactose was consumed by the

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engineered strain D452L-gwf (Fig. 3b). Thus, the final yield of 2-FL from lactose was 0.229

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mol/mol. The production of 2-FL from yeast could not compete with that from E. coli (1.23

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g/L − 9.12 g/L) 15-19. Nonetheless, we demonstrated that 2-FL can be produced by engineered

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yeast expressing Lac12, Gmd, WcaG, and FucT2.



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L-Fucose production in engineered S. cerevisiae. To further investigate whether or not 2-FL

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production by engineered yeast was hindered by the accumulation of 2-FL, α-L-fucosidase

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from X. manihotis 32 was introduced into the D452L-gwf strain. In vitro activity of α-L-

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fucosidase was confirmed by incubating 2-FL with the cell lysates of the D452L-gwf-fuco

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strain expressing α-L-fucosidase and the D452L-gwf strain carrying an empty plasmid (Fig.

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4). The cell lysate from the D452L-gwf-fuco strain hydrolyzed 2-FL into L-fucose and lactose,

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while the control strain did not (Fig. 4a and 4b). Because direct L-fucose production from

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GDP-L-fucose by α-L-fucosidase can interfere with our intended experiments, we also

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examined if α-L-fucosidase can hydrolyze GDP-L-fucose. As expected, α-L-fucosidase could

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not hydrolyze GDP-L-fucose (Supporting Information Fig. S3). L-fucose and 2-FL were

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measured by high-performance liquid chromatography (HPLC) and verified by gas

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chromatography/mass spectrometry (GC/MS)37 (Fig. 4c-e) . α-L-fucosidase hydrolyzed 0.06

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g/L of 2-FL per mg DCW within 1 h in vitro. These results confirmed that α-L-fucosidase was

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functionally expressed in S. cerevisiae.

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After confirming the activity of α-L-fucosidase in S. cerevisiae in vitro, we performed

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yeast fermentation and monitored the production of 2-FL and L-fucose (Fig. 5). The D452L-

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gwf-fuco strain expressing α-L-fucosidase consumed lactose more slowly during the

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fermentation than the control strain D452L-gwf with an empty plasmid (Fig. 5a). This reduced

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lactose consumption might be attributed to the recycling of lactose in the cell through

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hydrolysis of 2-FL by α-L-fucosidase (Fig. 1). The D452L-gwf with an empty plasmid

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produced 0.51 g/L of 2-FL without L-fucose production. However, the α-L-fucosidase

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expressing D452L-gwf-fuco strain produced 0.41 g/L of L-fucose extracellularly without 2-FL

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production (Fig. 5b and 5c). L-fucose was not detected even in the lysate from the D452L-

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gwf-fuco cells; indicating that L-fucose could be secreted into culture broth efficiently. As L-

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fucose can be generated only through hydrolysis of 2-FL, 0.41 g/L of L-fucose production can

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be interpreted that the D452L-gwf-fuco could potentially have a capacity to produce up to 1.22

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g/L of 2-FL if the produced L-fucose remained in the 2-FL as molecular weights of 2-FL and

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L-fucose are 488 and 164 g/mol, respectively. These results suggest that 2-FL production

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could be further improved with efficient export of intracellular 2-FL.

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Previously, microbial production of 2-FL has been attempted intensively using E. coli as

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a host strain 15-19. The 2-FL titer from engineered E. coli harboring the de novo pathway

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reached 9.12 g/L in batch culture 17, and the highest 2-FL titer of 23.1 g/L was achieved by a

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fed-batch culture of an engineered E. coli strain harboring the salvage pathway 16. While high

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titers were obtained with engineered E. coli, there are concerns of using a non-GRAS

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microorganism for the production of food and cosmetic ingredients. Also, bacterial

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fermentation always faces the risk of bacteriophage infection, which can be catastrophic for

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large-scale fermentation. As such, 2-FL production by engineered S. cerevisiae containing the

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de novo pathway was attempted. In this study, we demonstrated the feasibility of 2-FL

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production using a safe and food grade microorganism — S. cerevisiae.

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As mentioned above, one possible reason for inefficient 2-FL production by engineered

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yeast might be due to the limitation of 2-FL secretion. We can speculate that inefficient

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secretion might increase the intracellular concentration of 2-FL and the elevated 2-FL levels

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could potentially cause feedback inhibition on the 2-FL synthesis pathway. In order to

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examine whether the export of intracellular 2-FL to a culture medium is indeed a limiting

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factor of 2-FL production by engineered yeast, we introduced α-L-fucosidase, which

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hydrolyzes 2-FL into lactose and L-fucose, into a 2-FL producing yeast. The intracellular



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hydrolysis of 2-FL into lactose and L-fucose will eliminate 2-FL buildup so that the maximum

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potential of the 2-FL synthesis pathway can be reached. Our results showed that the strain

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D452L-gwf-fuco expressing α-L-fucosidase produced 0.41 g/L of L-fucose, while the control

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strain D452L-gwf (with an empty plasmid) produced 0.51 g/L of 2-FL without L-fucose

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production (Fig. 5b and 5c). 0.41 g/L of L-fucose production indicated that the engineered

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yeast D452L-gwf-fuco has a potential to generate 1.22 g/L of 2-FL, since L-fucose can only be

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generated through hydrolysis of 2-FL. These results suggest that intracellularly accumulated 2-

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FL may have inhibited the enzymes in the 2-FL biosynthesis pathway of S. cerevisiae, and 2-

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FL production could be further improved if efficient export of intracellular 2-FL is facilitated.

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Another potential cause of the inefficient 2-FL production by engineered yeast might be

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due to a mismatch of intracellular lactose and GDP-L-fucose concentrations. Since lactose was

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efficiently transported into the yeast cells by Lac12, the intracellular lactose concentration

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reached 0.11 g lactose/g cell within 6 h, (i.e. 10 wt% of yeast biomass was lactose). However,

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the intracellular GDP-L-fucose concentration was 1.56 mg GDP-L-fucose/g cell, which was

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70-fold lower than the intracellular lactose concentration. Furthermore, intracellular GDP-L-

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fucose levels may not be high enough for the fucosylation reaction of lactose to occur in order

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to produce 2-FL. The excessive intracellular lactose is toxic to engineered yeast. Lodi et al.

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demonstrated that lactose could be toxic to the mutant K. lactis lacking β-galactosidase gene

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due to an excessive accumulation of lactose in the cytosol 35. We also noticed a similar toxic

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effect caused by lactose in our engineered yeast strains carrying Lac12 transporter without β-

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galactosidase (Supporting Information Fig. S1 and S2). As such, we tested different lactose

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concentrations on 2-FL production (Supporting Information Fig. S4) and chose 3 g/L of

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lactose for our current study. Even with only 3 g/L of lactose in the medium, almost 10 wt% of

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the yeast cell contained lactose, indicating that Lac12 is a very efficient lactose transporter.

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However, for further enhancement of 2-FL production, Lac12 activity must also be carefully

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adjusted in order to both maintain a sufficient supply of lactose and to minimize toxicity

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effects.

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The titer of 2-FL (0.4 − 0.5 g/L) by the engineered yeast in batch culture was much

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lower than those of 2-FL (1.23 g/L − 9.12 g/L) by engineered E. coli 15-19. There are limited

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capabilities of S. cerevisiae regarding expression and folding of bacterial enzymes such as D‐

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psicose 3- epimerase (DPEase) from Agrobacterium tumefaciens38, L-arabinose isomerase (L-

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AI)39, and xylose isomerase40 from E. coli have also been reported. This is likely another cause

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of lower 2-FL production in yeast than bacteria. Future work will focus on screening more

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suitable enzymes for yeast expression or introducing GroE chaperonins to assist functional

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expression of bacterial enzymes in yeast 38

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CONCLUSIONS In conclusion, we successfully achieved 2-FL production by engineered S. cerevisiae

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through de novo pathway. The production of L-fucose was also achieved after introducing α-L-

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fucosidase into the 2-FL producing engineered yeast. While the titer (0.51 g/L) of 2-FL

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obtained by engineered yeast is not as high as those reported by engineered E. coli, balancing

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intracellular substrate ratios, alleviating lactose toxicity, and facilitating efficient 2-FL

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secretion will be further investigated in order to obtain higher titers of 2-FL and L-fucose by

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engineered S. cerevisiae. For future studies, alleviation of lactose toxicity, enhancement of

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intracellular GDP-L-fucose level, and usage of 2-FL-specific transporters should be focused

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on in order to create powerful engineered yeast strains for HMO production.



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MATERIALS AND METHODS

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Strains and Media. E. coli Top10 [F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80lacZ∆M15 ∆lacX74

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recA1 araD139 ∆(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG] was used for construction

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and propagation of plasmids. E. coli was grown in lysogeny broth (LB, 5 g/L yeast extract, 10 g/L

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tryptone, 10 g/L NaCl, pH 7.0) at 37 °C with ampicillin (100 µg/mL) added for selection when

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required. S. cerevisiae D452-2 (MATalpha, leu2, his3, ura3, and can1) was used as the host strain

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for 2-FL and L-fucose production. The yeast strains were grown on YP medium (10 g/L yeast

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extract, 20 g/L peptone) containing 20 g/L glucose at 30 °C. The yeast strains that were

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transformed with plasmids containing antibiotic markers were propagated on YPD (YP with 20 g/L

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of glucose) plates supplemented with the corresponding antibiotics such as Hygromycin (300

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µg/mL). Synthetic complete medium (SC, 1.7 g/L of yeast nitrogen base with 5 g/L of ammonium

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sulfate and appropriate amino acids, pH 6.5) containing 20 g/L of glucose (SCD) was used for

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maintaining plasmids in auxotrophic strains.

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Plasmids and strains construction. To enable S. cerevisiae to assimilate lactose, LAC12 encoding

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lactose permease 34 was cloned into pRS423-pGPD plasmid. The LAC12 gene fragment was

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amplified by polymerase chain reactions (PCR) from the genomic DNA of K. lactis (NRRL: Y-

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8279) using primer pairs (LAC12-F and LAC12-R). The PCR product and pRS423-pGPD were

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digested by SpeI and SalI, and ligated to construct plasmid pRS423-pGPD-LAC12. The

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constitutive expression cassette of LAC12 was then amplified from pRS423-pGPD-LAC12 using

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primer pairs of CS8-IU and CS8-ID, and integrated into the CS8 site 41 of yeast strain D452-2 for

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stable expression. The resulting strain was designated as D452L.


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For de novo synthesis of GDP-L-fucose, gmd and wcaG genes were obtained by PCR

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by using the genomic DNA of E. coli K-12 as a template. Two PCR primers, gmd-F and gmd-

274

R, were used for amplification of gmd gene. After digestion of the amplified gmd gene

275

fragment and pRS423-pGPD plasmid with SpeI and ClaI, they were ligated to construct

276

plasmid pRS423-pGPD-gmd. Similarly, wcaG gene was amplified by two PCR primers

277

(wcaG-F and wcaG-R). The wcaG gene fragment and pRS425-pGPD plasmid were digested

278

with BamHI and HindIII, and ligated to construct plasmid pRS425-pGPD-wcaG. Plasmids

279

pRS423-pGPD-gmd and pRS425-pGPD-wcaG were then transformed into strain D452L to

280

make strain D452L-gw.

281

To express α1,2-fucosyltransferase, fucT2 gene from H. pylori UA802 was codon-

282

optimized for S. cerevisiae and synthesized using the gBlocks service from Integrated DNA

283

Technologies (IDT) (Skokie, IL). The fucT2 gene was then amplified by primers fucT2_F and

284

fucT2_R using the synthesized DNA as a template. The fucT2 gene fragment and pRS426-

285

pGPD plasmid were digested with BamHI and ClaI, and ligated to construct plasmid pRS426-

286

pGPD-fucT2. The plasmid pRS426-pGPD-fucT2 was then transformed into strain D452L-gw

287

and the resulting strain was named as D452-gwf.

288

The gBlocks® fragment of the gene encoding α-L-fucosidase from X. manihotis was

289

synthesized from IDT, Inc. (Skokie, IL). The synthesized fragment was blunt ligated with

290

plasmid pRS42H-pGPD digested by SmaI. The resulting plasmid was designated as pRS42H-

291

pGPD-fuco. Strain D452-gwf-fuco was constructed by introducing plasmid pRS42H-pGPD-

292

fuco into D452-gwf strain. Primers, plasmids, and strains used in this work are listed in Table

293

1 and Tables S1 and S2 (Supporting Information) respectively. All constructed plasmids were

294

confirmed by DNA sequencing.



295

Yeast culture, fermentation, and metabolite analysis. To measure intracellular lactose, 1

296

mL of yeast cells were grown on YPD overnight, collected and incubated with 3 g/L of lactose

297

in liquid YP medium. The mixture was cultured at 30 °C for 6 h at 250 rpm. The yeast cells

298

were collected and washed twice to remove the entire medium component. The cells were

299

suspended in 500 µL of distilled water and boiled for 10 min to release intracellular lactose.

300

Intracellular lactose was measured by HPLC (Agilent Technologies 1200 Series, Agilent

301

Technologies, Santa Clara, CA). The HPLC was equipped with a Rezex ROA-Organic Acid

302

H+ (8%) column (Phenomenex Inc., Torrance, CA) and a refractive index detector (RID). The

303

column was eluted with 0.005 N H2SO4 at a flow rate of 0.6 mL/min at 50 °C.

304

Page 14 of 31

To measure intracellular GDP-L-fucose, 500 µL of yeast cell culture on YPD for 50 h

305

was collected and washed twice. Then, the cell pellets were resuspended in 500 µL of distilled

306

water. To release intracellular metabolites, the cells were disrupted by continuous beating with

307

glass beads for 40 min. Yeast cell lysis was achieved by boiling the cells for 2 min and

308

centrifuging for 10 min at 15,000 rpm to remove excess debris. The supernatant was injected

309

into a HPLC system with a diode array detector (Beckman Coulter System Gold, Pasadena,

310

CA) using a CAPCELL PAK C18 MG column (250×4.6 mm, Shiseido, Tokyo, Japan). The

311

column was eluted at a flow rate of 0.6 mL/min with 98% (v/v) of 20 mM triethylamineacetate

312

at pH 6.0 and 2% of acetonitrile. GDP-L-fucose was detected by absorbance at 254 nm.

313

To produce 2-FL and L-fucose, fermentation was performed by inoculating overnight

314

pre-culture (5 mL of SCD medium without appropriate amino acid for maintaining plasmids)

315

into 20 mL of YPD40L3 (YP medium with 40 g/L of glucose and 3 g/L of lactose) in a 125-

316

mL Erlenmeyer flask with an initial optical density at 600 nm (OD600) = 1.0 and incubated at

317

30 °C and 250 rpm. OD600 was monitored by a spectrophotometer (Biomate 5, Thermo Fisher


Page 15 of 31 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


318

Scientific, Waltham, MA). Extracellular metabolites such as glucose, glycerol, acetate,

319

ethanol, lactose, and 2-FL were measured by HPLC with a Rezex ROA-Organic Acid H+ (8%)

320

column (Phenomenex) and a RID. The column was eluted with 0.005 N H2SO4 at a flow rate

321

of 0.6 mL/min at 50 °C. To measure total (intracellular and extracellular) 2-FL, the

322

fermentation broth containing yeast cells was boiled for 10 min to release all the intracellular

323

2-FL and centrifuged at 15,000 rpm for 10 min. The supernatant was then analyzed by HPLC.

324

Confirmation of α-L-fucosidase enzymatic activity in vitro. Strain D452L-gwf-fuco

325

containing α-L-fucosidase and D452L-gwf with empty plasmid pRS42H acting as the control

326

strain was cultured in YPD medium containing 300 µg/mL of hygromycin in order to maintain

327

the plasmid. 5 mL of the yeast cell cultures were taken when the yeast OD600 reached 10. The

328

yeast cells were then collected by centrifugation at 15,000 rpm at 4 °C for 2 min and

329

suspended in 500 uL of 50 mM Tris-HCl (pH 7.5) buffer. The yeast cells were subjected to

330

cell lysis by glass bead beating using Fast Prep-24 homogenizer (MP Biomedicals, Solon, OH)

331

at 4 °C. After centrifugation, the supernatant was incubated with 2 g/L of 2-FL at 30 °C for 24

332

h. The samples from different time points were analyzed using HPLC, and 2-FL and L-fucose

333

were identified through GC/MS.

334

Identification of 2-FL and L-fucose using GC/MS. To identify L-fucose and 2-FL, the

335

samples were analyzed using GC/MS 37. 20 µL of supernatant was dried in a centrifugal

336

vacuum evaporator. For chemical derivatization, 10 µL of 40 mg/mL methoxyamine

337

hydrochloride in pyridine (Sigma-Aldrich, St. Louis, MO) was added to the dried sample and

338

incubated at 30 °C. After 90 min, 45 µL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide

339

(Sigma-Aldrich) was added to the sample and incubated at 37 °C for 30 min. The chemically

340

derivatized samples were then analyzed using an Agilent 7890A GC/5975C MSD system



Page 16 of 31

341

(Agilent Technologies) equipped with an HP-5ms column (30 m in length, 0.25 mm in

342

diameter, and 0.25 m in film thickness; Agilent Technologies) and a 10-m guard column. The

343

derivatized sample (1 µL) was injected into the GC column in a splitless mode. The oven

344

temperature was programmed to be initially at 80 °C for 1 min and then ramped to 300 °C at

345

10 °C/min for 1 min. Electron ionization was performed at 70 eV. The temperatures of ion

346

source and transfer line were 250 °C and 280 °C, respectively. The mass scan range used was

347

85–700 m/z.

348

AUTHOR INFORMATION

349

Corresponding Author

350

*E-mail: [email protected].

351

Present Addresses: 1206 West Gregory Drive, Urbana, IL 61801

352

ORCID:

353

Yong-Su Jin: 0000-0002-4464-9536

354

Jing-Jing Liu: 0000-0002-2302-2726

355

Suryang Kwak: 0000-0002-5202-8326

356

Author Contributions

357

Y.-S.J. and J.-J.L. developed the idea of this work. J.-J.L., S.K., and P.P. designed the experiments. J.-J.L., S.K.,

358

P.P., J.L., S.Y., E.J.Y., and H.L. performed the experiments. J.-J. L, S.K., P.P., K.H.K., and Y.-S.J. wrote the

359

manuscript. The final manuscript was approved by all of the authors.

360 361 362 363

ACKNOWLEDGEMENTS This project was supported by funding from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-


Page 17 of 31 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


364

2015R1D1A1A01060488). KHK acknowledges a grant support from the National Research

365

Foundation of Korea (NRF-2017R1A2B2005628). EJY was supported by the Research Fellow

366

Grant from the National Research Foundation of Korea (2017R1A6A3A11035069) and by the

367

Research Fellow Grant from Korea University. We thank Christine Anne Atkinson for

368

proofreading the manuscript.

369 370

REFERENCES

371

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fermentation by engineered Saccharomyces cerevisiae capable of fermenting cellobiose, J

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of GDP-L-fucose, L-fucose donor for fucosyloligosaccharide synthesis, in recombinant

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Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing

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the first step in the de novo synthesis of GDP-L-fucose, Proc Natl Acad Sci U S A 94,

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expression of L-fucokinase/guanosine 5'-diphosphate-L-fucose pyrophosphorylase from

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Bacteroides fragilis in Saccharomyces cerevisiae for the production of nucleotide sugars

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Research 334, 97-103.

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[38] Xia, P. F., Zhang, G. C., Liu, J. J., Kwak, S., Tsai, C. S., Kong, II, Sung, B. H., Sohn, J. H.,

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Wang, S. G., and Jin, Y. S. (2016) GroE chaperonins assisted functional expression of

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bacterial enzymes in Saccharomyces cerevisiae, Biotechnol Bioeng 113, 2149-2155.

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[39] Wisselink, H. W., Toirkens, M. J., del Rosario Franco Berriel, M., Winkler, A. A., van Dijken,

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Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus

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thermophilus xylA gene, which expresses an active xylose (glucose) isomerase, Applied

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and Environmental Microbiology 62, 4648-4651.

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[41] Liu, J. J., Kong, II, Zhang, G. C., Jayakody, L. N., Kim, H., Xia, P. F., Kwak, S., Sung, B. H.,

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Sohn, J. H., Walukiewicz, H. E., Rao, C. V., and Jin, Y. S. (2016) Metabolic Engineering

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of Probiotic Saccharomyces boulardii, Appl Environ Microbiol 82, 2280-2287.

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[42] Hosaka, K., Nikawa, J.-i., Kodaki, T., and Yamashita, S. (1992) A Dominant Mutation That

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Alters the Regulation of INO1 Expression in Saccharomyces cerevisiae, The

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Page 23 of 31 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

495


Figure captions

496 497

Fig. 1. Schematic diagram of 2-FL and L-fucose production in engineered S. cerevisiae. Lac12,

498

lactose permease; Gmd, GDP-mannose 4,6-dehydratase; WcaG, GDP-4-keto-6-deoxymannose 3,5-

499

epimerase 4-reductase; FucT2, alpha-1,2-fucosyltransferase; α-fucosidase, α-L-fucosidase; 2-FL,

500

2’-fucosyllactose.

501

Fig. 2. Lactose transport and GDP-L-fucose production by engineered yeast. (a), D452L

502

carrying lactose permease (Lac12) transported lactose into the cell; (b), GDP-L-fucose was

503

produced by introducing gmd and wcaG into D452L strain. D452-2, parental strain as a control;

504

D452L, D452-2 with Lac12 expression; D452L-gw, D452L with Gmd and WcaG expression. *ND,

505

not detected; (b) *, unknown peak from medium. Results are the mean of duplicated experiments;

506

error bars indicate standard deviations.

507

Fig. 3. 2-FL production by engineered yeast D452L-gwf. The concentrations of glucose, ethanol,

508

lactose, acetate, glycerol, and 2-FL were monitored by HPLC. (a), glucose consumption, ethanol

509

production and consumption, and yeast cell growth. (b), lactose consumption, acetate and glycerol

510

production. (c), the concentrations of total 2-FL and extracellular 2-FL. (d), the ratio of

511

extracellular 2-FL over the course of fermentation. Results are the mean of duplicated experiments;

512

error bars indicate standard deviations and are not visible when smaller than the symbol size.

513

Fig. 4. Confirmation of α-L-fucosidase activity in S. cerevisiae in vitro. (a), incubation of 2-FL

514

with cell lysate of strain D452L-gwf with empty plasmid as a control; (b), incubation of 2-FL with

515

cell lysate of strain D452L-gwf-fuco with α-L-fucosidase expression; (c), the HPLC

516

chromatograph of cell lysate of D452L-gwf-fuco incubated with 2-FL. 2-FL, lactose, and L-fucose

517

were indicated. *, unknown peak from medium; (d) and (e) are GC/MS confirmation of L-fucose



Page 24 of 31

518

and 2-FL, respectively. Results are the mean of duplicated experiments; error bars indicate

519

standard deviations and are not visible when smaller than the symbol size.

520

Fig. 5. L-fucose production by introducing α-L-fucosidase into a 2-FL producing yeast strain.

521

(a), lactose consumption. (b), 2-FL production. (c), L-fucose production. D452L-gwf, 2-FL

522

producing strain; D452L-gwf-fuco, D452L-gwf strain with expression of α-L-fucosidase. Results

523

are the mean of duplicated experiments; error bars indicate standard deviations and are not visible

524

when smaller than the symbol size.

525


Page 25 of 31 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

526


Fig. 1

527 528 529

Fig. 1. Schematic diagram of 2-FL and L-fucose production in engineered S. cerevisiae. Lac12,

530

lactose permease; Gmd, GDP-mannose 4,6-dehydratase; WcaG, GDP-4-keto-6-deoxymannose 3,5-

531

epimerase 4-reductase; FucT2, alpha-1,2-fucosyltransferase; α-fucosidase, α-L-fucosidase; 2-FL,

532

2’-fucosyllactose.

533



534

Page 26 of 31

Fig.2

535

536 537 538

Fig. 2. Lactose transport and GDP-L-fucose production by engineered yeast. (a), D452L

539

carrying lactose permease (Lac12) transported lactose into the cell; (b), GDP-L-fucose was

540

produced by introducing gmd and wcaG into D452L strain. D452-2, parental strain as a control;

541

D452L, D452-2 with Lac12 expression; D452L-gw, D452L with Gmd and WcaG expression. *ND,

542

not detected; (b) *, unknown peak from medium. Results are the mean of duplicated experiments;

543

error bars indicate standard deviations.

544 545


Page 27 of 31 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

546


Fig. 3

547

548 549 550

Fig. 3. 2-FL production by engineered yeast D452L-gwf. The concentrations of glucose, ethanol,

551

lactose, acetate, glycerol, and 2-FL were monitored by HPLC. (a), Glucose consumption, ethanol

552

production and consumption, and yeast cell growth. (b), Lactose consumption, acetate and glycerol

553

production. (c), The concentrations of total 2-FL and extracellular 2-FL. (d), The ratio of

554

extracellular 2-FL over the course of fermentation. Results are the mean of duplicated experiments;

555

error bars indicate standard deviations and are not visible when smaller than the symbol size.

556



557

Page 28 of 31

Fig. 4

558

559 560 561

Fig. 4. Confirmation of α-L-fucosidase activity in S. cerevisiae in vitro. (a), Incubation of 2-FL

562

with cell lysate of strain D452L-gwf with empty plasmid as a control; (b), Incubation of 2-FL with

563

cell lysate of strain D452L-gwf-fuco with α-L-fucosidase expression; (c), The HPLC

564

chromatograph of cell lysate of D452L-gwf-fuco incubated with 2-FL. 2-FL, lactose, and L-fucose

565

were indicated. *, unknown peak from medium; (d) and (e) are GC/MS confirmation of L-fucose

566

and 2-FL, respectively. Results are the mean of duplicated experiments; error bars indicate

567

standard deviations and are not visible when smaller than the symbol size.

568 569


Page 29 of 31 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

570


Fig. 5

571

572 573 574

Fig. 5. L-fucose production by introducing α-L-fucosidase into a 2-FL producing yeast strain.

575

(a), Lactose consumption. (b), 2-FL production. (c), L-fucose production. D452L-gwf, 2-FL

576

producing strain; D452L-gwf-fuco, D452L-gwf strain with expression of α-L-fucosidase. Results

577

are the mean of duplicated experiments; error bars indicate standard deviations and are not visible

578

when smaller than the symbol size.

579 580



Page 30 of 31

581 582

Table 1. Strains used in this study Strain name

Description of strains

Source

D452-2

MATα leu2 ura3 his3 can1

42

D452L

D452-2 with CS8-LAC12 integration

This study

D452L-gw

D452L with pRS423-pGPD-gmd and pRS425-

This study

pGPD-wcaG D452L-gwf

D452L-gw with pRS426-pGPD-fucT2

This study

D452L-gwf-fuco

D452L-gwf with pRS42H-pGPD-fuco

This study

583 584


Page 31 of 31 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


585 586

Biosynthesis of a functional human milk oligosaccharide, 2′-Fucosyllactose, and L-fucose,

587

using engineered Saccharomyces cerevisiae

588 589

Jing-Jing Liu a, Suryang Kwak a,b, Panchalee Pathanibul b, Jaewon Lee b, Sora Yu c, Eun Ju Yun a,c,

590

Hayoon Lim a, Kyoung Heon Kim c, Yong-Su Jin a,b,*

591 592

593 594 595

For Table of Contents Use Only

596


Biosynthesis of a functional human milk oligosaccharide, 2

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