CHEMBIOCHEM COMMUNICATIONS DOI: 10.1002/cbic.201402070
Synthesis of the Human Milk Oligosaccharide Lacto-NTetraose in Metabolically Engineered, Plasmid-Free E. coli Florian Baumgrtner,[a] Jrgen Conrad,[b] Georg A. Sprenger,[a] and Christoph Albermann*[a] Human milk oligosaccharides (HMOs) constitute the third most abundant solid component of human milk. HMOs have been demonstrated to show positive effects on the infant’s wellbeing. Despite numerous studies, more physiological analyses of single compounds are needed to fully elucidate these effects. Although being one of the most abundant core structures in human milk, the HMO lacto-N-tetraose (LNT) is not available at reasonable prices. In this study, we demonstrate the construction of the first E. coli strain capable of producing LNT in vivo. The strain was constructed by chromosomally integrating the genes lgtA and wbgO, encoding b-1,3-N-acetylglucosaminyltransferase and b-1,3-galactosyltransferase. In shakeflask cultivations, the strain yielded a total concentration of 219.1 3.5 mg L 1 LNT (LNT in culture broth and the cell pellet). After recovery of LNT, structural analysis by NMR spectroscopy confirmed the molecule structure.
Human milk is generally considered to be the best diet for the well-being of infants.[1] At approximately 5 to 15 g L 1, human milk contains a remarkable content of free oligosaccharides, much higher than in the milk of mammals such as cow, sheep, and goat.[2] The core structures of human milk oligosaccharides (HMOs) show a lactose residue at the reducing end, which can be extended by lacto-N-biose or N-acetyllactosamine units. These core structures can be further decorated by addition of fucosyl or sialyl residues, thereby resulting in over 200 different structures identified to date.[3, 4] As recently reviewed,[2] HMOs are central to the difference between formula milk and breast milk. Several studies have described well analyzed prebiotic effects or the blockade of pathogen lectins, thereby resulting in the wash-out of pathogens.[2] Further beneficial properties of HMOs have been proposed, such as modulation of intestinal epithelial cells or of lymphocyte maturation and reduction of leucocyte rolling and adhesion.[5–7] However, further studies of these properties demand single-compound HMOs. Currently, these are mainly extracted from human milk in a complex procedure because of the large numbers of structurally different HMOs. Efforts towards the biotechnological production of several HMOs have been described.[8] But despite being one of [a] F. Baumgrtner, Prof. Dr. G. A. Sprenger, Dr. C. Albermann Institute of Microbiology, University of Stuttgart Allmandring 31, 70569 Stuttgart (Germany) E-mail: [email protected] [b] Dr. J. Conrad Bioorganic Chemistry, Institute of Chemistry, University of Hohenheim Garbenstrasse 30, 70599 Stuttgart (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402070.
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the most abundant HMO core structures in human milk, lactoN-tetraose (LNT, Scheme 1) is not available in large quantities and at reasonable prices for research. Currently, LNT is mainly prepared by a multistep extraction procedure from human milk.[6, 9–11] Both chemical and enzymatic synthesis procedures
Scheme 1. Chemical structures of oligosaccharides LNT II (b-d-GlcNAc1-3-bd-Gal1-4-d-Glc) and LNT (b-d-Gal1-3-b-d-GlcNAc1-3-b-d-Gal1-4-d-Glc).
for LNT have been published.[12, 13] However, these methods are not yet satisfactory, as the chemical synthesis requires several steps of protection and deprotection of reactive groups, and enzymatic synthesis suffers from unfavorable equilibria and low regioselectivities of the transglycosylation reactions, thereby leading to low efficiency. The in vitro synthesis of benzyl blacto-N-tetraoside from a benzyl b-lacto-N-trioside and the nucleotide-activated sugar UDP-galactose was demonstrated by using the regio- and stereoselective Leloir b-1,3-galactosyltransferase WbgO from Escherichia coli O55:H7.[14] The in vivo synthesis of the structural isomer of LNT, lacto-N-neotetraose (LNnT), was demonstrated by high-cell-density cultivation of E. coli cells expressing plasmid-borne genes for Leloir glycosyltransferases.[15, 16] These cells provided intracellular pools of the required nucleotide-activated sugars UDP-N-acetylglucosamine and UDP-galactose and were fed with lactose. Here we demonstrate that expression of the gene for WbgO in E. coli K-12, which is capable of the synthesis of lacto-N-triose II (LNT II, Scheme 1), leads to the formation of LNT from lactose and the low-cost carbon sources glycerol or glucose in shake-flask cultivation. For the construction of the LNT-producing strain, E. coli K-12 LJ110 was chosen as parent strain.[17] This plasmid-free strain was modified by knock-out of sugar degradation loci through introduction of an expression cassettes by homologous recombination.[18, 19] To construct the strain, lacZ (encoding b-galactosidase) of E. coli strain LJ110 was removed, and the strain was equipped with the b-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis to allow synthesis of LNT II.[20, 21] The resulting strain was supplemented with wbgO (encoding b-1,3-galactosyltransferase WbgO; Scheme 2).[14] As the use of antibiotics is undesirable in the ChemBioChem 2014, 15, 1896 – 1900
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Scheme 2. In vivo synthesis of LNT II and LNT in recombinant E. coli cells with Leloir glycosyltransferases and intracellular nucleotide-activated sugars. The lacZ gene was deleted to prevent metabolism of lactose. Arrows with dashed lines indicate that the majority of these products are found in the culture supernatant.
production of food- or pharmaceutical-related compounds, we decided against the use of antibiotic-resistance-encoding plasmids as vectors for recombinant genes in the final production strains. Instead, chromosomal integration of genes was chosen. Although leading to lower gene copy number, this has the advantage of genetic stability, as gene loss (segregational instability of plasmids) is avoided.[18, 19, 22] Leloir glycosyltransferases were used for intracellular synthesis of LNT. These glycosyltransferases use the nucleotide-activated sugars UDP-N-acetylglucosamine and UDP-galactose, which are endogenous to the E. coli host. To enable the intracellular synthesis of LNT II, the N. meningitidis gene lgtA (encoding b-1,3-N-acetylglucosaminyltransferase)[20, 21] was custom
www.chembiochem.org synthesized with codon optimization. The gene was cloned into an expression vector with isopropyl-b-d-thiogalactopyranoside (IPTG)-inducible Ptac-promoter, to yield pJFK-lgtA (Table 1). After equipping the vector with an FRT-flanked chloramphenicol resistance gene downstream of lgtA, the expression cassette, including the Ptac promoter, a ribosome binding site (Shine-Dalgarno sequence), the gene of interest (lgtA), a FRT-cat-FRT resistance marker, and a transcription terminator sequence from rrnB, was amplified by PCR. The cassette was then chromosomally integrated into the lacZYA locus to eliminate metabolism and possible acetylation of lactose by LacZ and LacA, respectively. Lactose uptake was retained by complementing the resulting strain LJ-A-cat (Table 1) with an E. coli K12 lacY gene under the control of a Ptac promoter. To do so, lacY was first cloned into the expression vector pJF119EH, followed by the cloning of an FRT-kan-FRT resistance cassette to obtain pJF-lacY-FRT-kan-FRT. After DNA amplification, the expression cassette was chromosomally integrated[18] into the fucIK gene locus, thereby resulting in strain LJ-AY-cat-kan. Transformation of this strain with the FLP-recombinase-expressing plasmid pCP20[23] removed the antibiotic resistance gene cassettes and yielded strain LJ-AY, which was used for further chromosomal integrations (Table 1). Scheme 2 describes the intended intracellular pathway from lactose to LNT. To study the formation of HMOs, shake-flask cultivations in minimal medium with lactose and one of the two common carbon sources (glycerol or glucose) were performed with various strains. For our purposes, glucose as carbon source has the undesirable effect of preventing the concomitant uptake of lactose through LacY (due to inhibition of LacY by unphos-
Table 1. Bacterial strains and plasmids used in this study. Relevant genotype or sequences
Source or ref. laboratory strain
E. coli LJ110 E. coli LJ-A-cat E. coli LJ-AY-cat-kan E. coli LJ-AY E. coli LJ-AYO-cat Plasmids pJF119EH pKD46 pCP20
F , f80d, lacZDM15, endA1, recA1, hsdR17(rK mK ), supE44, thi-1, gyrA96, relA1, D(lacZYAargF)U169 E. coli W3110 fnr + E. coli LJ110, lacZYA::lgtA-FRT-cat-FRT E. coli LJ110, lacZYA::lgtA-FRT-cat-FRT fucIK::lacY-FRT-kan-FRT E. coli LJ110, lacZYA::lgtA-FRT fucIK::lacY-FRT E. coli LJ110, lacZYA::lgtA-FRT fucIK::lacY-FRT xylAB::wbgO-FRT-cat-FRT cloning vector, Ptac, AmpR ParaB g b exo (red recombinase), AmpR FLP + , l cI857 + , l pR Repts, AmpR, CmR
pCAS30-FRT-cat-FRT pJF-crtY-FRT-kan-FRT pJF-futC pF81kan pJF119EH-kan pJFK-futC pJFK-lgtA pJF-lacY pJFK-wbgO pJFK-lgtA-FRT-cat-FRT pJF-lacY-FRT-kan-FRT pJFK-wbgO-FRT-cat-FRT
pJF119DN, Pantoea ananatis crtE gene, FRT-sites, AmpR,CmR pJF119DN, P. ananatis crtY gene, FRT-sites, AmpR,KmR pJF119DN, Helicobacter pylori futC gene, AmpR pJF119EH, pheA(fbr), aroF, aroB, aroL, KanR pJF119EH, exchange of AmpR to KanR pJF-futC, exchange of AmpR to KanR pJFK-futC, exchange of futC with codon-optimized N. meningitidis lgtA gene, KanR pJF119EH, E. coli lacY gene, AmpR pJFK-futC, exchange of futC with codon-optimized E. coli O55:H7 wbgO gene, KanR pJFK-lgtA, FRT-sites, KanR, CmR pJF-lacY, FRT-sites, AmpR, KanR pJFK-wbgO, FRT-sites, KanR, CmR
Frste et al.[24] Datsenko and Wanner[19] Cherepanov and Wackernagel[23] Vallon et al.[25] Albermann et al.[18] Baumgrtner et al.[22] Weiner et al.[26] this study this study this study this study this study this study this study this study
Strains E. coli DH5a
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Zeppenfeld et al.[17] this study this study this study this study
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phorylated Enzyme IIAGlc).[27] However, utilization of glycerol might lead to lower product yields because of the need for gluconeogenic steps towards UDP-N-acetylglucosamine and UDP-galactose. Therefore, glucose and glycerol were compared as possible main carbon sources for growth and the provision of metabolites. In both cases, lactose was fed as the acceptor for the glycosyltransferase LgtA. Recombinant cells were incubated in minimal medium in shake flasks and grown at 30 8C. After reaching the exponential growth phase, cells were induced with IPTG (0.5 mm), and lactose (2 g L 1) was added. As expected, the lacY-deficient strain LJ-A-cat was neither capable of lactose utilization for growth, nor of LNT II-production. Strain LJ-AY-cat-kan (Table 1, chromosomally integrated lacY under a Ptac promoter) was capable of lactose utilization for the synthesis of LNT II. With glucose as main carbon source, this strain showed an exponential growth rate (mmax) of 0.33 h 1, total LNT II formation of 1.906 0.201 g L 1 (13 % in the cell pellet), and an LNT II yield of 641.1 11.3 mg gCDW 1 (cell dry weight; Table 2) 24 h after lactose supplementation and induction with IPTG. Cultures in medium with glycerol as the carbon source showed a lower mmax (0.28 h 1), but a higher cell density and a higher productivity, with an LNT II concentration of 2.465 0.057 g L 1 (11 % in the cell pellet) and a yield of 997.7 1.7 mg gCDW 1 after 24 h. Thus, glycerol showed a higher potential for in vivo synthesis of LNT II, with final LNT II concentrations about 30 % higher than with glucose. The molar LNT II yield from lactose was 77.34 1.78 % for glycerol and 59.82 6.30 % for glucose (Table 2). For the intracellular conversion of LNT II to LNT, a synthetic and codon-optimized version of wbgO (b-1,3-galactosyltransferase) from E. coli O55:H7 was chromosomally integrated into the xylAB locus as described for lgtA,[14] thus yielding strain LJAYO-cat. This strain was cultivated under the same conditions as for LJ-AY-cat-kan to yield the tetrasaccharide LNT. LJ-AYO-cat exhibited growth rates comparable to those of LJ-AY-cat-kan (mmax 0.33 and 0.28 h 1 with glucose and glycerol, respectively). We examined the production of LNT II and LNT in both media. LJ-AYO-cat also synthesized more LNT II in medium with glycerol: 1.890 0.067 g L 1 (13 % in the cell pellet) in cultures with glucose and 2.090 0.022 g L 1 (10 % in the cell pellet) with glycerol, and LNT II yields of 570.6 15.2 and 66.2 1.6 mg gCDW 1, respectively. Whereas LNT II production was higher in medium with glycerol, LNT concentration and yield
were higher in medium with glucose: 219.1 3.5 mg L 1 LNT (47 % in the cell pellet) 24 h after induction, compared to 162.1 6.2 mg L 1 (25 % in the cell pellet) with glycerol. This corresponds to yields of 66.2 1.6 mg gCDW 1 for medium with glucose and 58.5 3.0 mg gCDW 1 for medium with glycerol (Table 2). Thus, we show for the first time that LNT can be synthesized by recombinant E. coli cells. Under the applied conditions, glucose proved to be the more favorable carbon source for LNT synthesis. But as LNT II is not fully converted into LNT by LJ-AYO-cat, there might be limitation in the activity of WbgO or in the supply of the donor substrate UDP-galactose. Studies of lactose uptake and UDP-sugar formation might help elucidate the impacts of the inducer exclusion by glucose,[27] or the need for strengthening gluconeogenic steps with glycerol as the carbon source. Structural analysis of oligosaccharides synthesized in a whole-cell system is of particular importance to exclude nonspecific reactions or modifications of the oligosaccharides by endogenous cellular components. In order to verify the structures of LNT II and LNT produced in vivo, the products were isolated from culture broth and the cell pellet, respectively. The MS analysis showed m/z ratios of the sodium adducts of 568.2 and 730.2 for LNT II and LNT, respectively (Figure S1 in the Supporting Information); this is in good agreement with the calculated masses of these compounds. To analyze the configuration of LNT further, extensive NMR analysis was performed, including 1H, 13C, TOCSY1D, eCOSY, coupled and decoupled band-selective gHSQC, as well as gHMBC, gC2H2BC, and gHSQC-TOCSY spectra. In addition to allowing comparison to previously published NMR data by Bush et al., [28] Platzer and Davoust, [29] and Strecker et al.,[30] this also allowed autonomous verification of the structure and the annotation of previously undefined proton coupling constants, especially from the coupled band-selective gHSQC spectra. Chemical shifts and couplings of LNT II and LNT are listed in Tables S2 to S5. Thus, the structures of LNT II and LNT were unambiguously verified. For the in vivo synthesis of LNnT, Priem et al. described the formation of elongated LNnT, by repetitive reactions of N-acetylglucosaminyl- and galactosyl-transferases.[21] Such elongation beyond the tetrasaccharide was not observed during the in vivo synthesis of LNT. This might be explained by a lower activity of the N-acetylglucosaminyltransferase LgtA for the sub-
Table 2. Total product concentrations and yields in shake-flask cultivations 24 h after induction, quantified by HPLC.[a] Strain E. coli LJ-AY-cat-kan E. coli LJ-AYO-cat
Carbon source
Conc. [g L 1]
Lacto-N-triose II Yield [mg gCDW 1][b] Yield [mg glactose 1][c]
Conc. [mg L 1]
Lacto-N-tetraose Yield [mg gCDW 1] Yield [mg glactose 1][c]
1% 1% 1% 1%
1.906 0.201 2.465 0.057 1.890 0.067 2.090 0.022
641.1 11.3 997.7 1.7 570.6 15.2 753.7 17.6
– – 219.1 3.5 162.1 6.2
– – 66.2 1.6 58.5 3.0
glucose glycerol glucose glycerol
953.2 100.4 1232.6 28.3 944.8 33.5 1045.2 11.1
– – 109.6 1.7 81.1 3.1
[a] Cultivation at 30 8C and 90 rpm in minimal medium with 1 % glucose or 1 % glycerol as the main carbon source, and 0.2 % lactose as the source for product formation (concentrations and yields 24 h after induction with 0.5 mm IPTG from a minimum of two biological replicates). [b] Yield of LJ-AY-catkan determined by calculating cell dry mass concentrations (OD600), with correlation factors of E. coli strain LJ-AYO-cat (0.37 and 0.30 g L 1 for cultures in minimal medium with glucose and glycerol, respectively). [c] Product yields from lactose were calculated from the total lactose concentration (2 g L 1).
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CHEMBIOCHEM COMMUNICATIONS strate LNT compared to LNnT, as shown by in vitro assays,[20] or by a higher intracellular concentration of LNnT, which is not secreted into the culture broth.[21] In summary, we have constructed plasmid-free E. coli strains lacking b-galactosidase activity but displaying lactose uptake (LacZ LacY + ), by chromosomal integration as previously described.[18, 22] Additionally, the strains carry recombinant genes for b-1,3-N-acetylglucosaminyltransferase and b-1,3-galactosyltransferase (lgtA and wbgO), each under control of a Ptac promoter. Using nucleotide-activated sugars from endogenous pools for Leloir glycosyltransferases, this strain is the first E. coli strain capable of producing lacto-N-tetraose, in addition to lacto-N-triose II, allowing LNT-synthesis from low cost substrates like lactose and glucose or glycerol. Thus, we describe here a simple synthesis of LNT by utilizing a genetically stable bacterial strain and inexpensive substrates, to make LNT more widely available for research.
Experimental Section Chemicals, media, and bacterial strains: All strains were based on the laboratory strain E. coli LJ110.[17] E. coli DH5a was used for plasmid construction. Unless stated otherwise, E. coli strains were cultivated on lysogeny broth (LB) agar plates at 37 8C or in LB liquid medium at 37 8C with shaking (160 rpm). Antibiotics for strain construction were chloramphenicol (50 mg mL 1), kanamycin (50 mg mL 1), or ampicillin (100 mg mL 1). Difco MacConkey agar base was purchased from Otto Nordwald (Hamburg, Germany). Standards lacto-N-tetraose (purity > 95 %) and lacto-N-neotetraose (purity > 95 %) were purchased from IsoSep (Tullinge, Sweden). E. coli b-galactosidase (1500 U mL 1) was from Roche. All other chemicals and reagents were purchased from either Sigma–Aldrich or Carl Roth and were of the highest purity available. Construction of plasmids and strains: All strains and plasmids are listed in Table 1. Expression vectors with ampicillin resistance were based on the previously described plasmid pJF119EH.[24] Vectors with kanamycin resistance were derived from the expression plasmids pJF-futC and pJF119EH,[22, 24] by replacing the b-lactamase gene with a fragment with a kanamycin-resistance gene from pF81kan[26] at RcaI restriction sites. The resulting plasmids were termed pJFK-futC and pJF119EH-kan, respectively. Synthetic, codon-optimized open reading frames for lgtA and wbgO were ordered from Life Technologies and cloned separately into the NdeI and BamHI sites of pJFK after removing futC, to yield pJFK-lgtA and pJFK-wbgO. The open reading frame of lacY was amplified from chromosomal E. coli LJ110 (primers lacY-for lacY-rev in Table S1), and cloned into the EcoRI/BamHI sites of pJF119EH to yield pJFlacY. Plasmids pJFK-lgtA, pJFK-wbgO, and pJF-lacY were digested with HindIII and ligated with a HindIII-digested FRT-cat-FRT- or FRTkan-FRT-Fragment from pCAS30-FRT-cat-FRT or pJF-crtY-FRT-kanFRT[18, 25] to yield pJFK-lgtA-FRT-cat-FRT, pJFK-wbgO-FRT-cat-FRT and pJF-lacY-FRT-kan-FRT, respectively. Plasmid-free E. coli strains were based on E. coli LJ110, and were constructed by homologous recombination of expression cassettes into sugar degradation loci.[18] Expression cassettes lgtA-FRT-catFRT, wbgO-FRT-cat-FRT, and lacY-FRT-kan-FRT were amplified (primers in Table S1) and consecutively transferred to E. coli LJ110 strains carrying the l-red recombinase system on plasmid pKD46.[19] Colonies carrying successfully integrated genes were selected by antibiotic resistance and were assessed by their color on MacConkey agar sugar plates as described previously.[18, 22] Antibiot 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org ic resistance cassettes were removed by transient expression of FLP recombinase (encoded by plasmid pCP20) as previously described.[23] Shake-flask cultivations for production of LNT II and LNT: Synthesis of LNT II and LNT was assayed after cultivation of strains (at least two replicates) in 250 mL shake flasks with minimal medium (25 mL), with glycerol or glucose (carbon source) and chloramphenicol (to prevent contamination). Minimal medium was slightly modified from a previous composition:[22, 31] (NH4)2SO4 (2.68 g L 1), (NH4)2-H-citrate (1 g L 1), glycerol or glucose (10 g L 1), K2HPO4 (14.6 g L 1), MgSO4 (0.241 g L 1), MnSO4·H2O (10 mg L 1), Na2SO4 (2 g L 1), NaH2PO4·H2O (4 g L 1), NH4Cl (0.5 g L 1), thiamine (10 mg L 1), and trace element solution (3 mL L 1; CaCl2·2 H2O FeCl3·6 H2O (16.7 g L 1), Na2-EDTA (20.1 g L 1), (0.5 g L 1), 1 1 ZnSO4·7 H2O (0.18 g L ), MnSO4·H2O (0.1 g L ), CuSO4·5 H2O (0.16 g L 1), and CoCl2·6 H2O (0.18 g L 1)). Strains were cultivated in 250 mL shake flasks (25 mL culture volume) at 30 8C and 90 rpm to OD600 = 0.4–0.5, before inducing expression by addition of IPTG (0.5 mm), with lactose (2 g L 1) addition as the substrate for product formation. Samples for HPLC analysis were taken 24 h after induction and centrifuged (15 300 g, 2 min). Supernatants were stored at 20 8C and pellets were washed with saline (NaCl, 0.9 g L 1) and centrifuged again, before storage at 20 8C. CDWs of E. coli LJ-AYO-cat cultures were measured 24 h after induction by centrifugation (5869 g, 4 8C, 20 min) of culture (10 mL) and drying the cell pellet (22 h, 120 8C), before weighing. Measurements were taken from two separate cultures per carbon source (duplicate measurements per culture). Correlations of CDW to OD600 for LJ-AYO-cat were determined as 0.37 and 0.30 g L 1 for cultures in minimal medium with glucose and glycerol, respectively. These correlations were also used for the calculation of product yields per CDW of E. coli strain LJ-AY-cat-kan. Cleavage of lacto-N-neotetraose to obtain a lacto-N-triose II standard: To obtain LNT II as a standard for HPLC analysis, lacto-Nneotetraose (141 nmol) was digested with b-galactosidase (1.5 U) in Tris·HCl (25 mL, 100 mm, pH 7.5) at 37 8C overnight. The enzymatic reaction was followed by HPLC. HPLC analysis of oligosaccharides: Saccharides from shake-flask experiments were analyzed by HPLC. After centrifugation, sugars in culture supernatants were derivatized without prior treatment. Cell pellets of 2 mL culture were resuspended in water to a total volume of 150 mL, then lysed by incubation in boiling water (5 min) followed by centrifugation (20 800 g, 5 min). The resulting supernatant was analyzed for intracellular sugar content. To label the reducing sugars, samples (50 mL) or standards of lactose, LNT, or LNT II were incubated (2 h, 65 8C) with 2-aminobenzoic acid (25 mL; 2-aminobenzoic acid (350 mm) in DMSO (80 %, v/v) and acetic acid (20 %, v/v)) and 2-picoline borane (25 mL; 2-picoline borane (1 m) in DMSO).[32] After incubation, preparations were diluted (1:5) in HPLC mobile phase solvent A (see below) before analysis. HPLC analysis was performed on a Dionex HPLC instrument (Thermo Scientific; Chromeleon software, Gina autosampler, P580 pumps, RF2000 fluorescence detector). Products were separated on a Luna C18 (2) reversed-phase column (250 4.6 mm, 5 mm; Phenomenex, Aschaffenburg, Germany) at a flow-rate of 1 mL min 1 with mobile phase solvent A (acetonitrile (10.71 %, v/v), tetrahydrofuran (0.89 %, v/v), ortho-phosphoric acid (0.38 %, v/v) and 1-butylamine (0.27 %, v/v)), and solvent B (acetonitrile): 100 % solvent A (20 min), linear gradient to 100 % solvent B (4 min), linear gradient to 100 % solvent A (1 min), equilibration with 100 % solChemBioChem 2014, 15, 1896 – 1900
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vent A (5 min). The fluorescent detector was operated at lex = 230 nm and lem = 425 nm. Concentrations of lactose and lacto-N-tetraose were determined by comparison to derivatized, commercial standards (95 % purity assumed for commercial lacto-N-tetraose). Concentrations of lacto-Ntriose II were calculated by comparison of peak areas to those obtained from commercial lactose standards, with the assumption that the 2-aminobenzoic acid derivatization of glycans with different lengths gives comparable fluorescence signal intensities.[32] Comparison of derivatized standards of lactose and lacto-N-tetraose confirmed these results by showing concentration-to-emission ratios with a deviation below 2 %. Preparative synthesis and isolation of lacto-N-triose II and lactoN-tetraose: For the preparative production of oligosaccharides, minimal medium (750 mL) with glycerol (10 g L 1, for LNT II) or glucose (10 g L 1, for LNT) and chloramphenicol (50 mg L 1) was inoculated (starting OD600 = 0.05) with LJ-AY-cat-kan or LJ-AYO-cat, respectively, in 3 L baffled shake flasks. Shake flasks were incubated at 30 8C with 90 rpm shaking. At OD600 = 0.4–0.5, IPTG (0.5 mm) and lactose (0.3 %) were added. Cells were separated from culture supernatants by centrifugation, (2831 g, 4 8C, 15 min) 48 h after induction. LNT II was recovered from culture supernatants; LNT was recovered from cell pellets. Pellets were resuspended in H2O, incubated (100 8C, 20 min) and centrifuged (6371 g, 4 8C, 15 min). The resulting supernatant was used for isolation of LNT. LNT II and LNT were recovered by using activated charcoal chromatography and gel-filtration as previously described.[22] Purifications through an activated charcoal chromatography and gel-filtration chromatography yielded 323.9 mg of LNT II and 102.1 mg of LNT. Structural analysis of lacto-N-triose II and lacto-N-tetraose: Purified products were analyzed by mass spectrometry on a microTOFQ system (Bruker; mass spectrum of LNT in Figure S1). NMR spectra were recorded in D2O on a Unitiy Inova 500 MHz spectrometer (Varian) at 296 K. 1H and 13C spectra were referenced to residual H2O and deuterated methanol (d = 4.70 and 49 ppm, respectively). 1 H, 13C, eCOSY, TOCSY1D, band-selective gHSQC (with and without decoupling), gHSQC-TOCSY, gHMBC, and gC2H2BC spectra were recorded by using CHEMPACK 4.0 pulse sequences (Varian Vnmrj 2.1B software). Recorded NMR spectra of LNT are shown in the Supporting Information.
Acknowledgements The authors gratefully acknowledge Katrin Wohlbold (Institute of Organic Chemistry, University of Stuttgart) for MS measurements and Prof. Dr. Uwe Beifuß (Institute of Chemistry, University of Hohenheim) for enabling NMR analysis on a Varian Unitiy Inova 500 MHz spectrometer. This work was supported by contract research “Glycomics/Glycobiology” of the Baden-Wrttemberg Stiftung.
[1] L. Bode, Nutr. Rev. 2009, 67, S183 – S191. [2] L. Bode, Glycobiology 2012, 22, 1147 – 1162. [3] M. R. Ninonuevo, Y. Park, H. Yin, J. Zhang, R. E. Ward, B. H. Clowers, J. B. German, S. L. Freeman, K. Killeen, R. Grimm, C. B. Lebrilla, J. Agric. Food Chem. 2006, 54, 7471 – 7480. [4] C. Kunz, S. Rudloff, W. Baier, N. Klein, S. Strobel, Annu. Rev. Nutr. 2000, 20, 699 – 722. [5] T. Eiwegger, B. Stahl, J. Schmitt, G. Boehm, M. Gerstmayr, J. Pichler, E. Dehlink, C. Loibichler, R. Urbanek, Z. Szpfalusi, Pediatr. Res. 2004, 56, 536 – 540. [6] S. Kuntz, S. Rudloff, C. Kunz, Br. J. Nutr. 2008, 99, 462 – 471. [7] L. Bode, C. Kunz, M. Muhly-Reinholz, K. Mayer, W. Seeger, S. Rudloff, Thromb. Haemostasis 2004, 92, 1402 – 1410. [8] N. S. Han, T.-J. Kim, Y.-C. Park, J. Kim, J.-H. Seo, Biotechnol. Adv. 2012, 30, 1268 – 1278. [9] Y. Bao, C. Chen, D. S. Newburg, Anal. Biochem. 2013, 433, 28 – 35. [10] S. Thurl, B. Mller-Werner, G. Sawatzki, Anal. Biochem. 1996, 235, 202 – 206. [11] P. Chaturvedi, C. D. Warren, G. M. Ruiz-Palacios, L. K. Pickering, D. S. Newburg, Anal. Biochem. 1997, 251, 89 – 97. [12] M. R. E. Aly, E.-S. I. Ibrahim, E.-S. H. El Ashry, R. R. Schmidt, Carbohydr. Res. 1999, 316, 121 – 132. [13] T. Murata, T. Inukai, M. Suzuki, M. Yamagishi, T. Usui, Glycoconjugate J. 1999, 16, 189 – 195. [14] X.-w. Liu, C. Xia, L. Li, W.-y. Guan, N. Pettit, H.-c. Zhang, M. Chen, P. G. Wang, Bioorg. Med. Chem. 2009, 17, 4910 – 4915. [15] C. Dumon, C. Bosso, J. P. Utille, A. Heyraud, E. Samain, ChemBioChem 2006, 7, 359 – 365. [16] C. Dumon, B. Priem, S. L. Martin, A. Heyraud, C. Bosso, E. Samain, Glycoconjugate J. 2001, 18, 465 – 474. [17] T. Zeppenfeld, C. Larisch, J. W. Lengeler, K. Jahreis, J. Bacteriol. 2000, 182, 4443 – 4452. [18] C. Albermann, N. Trachtmann, G. A. Sprenger, Biotechnol. J. 2010, 5, 32 – 38. [19] K. A. Datsenko, B. L. Wanner, Proc. Natl. Acad. Sci. USA 2000, 97, 6640 – 6645. [20] O. Blixt, I. van Die, T. Norberg, D. H. van den Eijnden, Glycobiology 1999, 9, 1061 – 1071. [21] B. Priem, M. Gilbert, W. W. Wakarchuk, A. Heyraud, E. Samain, Glycobiology 2002, 12, 235 – 240. [22] F. Baumgrtner, L. Seitz, G. A. Sprenger, C. Albermann, Microb. Cell Fact. 2013, 12, 40. [23] P. P. Cherepanov, W. Wackernagel, Gene 1995, 158, 9 – 14. [24] J. P. Frste, W. Pansegrau, R. Frank, H. Blçcker, P. Scholz, M. Bagdasarian, E. Lanka, Gene 1986, 48, 119 – 131. [25] T. Vallon, S. Ghanegaonkar, O. Vielhauer, A. Mller, C. Albermann, G. A. Sprenger, M. Reuss, K. Lemuth, Appl. Microbiol. Biotechnol. 2008, 81, 175 – 182. [26] M. Weiner, C. Albermann, K. Gottlieb, G. A. Sprenger, D. Weuster-Botz, Biochem. Eng. J. 2014, 83, 62 – 69. [27] S. O. Nelson, J. K. Wright, P. W. Postma, EMBO J. 1983, 2, 715 – 720. [28] C. A. Bush, M. M. Panitch, V. K. Dua, T. E. Rohr, Anal. Biochem. 1985, 145, 124 – 136. [29] N. Platzer, D. Davoust, Carbohydr. Res. 1989, 191, 191 – 207. [30] G. Strecker, J.-M. Wieruszeski, J.-C. Michalski, J. Montreuil, Glycoconjugate J. 1989, 6, 67 – 83. [31] B. Wilms, A. Hauck, M. Reuss, C. Syldatk, R. Mattes, M. Siemann, J. Altenbuchner, Biotechnol. Bioeng. 2001, 73, 95 – 103. [32] L. R. Ruhaak, E. Steenvoorden, C. A. M. Koeleman, A. M. Deelder, M. Wuhrer, Proteomics 2010, 10, 2330 – 2336.
Conflict of interest statement: Parts of the presented results have been filed as a patent application. Keywords: biosynthesis · chromosomal integration glycosyltransferases · oligosaccharides · lacto-N-tetraose
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Received: March 3, 2014 Published online on July 17, 2014
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Synthesis of the Human Milk Oligosaccharide Lacto-NTetraose in Metabolically Engineered, Plasmid-Free E. coli Florian Baumgrtner,[a] Jrgen Conrad,[b] Georg A. Sprenger,[a] and Christoph Albermann*[a] Human milk oligosaccharides (HMOs) constitute the third most abundant solid component of human milk. HMOs have been demonstrated to show positive effects on the infant’s wellbeing. Despite numerous studies, more physiological analyses of single compounds are needed to fully elucidate these effects. Although being one of the most abundant core structures in human milk, the HMO lacto-N-tetraose (LNT) is not available at reasonable prices. In this study, we demonstrate the construction of the first E. coli strain capable of producing LNT in vivo. The strain was constructed by chromosomally integrating the genes lgtA and wbgO, encoding b-1,3-N-acetylglucosaminyltransferase and b-1,3-galactosyltransferase. In shakeflask cultivations, the strain yielded a total concentration of 219.1 3.5 mg L 1 LNT (LNT in culture broth and the cell pellet). After recovery of LNT, structural analysis by NMR spectroscopy confirmed the molecule structure.
Human milk is generally considered to be the best diet for the well-being of infants.[1] At approximately 5 to 15 g L 1, human milk contains a remarkable content of free oligosaccharides, much higher than in the milk of mammals such as cow, sheep, and goat.[2] The core structures of human milk oligosaccharides (HMOs) show a lactose residue at the reducing end, which can be extended by lacto-N-biose or N-acetyllactosamine units. These core structures can be further decorated by addition of fucosyl or sialyl residues, thereby resulting in over 200 different structures identified to date.[3, 4] As recently reviewed,[2] HMOs are central to the difference between formula milk and breast milk. Several studies have described well analyzed prebiotic effects or the blockade of pathogen lectins, thereby resulting in the wash-out of pathogens.[2] Further beneficial properties of HMOs have been proposed, such as modulation of intestinal epithelial cells or of lymphocyte maturation and reduction of leucocyte rolling and adhesion.[5–7] However, further studies of these properties demand single-compound HMOs. Currently, these are mainly extracted from human milk in a complex procedure because of the large numbers of structurally different HMOs. Efforts towards the biotechnological production of several HMOs have been described.[8] But despite being one of [a] F. Baumgrtner, Prof. Dr. G. A. Sprenger, Dr. C. Albermann Institute of Microbiology, University of Stuttgart Allmandring 31, 70569 Stuttgart (Germany) E-mail: [email protected] [b] Dr. J. Conrad Bioorganic Chemistry, Institute of Chemistry, University of Hohenheim Garbenstrasse 30, 70599 Stuttgart (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402070.
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the most abundant HMO core structures in human milk, lactoN-tetraose (LNT, Scheme 1) is not available in large quantities and at reasonable prices for research. Currently, LNT is mainly prepared by a multistep extraction procedure from human milk.[6, 9–11] Both chemical and enzymatic synthesis procedures
Scheme 1. Chemical structures of oligosaccharides LNT II (b-d-GlcNAc1-3-bd-Gal1-4-d-Glc) and LNT (b-d-Gal1-3-b-d-GlcNAc1-3-b-d-Gal1-4-d-Glc).
for LNT have been published.[12, 13] However, these methods are not yet satisfactory, as the chemical synthesis requires several steps of protection and deprotection of reactive groups, and enzymatic synthesis suffers from unfavorable equilibria and low regioselectivities of the transglycosylation reactions, thereby leading to low efficiency. The in vitro synthesis of benzyl blacto-N-tetraoside from a benzyl b-lacto-N-trioside and the nucleotide-activated sugar UDP-galactose was demonstrated by using the regio- and stereoselective Leloir b-1,3-galactosyltransferase WbgO from Escherichia coli O55:H7.[14] The in vivo synthesis of the structural isomer of LNT, lacto-N-neotetraose (LNnT), was demonstrated by high-cell-density cultivation of E. coli cells expressing plasmid-borne genes for Leloir glycosyltransferases.[15, 16] These cells provided intracellular pools of the required nucleotide-activated sugars UDP-N-acetylglucosamine and UDP-galactose and were fed with lactose. Here we demonstrate that expression of the gene for WbgO in E. coli K-12, which is capable of the synthesis of lacto-N-triose II (LNT II, Scheme 1), leads to the formation of LNT from lactose and the low-cost carbon sources glycerol or glucose in shake-flask cultivation. For the construction of the LNT-producing strain, E. coli K-12 LJ110 was chosen as parent strain.[17] This plasmid-free strain was modified by knock-out of sugar degradation loci through introduction of an expression cassettes by homologous recombination.[18, 19] To construct the strain, lacZ (encoding b-galactosidase) of E. coli strain LJ110 was removed, and the strain was equipped with the b-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis to allow synthesis of LNT II.[20, 21] The resulting strain was supplemented with wbgO (encoding b-1,3-galactosyltransferase WbgO; Scheme 2).[14] As the use of antibiotics is undesirable in the ChemBioChem 2014, 15, 1896 – 1900
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Scheme 2. In vivo synthesis of LNT II and LNT in recombinant E. coli cells with Leloir glycosyltransferases and intracellular nucleotide-activated sugars. The lacZ gene was deleted to prevent metabolism of lactose. Arrows with dashed lines indicate that the majority of these products are found in the culture supernatant.
production of food- or pharmaceutical-related compounds, we decided against the use of antibiotic-resistance-encoding plasmids as vectors for recombinant genes in the final production strains. Instead, chromosomal integration of genes was chosen. Although leading to lower gene copy number, this has the advantage of genetic stability, as gene loss (segregational instability of plasmids) is avoided.[18, 19, 22] Leloir glycosyltransferases were used for intracellular synthesis of LNT. These glycosyltransferases use the nucleotide-activated sugars UDP-N-acetylglucosamine and UDP-galactose, which are endogenous to the E. coli host. To enable the intracellular synthesis of LNT II, the N. meningitidis gene lgtA (encoding b-1,3-N-acetylglucosaminyltransferase)[20, 21] was custom
www.chembiochem.org synthesized with codon optimization. The gene was cloned into an expression vector with isopropyl-b-d-thiogalactopyranoside (IPTG)-inducible Ptac-promoter, to yield pJFK-lgtA (Table 1). After equipping the vector with an FRT-flanked chloramphenicol resistance gene downstream of lgtA, the expression cassette, including the Ptac promoter, a ribosome binding site (Shine-Dalgarno sequence), the gene of interest (lgtA), a FRT-cat-FRT resistance marker, and a transcription terminator sequence from rrnB, was amplified by PCR. The cassette was then chromosomally integrated into the lacZYA locus to eliminate metabolism and possible acetylation of lactose by LacZ and LacA, respectively. Lactose uptake was retained by complementing the resulting strain LJ-A-cat (Table 1) with an E. coli K12 lacY gene under the control of a Ptac promoter. To do so, lacY was first cloned into the expression vector pJF119EH, followed by the cloning of an FRT-kan-FRT resistance cassette to obtain pJF-lacY-FRT-kan-FRT. After DNA amplification, the expression cassette was chromosomally integrated[18] into the fucIK gene locus, thereby resulting in strain LJ-AY-cat-kan. Transformation of this strain with the FLP-recombinase-expressing plasmid pCP20[23] removed the antibiotic resistance gene cassettes and yielded strain LJ-AY, which was used for further chromosomal integrations (Table 1). Scheme 2 describes the intended intracellular pathway from lactose to LNT. To study the formation of HMOs, shake-flask cultivations in minimal medium with lactose and one of the two common carbon sources (glycerol or glucose) were performed with various strains. For our purposes, glucose as carbon source has the undesirable effect of preventing the concomitant uptake of lactose through LacY (due to inhibition of LacY by unphos-
Table 1. Bacterial strains and plasmids used in this study. Relevant genotype or sequences
Source or ref. laboratory strain
E. coli LJ110 E. coli LJ-A-cat E. coli LJ-AY-cat-kan E. coli LJ-AY E. coli LJ-AYO-cat Plasmids pJF119EH pKD46 pCP20
F , f80d, lacZDM15, endA1, recA1, hsdR17(rK mK ), supE44, thi-1, gyrA96, relA1, D(lacZYAargF)U169 E. coli W3110 fnr + E. coli LJ110, lacZYA::lgtA-FRT-cat-FRT E. coli LJ110, lacZYA::lgtA-FRT-cat-FRT fucIK::lacY-FRT-kan-FRT E. coli LJ110, lacZYA::lgtA-FRT fucIK::lacY-FRT E. coli LJ110, lacZYA::lgtA-FRT fucIK::lacY-FRT xylAB::wbgO-FRT-cat-FRT cloning vector, Ptac, AmpR ParaB g b exo (red recombinase), AmpR FLP + , l cI857 + , l pR Repts, AmpR, CmR
pCAS30-FRT-cat-FRT pJF-crtY-FRT-kan-FRT pJF-futC pF81kan pJF119EH-kan pJFK-futC pJFK-lgtA pJF-lacY pJFK-wbgO pJFK-lgtA-FRT-cat-FRT pJF-lacY-FRT-kan-FRT pJFK-wbgO-FRT-cat-FRT
pJF119DN, Pantoea ananatis crtE gene, FRT-sites, AmpR,CmR pJF119DN, P. ananatis crtY gene, FRT-sites, AmpR,KmR pJF119DN, Helicobacter pylori futC gene, AmpR pJF119EH, pheA(fbr), aroF, aroB, aroL, KanR pJF119EH, exchange of AmpR to KanR pJF-futC, exchange of AmpR to KanR pJFK-futC, exchange of futC with codon-optimized N. meningitidis lgtA gene, KanR pJF119EH, E. coli lacY gene, AmpR pJFK-futC, exchange of futC with codon-optimized E. coli O55:H7 wbgO gene, KanR pJFK-lgtA, FRT-sites, KanR, CmR pJF-lacY, FRT-sites, AmpR, KanR pJFK-wbgO, FRT-sites, KanR, CmR
Frste et al.[24] Datsenko and Wanner[19] Cherepanov and Wackernagel[23] Vallon et al.[25] Albermann et al.[18] Baumgrtner et al.[22] Weiner et al.[26] this study this study this study this study this study this study this study this study
Strains E. coli DH5a
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phorylated Enzyme IIAGlc).[27] However, utilization of glycerol might lead to lower product yields because of the need for gluconeogenic steps towards UDP-N-acetylglucosamine and UDP-galactose. Therefore, glucose and glycerol were compared as possible main carbon sources for growth and the provision of metabolites. In both cases, lactose was fed as the acceptor for the glycosyltransferase LgtA. Recombinant cells were incubated in minimal medium in shake flasks and grown at 30 8C. After reaching the exponential growth phase, cells were induced with IPTG (0.5 mm), and lactose (2 g L 1) was added. As expected, the lacY-deficient strain LJ-A-cat was neither capable of lactose utilization for growth, nor of LNT II-production. Strain LJ-AY-cat-kan (Table 1, chromosomally integrated lacY under a Ptac promoter) was capable of lactose utilization for the synthesis of LNT II. With glucose as main carbon source, this strain showed an exponential growth rate (mmax) of 0.33 h 1, total LNT II formation of 1.906 0.201 g L 1 (13 % in the cell pellet), and an LNT II yield of 641.1 11.3 mg gCDW 1 (cell dry weight; Table 2) 24 h after lactose supplementation and induction with IPTG. Cultures in medium with glycerol as the carbon source showed a lower mmax (0.28 h 1), but a higher cell density and a higher productivity, with an LNT II concentration of 2.465 0.057 g L 1 (11 % in the cell pellet) and a yield of 997.7 1.7 mg gCDW 1 after 24 h. Thus, glycerol showed a higher potential for in vivo synthesis of LNT II, with final LNT II concentrations about 30 % higher than with glucose. The molar LNT II yield from lactose was 77.34 1.78 % for glycerol and 59.82 6.30 % for glucose (Table 2). For the intracellular conversion of LNT II to LNT, a synthetic and codon-optimized version of wbgO (b-1,3-galactosyltransferase) from E. coli O55:H7 was chromosomally integrated into the xylAB locus as described for lgtA,[14] thus yielding strain LJAYO-cat. This strain was cultivated under the same conditions as for LJ-AY-cat-kan to yield the tetrasaccharide LNT. LJ-AYO-cat exhibited growth rates comparable to those of LJ-AY-cat-kan (mmax 0.33 and 0.28 h 1 with glucose and glycerol, respectively). We examined the production of LNT II and LNT in both media. LJ-AYO-cat also synthesized more LNT II in medium with glycerol: 1.890 0.067 g L 1 (13 % in the cell pellet) in cultures with glucose and 2.090 0.022 g L 1 (10 % in the cell pellet) with glycerol, and LNT II yields of 570.6 15.2 and 66.2 1.6 mg gCDW 1, respectively. Whereas LNT II production was higher in medium with glycerol, LNT concentration and yield
were higher in medium with glucose: 219.1 3.5 mg L 1 LNT (47 % in the cell pellet) 24 h after induction, compared to 162.1 6.2 mg L 1 (25 % in the cell pellet) with glycerol. This corresponds to yields of 66.2 1.6 mg gCDW 1 for medium with glucose and 58.5 3.0 mg gCDW 1 for medium with glycerol (Table 2). Thus, we show for the first time that LNT can be synthesized by recombinant E. coli cells. Under the applied conditions, glucose proved to be the more favorable carbon source for LNT synthesis. But as LNT II is not fully converted into LNT by LJ-AYO-cat, there might be limitation in the activity of WbgO or in the supply of the donor substrate UDP-galactose. Studies of lactose uptake and UDP-sugar formation might help elucidate the impacts of the inducer exclusion by glucose,[27] or the need for strengthening gluconeogenic steps with glycerol as the carbon source. Structural analysis of oligosaccharides synthesized in a whole-cell system is of particular importance to exclude nonspecific reactions or modifications of the oligosaccharides by endogenous cellular components. In order to verify the structures of LNT II and LNT produced in vivo, the products were isolated from culture broth and the cell pellet, respectively. The MS analysis showed m/z ratios of the sodium adducts of 568.2 and 730.2 for LNT II and LNT, respectively (Figure S1 in the Supporting Information); this is in good agreement with the calculated masses of these compounds. To analyze the configuration of LNT further, extensive NMR analysis was performed, including 1H, 13C, TOCSY1D, eCOSY, coupled and decoupled band-selective gHSQC, as well as gHMBC, gC2H2BC, and gHSQC-TOCSY spectra. In addition to allowing comparison to previously published NMR data by Bush et al., [28] Platzer and Davoust, [29] and Strecker et al.,[30] this also allowed autonomous verification of the structure and the annotation of previously undefined proton coupling constants, especially from the coupled band-selective gHSQC spectra. Chemical shifts and couplings of LNT II and LNT are listed in Tables S2 to S5. Thus, the structures of LNT II and LNT were unambiguously verified. For the in vivo synthesis of LNnT, Priem et al. described the formation of elongated LNnT, by repetitive reactions of N-acetylglucosaminyl- and galactosyl-transferases.[21] Such elongation beyond the tetrasaccharide was not observed during the in vivo synthesis of LNT. This might be explained by a lower activity of the N-acetylglucosaminyltransferase LgtA for the sub-
Table 2. Total product concentrations and yields in shake-flask cultivations 24 h after induction, quantified by HPLC.[a] Strain E. coli LJ-AY-cat-kan E. coli LJ-AYO-cat
Carbon source
Conc. [g L 1]
Lacto-N-triose II Yield [mg gCDW 1][b] Yield [mg glactose 1][c]
Conc. [mg L 1]
Lacto-N-tetraose Yield [mg gCDW 1] Yield [mg glactose 1][c]
1% 1% 1% 1%
1.906 0.201 2.465 0.057 1.890 0.067 2.090 0.022
641.1 11.3 997.7 1.7 570.6 15.2 753.7 17.6
– – 219.1 3.5 162.1 6.2
– – 66.2 1.6 58.5 3.0
glucose glycerol glucose glycerol
953.2 100.4 1232.6 28.3 944.8 33.5 1045.2 11.1
– – 109.6 1.7 81.1 3.1
[a] Cultivation at 30 8C and 90 rpm in minimal medium with 1 % glucose or 1 % glycerol as the main carbon source, and 0.2 % lactose as the source for product formation (concentrations and yields 24 h after induction with 0.5 mm IPTG from a minimum of two biological replicates). [b] Yield of LJ-AY-catkan determined by calculating cell dry mass concentrations (OD600), with correlation factors of E. coli strain LJ-AYO-cat (0.37 and 0.30 g L 1 for cultures in minimal medium with glucose and glycerol, respectively). [c] Product yields from lactose were calculated from the total lactose concentration (2 g L 1).
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CHEMBIOCHEM COMMUNICATIONS strate LNT compared to LNnT, as shown by in vitro assays,[20] or by a higher intracellular concentration of LNnT, which is not secreted into the culture broth.[21] In summary, we have constructed plasmid-free E. coli strains lacking b-galactosidase activity but displaying lactose uptake (LacZ LacY + ), by chromosomal integration as previously described.[18, 22] Additionally, the strains carry recombinant genes for b-1,3-N-acetylglucosaminyltransferase and b-1,3-galactosyltransferase (lgtA and wbgO), each under control of a Ptac promoter. Using nucleotide-activated sugars from endogenous pools for Leloir glycosyltransferases, this strain is the first E. coli strain capable of producing lacto-N-tetraose, in addition to lacto-N-triose II, allowing LNT-synthesis from low cost substrates like lactose and glucose or glycerol. Thus, we describe here a simple synthesis of LNT by utilizing a genetically stable bacterial strain and inexpensive substrates, to make LNT more widely available for research.
Experimental Section Chemicals, media, and bacterial strains: All strains were based on the laboratory strain E. coli LJ110.[17] E. coli DH5a was used for plasmid construction. Unless stated otherwise, E. coli strains were cultivated on lysogeny broth (LB) agar plates at 37 8C or in LB liquid medium at 37 8C with shaking (160 rpm). Antibiotics for strain construction were chloramphenicol (50 mg mL 1), kanamycin (50 mg mL 1), or ampicillin (100 mg mL 1). Difco MacConkey agar base was purchased from Otto Nordwald (Hamburg, Germany). Standards lacto-N-tetraose (purity > 95 %) and lacto-N-neotetraose (purity > 95 %) were purchased from IsoSep (Tullinge, Sweden). E. coli b-galactosidase (1500 U mL 1) was from Roche. All other chemicals and reagents were purchased from either Sigma–Aldrich or Carl Roth and were of the highest purity available. Construction of plasmids and strains: All strains and plasmids are listed in Table 1. Expression vectors with ampicillin resistance were based on the previously described plasmid pJF119EH.[24] Vectors with kanamycin resistance were derived from the expression plasmids pJF-futC and pJF119EH,[22, 24] by replacing the b-lactamase gene with a fragment with a kanamycin-resistance gene from pF81kan[26] at RcaI restriction sites. The resulting plasmids were termed pJFK-futC and pJF119EH-kan, respectively. Synthetic, codon-optimized open reading frames for lgtA and wbgO were ordered from Life Technologies and cloned separately into the NdeI and BamHI sites of pJFK after removing futC, to yield pJFK-lgtA and pJFK-wbgO. The open reading frame of lacY was amplified from chromosomal E. coli LJ110 (primers lacY-for lacY-rev in Table S1), and cloned into the EcoRI/BamHI sites of pJF119EH to yield pJFlacY. Plasmids pJFK-lgtA, pJFK-wbgO, and pJF-lacY were digested with HindIII and ligated with a HindIII-digested FRT-cat-FRT- or FRTkan-FRT-Fragment from pCAS30-FRT-cat-FRT or pJF-crtY-FRT-kanFRT[18, 25] to yield pJFK-lgtA-FRT-cat-FRT, pJFK-wbgO-FRT-cat-FRT and pJF-lacY-FRT-kan-FRT, respectively. Plasmid-free E. coli strains were based on E. coli LJ110, and were constructed by homologous recombination of expression cassettes into sugar degradation loci.[18] Expression cassettes lgtA-FRT-catFRT, wbgO-FRT-cat-FRT, and lacY-FRT-kan-FRT were amplified (primers in Table S1) and consecutively transferred to E. coli LJ110 strains carrying the l-red recombinase system on plasmid pKD46.[19] Colonies carrying successfully integrated genes were selected by antibiotic resistance and were assessed by their color on MacConkey agar sugar plates as described previously.[18, 22] Antibiot 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org ic resistance cassettes were removed by transient expression of FLP recombinase (encoded by plasmid pCP20) as previously described.[23] Shake-flask cultivations for production of LNT II and LNT: Synthesis of LNT II and LNT was assayed after cultivation of strains (at least two replicates) in 250 mL shake flasks with minimal medium (25 mL), with glycerol or glucose (carbon source) and chloramphenicol (to prevent contamination). Minimal medium was slightly modified from a previous composition:[22, 31] (NH4)2SO4 (2.68 g L 1), (NH4)2-H-citrate (1 g L 1), glycerol or glucose (10 g L 1), K2HPO4 (14.6 g L 1), MgSO4 (0.241 g L 1), MnSO4·H2O (10 mg L 1), Na2SO4 (2 g L 1), NaH2PO4·H2O (4 g L 1), NH4Cl (0.5 g L 1), thiamine (10 mg L 1), and trace element solution (3 mL L 1; CaCl2·2 H2O FeCl3·6 H2O (16.7 g L 1), Na2-EDTA (20.1 g L 1), (0.5 g L 1), 1 1 ZnSO4·7 H2O (0.18 g L ), MnSO4·H2O (0.1 g L ), CuSO4·5 H2O (0.16 g L 1), and CoCl2·6 H2O (0.18 g L 1)). Strains were cultivated in 250 mL shake flasks (25 mL culture volume) at 30 8C and 90 rpm to OD600 = 0.4–0.5, before inducing expression by addition of IPTG (0.5 mm), with lactose (2 g L 1) addition as the substrate for product formation. Samples for HPLC analysis were taken 24 h after induction and centrifuged (15 300 g, 2 min). Supernatants were stored at 20 8C and pellets were washed with saline (NaCl, 0.9 g L 1) and centrifuged again, before storage at 20 8C. CDWs of E. coli LJ-AYO-cat cultures were measured 24 h after induction by centrifugation (5869 g, 4 8C, 20 min) of culture (10 mL) and drying the cell pellet (22 h, 120 8C), before weighing. Measurements were taken from two separate cultures per carbon source (duplicate measurements per culture). Correlations of CDW to OD600 for LJ-AYO-cat were determined as 0.37 and 0.30 g L 1 for cultures in minimal medium with glucose and glycerol, respectively. These correlations were also used for the calculation of product yields per CDW of E. coli strain LJ-AY-cat-kan. Cleavage of lacto-N-neotetraose to obtain a lacto-N-triose II standard: To obtain LNT II as a standard for HPLC analysis, lacto-Nneotetraose (141 nmol) was digested with b-galactosidase (1.5 U) in Tris·HCl (25 mL, 100 mm, pH 7.5) at 37 8C overnight. The enzymatic reaction was followed by HPLC. HPLC analysis of oligosaccharides: Saccharides from shake-flask experiments were analyzed by HPLC. After centrifugation, sugars in culture supernatants were derivatized without prior treatment. Cell pellets of 2 mL culture were resuspended in water to a total volume of 150 mL, then lysed by incubation in boiling water (5 min) followed by centrifugation (20 800 g, 5 min). The resulting supernatant was analyzed for intracellular sugar content. To label the reducing sugars, samples (50 mL) or standards of lactose, LNT, or LNT II were incubated (2 h, 65 8C) with 2-aminobenzoic acid (25 mL; 2-aminobenzoic acid (350 mm) in DMSO (80 %, v/v) and acetic acid (20 %, v/v)) and 2-picoline borane (25 mL; 2-picoline borane (1 m) in DMSO).[32] After incubation, preparations were diluted (1:5) in HPLC mobile phase solvent A (see below) before analysis. HPLC analysis was performed on a Dionex HPLC instrument (Thermo Scientific; Chromeleon software, Gina autosampler, P580 pumps, RF2000 fluorescence detector). Products were separated on a Luna C18 (2) reversed-phase column (250 4.6 mm, 5 mm; Phenomenex, Aschaffenburg, Germany) at a flow-rate of 1 mL min 1 with mobile phase solvent A (acetonitrile (10.71 %, v/v), tetrahydrofuran (0.89 %, v/v), ortho-phosphoric acid (0.38 %, v/v) and 1-butylamine (0.27 %, v/v)), and solvent B (acetonitrile): 100 % solvent A (20 min), linear gradient to 100 % solvent B (4 min), linear gradient to 100 % solvent A (1 min), equilibration with 100 % solChemBioChem 2014, 15, 1896 – 1900
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vent A (5 min). The fluorescent detector was operated at lex = 230 nm and lem = 425 nm. Concentrations of lactose and lacto-N-tetraose were determined by comparison to derivatized, commercial standards (95 % purity assumed for commercial lacto-N-tetraose). Concentrations of lacto-Ntriose II were calculated by comparison of peak areas to those obtained from commercial lactose standards, with the assumption that the 2-aminobenzoic acid derivatization of glycans with different lengths gives comparable fluorescence signal intensities.[32] Comparison of derivatized standards of lactose and lacto-N-tetraose confirmed these results by showing concentration-to-emission ratios with a deviation below 2 %. Preparative synthesis and isolation of lacto-N-triose II and lactoN-tetraose: For the preparative production of oligosaccharides, minimal medium (750 mL) with glycerol (10 g L 1, for LNT II) or glucose (10 g L 1, for LNT) and chloramphenicol (50 mg L 1) was inoculated (starting OD600 = 0.05) with LJ-AY-cat-kan or LJ-AYO-cat, respectively, in 3 L baffled shake flasks. Shake flasks were incubated at 30 8C with 90 rpm shaking. At OD600 = 0.4–0.5, IPTG (0.5 mm) and lactose (0.3 %) were added. Cells were separated from culture supernatants by centrifugation, (2831 g, 4 8C, 15 min) 48 h after induction. LNT II was recovered from culture supernatants; LNT was recovered from cell pellets. Pellets were resuspended in H2O, incubated (100 8C, 20 min) and centrifuged (6371 g, 4 8C, 15 min). The resulting supernatant was used for isolation of LNT. LNT II and LNT were recovered by using activated charcoal chromatography and gel-filtration as previously described.[22] Purifications through an activated charcoal chromatography and gel-filtration chromatography yielded 323.9 mg of LNT II and 102.1 mg of LNT. Structural analysis of lacto-N-triose II and lacto-N-tetraose: Purified products were analyzed by mass spectrometry on a microTOFQ system (Bruker; mass spectrum of LNT in Figure S1). NMR spectra were recorded in D2O on a Unitiy Inova 500 MHz spectrometer (Varian) at 296 K. 1H and 13C spectra were referenced to residual H2O and deuterated methanol (d = 4.70 and 49 ppm, respectively). 1 H, 13C, eCOSY, TOCSY1D, band-selective gHSQC (with and without decoupling), gHSQC-TOCSY, gHMBC, and gC2H2BC spectra were recorded by using CHEMPACK 4.0 pulse sequences (Varian Vnmrj 2.1B software). Recorded NMR spectra of LNT are shown in the Supporting Information.
Acknowledgements The authors gratefully acknowledge Katrin Wohlbold (Institute of Organic Chemistry, University of Stuttgart) for MS measurements and Prof. Dr. Uwe Beifuß (Institute of Chemistry, University of Hohenheim) for enabling NMR analysis on a Varian Unitiy Inova 500 MHz spectrometer. This work was supported by contract research “Glycomics/Glycobiology” of the Baden-Wrttemberg Stiftung.
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Conflict of interest statement: Parts of the presented results have been filed as a patent application. Keywords: biosynthesis · chromosomal integration glycosyltransferases · oligosaccharides · lacto-N-tetraose
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: March 3, 2014 Published online on July 17, 2014
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