Efficient chemoenzymatic synthesis of lipo-chitin oligosaccharides as plant growth promoters

Article abstract of DOI:10.1039/c5gc00623f

Lipo-chitin oligosaccharides (Nod and Myc LCOs) are molecules involved in symbiotic phenomena in the plant kingdom. They play a major role in the process of atmospheric nitrogen fixation and mineral soil nutrients uptake both in legumes and in non-legumes, and are active at extremely low concentrations down to the nano- and even picomolar range. These compounds contain various substitutions along the oligosaccharide backbone of the molecule including an essential fatty acid on the non-reducing unit and are considered as environmentally-friendly fertilizers. Currently, chemical synthesis cannot produce precursors of Nod and Myc LCOs at a large scale and an in vitro chemoenzymatic pathway is presented here as a new and efficient method for preparing quantities of these high-value oligosaccharides. VC1280 (Vibrio cholerae) is a chitin deacetylase (CD) capable of regioselectively cleaving an acetate from the non-reducing penultimate N-acetyl-d-glucosaminyl (GlcNAc) unit of chitin oligosaccharides (COs). This provides a free amino group which can be further N-acylated with a fatty-acid chain to give analogues of LCOs. Alternatively, the non-reducing GlcNAc unit can be removed by β-N-acetylglucosaminidase treatment, followed by N-acylation to give natural LCOs. VC1280 CD was produced in the periplasm of E. coli. Under the conditions used, 120 mg of the pure enzyme was recovered from 1 L of culture medium. For the first time, in vitro production of a library of natural LCOs as well as their analogues has been carried out at a preparative scale from biosourced chitin oligosaccharides constituting an approach of major interest for sustainable agriculture.

Full text of DOI:10.1039/c5gc00623f

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Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
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Efficient chemoenzymatic synthesis of lipoꢀ
chitinoligosaccharides plant growth promotors
R. Chambon,a G. Despras,b A Brossay,b B. Vauzeilles,b,c D. Urban,b J.ꢀM. Beau,b,c
S. Armand,a S. Cottaz,*a and S. Forta
Lipoꢀchitinoligosaccharides (Nod and Myc LCOs) are molecules involved in symbiotic phenomena in
the plant kingdom. They play a major role in the process of atmospheric nitrogen fixation and mineral
soil nutrients uptake both in legumes and in nonꢀlegumes, and are active at extremely low concentrations
down to the nanoꢀ and even picomolar range. These compounds contain various substitutions along the
oligosaccharidic backbone of the molecule including an essential fatty acid on the nonꢀreducing unit and
are considered as environmentallyꢀfriendly fertilizers. Currently, chemical synthesis cannot produce
precursors of Nod and Myc LCOs at a large scale and an in vitro chemoꢀenzymatic pathway is presented
here as a new and efficient method for preparing quantities of these highꢀvalue oligosaccharides.
VC1280 (Vibrio cholerae) is a chitin deacetylase (CD) able to regioselectively cleave an acetate from the
nonꢀreducing penultimate Nꢀacetyl Dꢀglucosaminyl (GlcNAc) unit of chitinoligosaccharides (COs). This
provides a free amino group which can be further Nꢀacylated with a fattyꢀacid chain to give analogues of
LCOs. Alternatively the nonꢀreducing GlcNAc unit can be removed by βꢀNꢀacetylglucosaminidase
treatment, followed by Nꢀacylation to give natural LCOs. VC1280 CD was produced in the periplasm of
E. coli. In the conditions used, 120 mg of pure enzyme was recovered from 1L of culture medium. For
the first time, in vitro production of a library of natural LCOs as well as analogues has been carried out at
preparative scale from biosourced chitinoligosaccharides constituting an approach of major interest for
sustainable agriculture.
to their respective host plant, thereby promoting plant growth
independently of the available soil nitrogen.
Introduction
Nodulation factors (Nod factors) are key signal molecules for nodule
development and bacterial invasion during initial stages in the
Rhizobiumꢀlegume symbiosis.1ꢀ3 They consist of lipoꢀ
chitinoligosaccharides (LCOs) and are produced by rhizobia,
including the genera Allorhizobium, Azorhizobium, Bradyrhizobium,
Mesorhizobium, Rhizobium and Sinorhizobium. Such symbioses
result in the formation of root nodules, new organs colonized by
differentiated bacteria, which fix atmospheric nitrogen and provide it
While Nod LCOs are involved in the Rhizobiumꢀlegume
symbiosis, another kind of symbiosis, between mycorrhizal fungi
and plants implicates LCOs.4 Arbuscular mycorrhiza is a root
endosymbiosis between fungi of the ancient phylum Glomeromycota
and terrestrial plants. The arbuscular mycorrhiza fungus G.
intraradices secretes a mixture of sulfated and nonꢀsulfated LCOs
(called MycꢀLCOs because they stimulate mycorrhiza formation in
legumes and nonꢀlegumes) that have structural similarities with
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rhizobial Nod LCOs. Nod and Myc LCOs are extremely active deacetylase (CD). CD (EC 3.5.1.ꢀ) are produced by marine bacteria,
molecules, down to the nanoꢀ even picomolar range, and quantities several fungi and insects. They hydrolyze the acetamido group of
required for fertilization are less than 1 mg per hectare of planted GlcNAc units of chitin or COs, generating GlcN units and acetic
seed!
acid. They are members of the carbohydrate esterase family 4 (CE4),
All these properties make Nod and Myc LCOs natural and as defined in the CAZy database (http://www.cazy.org).
biodegradable molecules with a great promise to improve the yield
of crops and reduce the use of fertilizers.
Vibrio cholerae CD is acting on the penultimate Nꢀ
acetylglucosaminyl (GlcNAc) unit of COs. It has been suggested that
Nod and Myc LCOs consist of an oligomeric backbone of βꢀ1,4ꢀ this enzyme acts in the same way as rhizobium NodB CD in the
linked Nꢀacetylꢀ ꢀglucosaminyl residues, Nꢀacylated with a fatty biosynthesis of Nod LCOs, albeit with a significant difference in the
D
acid chain at the nonꢀreducing terminal residue.4ꢀ6 Their structures location of the hydrolyzed acetamido group from the COs
differ in the number of GlcNAc residues present in the backbone.12 The objective of this work was to express and purify
chitinoligosaccharidic backbone, with a degree of polymerization Vibrio cholerae CD in E. coli to examine a preparative scale
(DP) of generally four and five glucosaminyl units. There are also production of Nod and Myc LCOs precursors, thus providing new
variations in the nature of the fatty acyl group, and in the substitution route to these important molecules and analogues as well by
of the reducing and nonꢀreducing terminal residues (e.g. Oꢀacetyl, Oꢀ subsequent fatty acid Nꢀacylation.
fucosyl, Oꢀcarbamoyl or Oꢀsulfate group).
The availability of LCOs and their chitinoligosaccharidic precursors
Experimental
is a critical issue. Rizhobia culture at industrial scale is a process
dedicated more for their use as inoculants rather than for extraction General
and purification of LCOs.7 Chemical synthesis has only enabled Reagents for bacterial media were from Euromedex (Mundolsheim,
access to minute quantities of such compounds so far, despite its France) and Invitrogen (CergyꢀPontoise, France). Reagents for
potential for large scale production thanks to considerable progress molecular biology were obtained from Invitrogen, Euromedex,
in synthetic methodologies.8 Precursors of Nod factors have been MachereyꢀNagel (Hoerd, France) and Thermo Scientific (Villebon
produced by highꢀcellꢀdensity culture of metabolically engineered sur Yvette, France). FACOSTM (a low molecular weight chitosan
E.coli.9 This latter approach is hampered by the difficulty to purify inferior to 2000 g.molꢀ1 with an acetylation degree of 10 %) was
mixtures of partially deꢀNꢀacetylated chitinoligosaccharides from the purchased from Kitto Life Co (KyongkiꢀDo, South Korea).
bacterial cells. As an alternative, we selected chitin as starting
renewable biomolecule. Chitin is one of the most abundant naturally
occurring polymers since it is mainly found in the cuticles of all
arthropods, the endoskeletons of all cephalopods, but also in the cell
walls of fungi and yeasts. Chitin is a linear copolymer of β(1→4)ꢀ
Chemicals and Aspergillus oryzae βꢀgalactosidase containing βꢀNꢀ
acetylglucosaminidase were purchased from SigmaꢀAldrich Chimie
(Saint QuentinꢀFallavier, France).
Thin Layer Chromatography
linked
Dꢀglucosamine (GlcN) and GlcNAc units in varying
Reactions were monitored by thin layer chromatography (TLC)
using Silica Gel 60 F254 precoated plates (E. Merck, Darmstadt) and
proportions.10 Chemical or enzymatic depolymerization of chitin is
hampered by its highly crystalline structure and water insolubility,
leading mainly to GlcNAc and chitinbiose (COꢀII)11 while higher
oligomers are obtained only in low yield. Partially deacetylated
chitin, namely chitosan, is water soluble and its enzymatic
hydrolysis efficiently provides oligosaccharides in a wide range of
molecular weights. These oligomers are produced industrially and
marketed for cosmetic application. Nꢀacetylation of chitosan
oligomers would provide an efficient way to produce
chitinoligosaccharides (COs) which could serve as precursors of
LCOs by in vitro selective deꢀNꢀacetylation catalyzed by chitin
propanol/H2O/ammonia (70:30:1) as
a
solvent. Detection of
carbohydrates was achieved by
charring with the
diphenylamine/aniline/orthophosphoric acid reagent.13
Analytical sizeꢀexclusion chromatography (HPSEC)
COs and CO(N)s11 were analysed by HPSEC. The system consisted
of a Shodex OH pack SBꢀG precolumn in front of a series of a
Shodex OH pack SBꢀ802 and Shodex OH pack SBꢀ802.5 columns
(8*300 mm), connected to a pump (Waters 510, Millipore). Samples
were solubilized in water. Elution was performed with a 100 mM
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sodium nitrate solution at a flow rate of 0.5 mL minꢀ1 and monitored pooled, dialyzed against 20 mM potassium phosphate buffer pH 8
by a refractometric detector (Waters 410, Millipore).
and stored at – 80 °C. The protein concentration was determined by
the Bradford method with BSA as a standard.14
Mass spectrometry analysis
The mass measurements of chitinoligosaccharides were performed
SDSꢀPAGE
using a MALDIꢀToF/ToF AutoFlex I (Bruker) for low resolution Protein samples were heated at 100 °C for 5 min in Laemmli buffer
analysis, and using a ESIꢀQToF (Xevo GS2ꢀS QToFꢀWaters) for (65 mM Tris/HCl, pH 6.8, 0.3 % SDS, 10 % glycerol, 5 % βꢀ
high resolution (HRESIMS) analysis (PSM facility, PCNꢀICMG, mercaptoethanol and 0.1 mg.mLꢀ1 bromophenol blue),15 followed by
Grenoble).
electrophoresis in a 10 % polyacrylamide gel. Gels were stained with
Instant Blue (Expedeon).
NMR procedures
Enzyme assays
NMR spectra were recorded on Bruker AV300, AV360 AV400 and
cryoꢀAV600 spectrometers, at 350 ºK (300 °K for cryoꢀAV600).
Chemical shifts (in ppm) were determined relative to residual nonꢀ
deuterated solvent as an internal reference. Coupling constant(s) in
hertz (Hz) were measured from oneꢀdimensional spectra and
multiplicities were abbreviated as following: d (doublet), t (triplet),
m (multiplet). In NMR data, Hꢀ1I and Cꢀ1I refers respectively to
anomeric proton and carbon at the reducing unit.
Chitin deacetylase (CD) activity was assayed by incubating
chitinbiose (COꢀII) with 1.5 ꢀM CD in 20 mM HEPES buffer pH 7
at 37°C. At regular time intervals, the amount of amino group was
quantified by using 3ꢀmethylꢀ2ꢀbenzothiazolinone hydrazone
hydrochloride reagent (MBTH).16 For the determination of kinetic
parameters, measurements of initial rates were made at several
substrate concentrations ranging from 0.2 to 3 mM. KM and kcat were
determined by nonꢀlinear regression using the Grafit program
(Erithacus Software)
Bacterial strains, plasmids and growth conditions
Plasmid pET26bꢀVC1280 was kindly provided by late Professor
Saul Roseman (Department of Biology, The John Hopkins
University, Baltimore, USA). Escherichia coli strain TOP10
(Invitrogen) was used as host for the plasmid propagation.
Escherichia coli strain BL21 (DE3) (Invitrogen) was used for the
largeꢀscale production of V. cholerae CD.
Preparation of COs
Commercial chitosan oligosaccharides (FACOSTM
)
were Nꢀ
acetylated by treatment with 5% acetic anhydride in (50:50) waterꢀ
methanol for 12h at 20°C followed by concentration in vacuo. The
mixture was dissolved in water and passed through a cationic
exchange resin DOWEX 50WX4 (H+ form) column to eliminate the
partially acetylated oligosaccharides. The nonꢀretained fraction
containing COs was neutralized with an anionic exchange resin
Amberlite IRA400 (OHꢀ form) and freezeꢀdried. COs were
individually separated by size exclusion chromatography (SEC)
consisting of a Biogel P4 colomn (1.5 x 1000 mm) connected to a
pump (Smartline semiꢀpreparative, Knauer) and with an inline
degasser (Knauer). Elution was performed with water at flow rate of
2.25 mL.minꢀ1, and monitored by a refractometric detector (IOTAꢀ2,
Precision Instrument (DIONEX)). COs purity was confirmed by
MALDIꢀToF MS and HPSEC analyzes.
Overexpression and purification of recombinant V. cholerae CD
E. coli strain BL21 (DE3) harboring the plasmid pET26bꢀVC1280
was grown at 37 °C in 1 L of LuriaꢀBertani broth medium
supplemented with 40 ꢀg.mLꢀ1 kanamycin to a cell density of 0.5 at
600 nm. The temperature was lowered to 20° C and protein
production was induced during 22 h by the addition of 50 µM
isopropyl βꢀDꢀthiogalactopyranoside. Cells were then harvested by
centrifugation at 8,450 x g for 10 min at 4° C and lysed by cell
disruption. After centrifugation at 50,000 x g for 30 min at 4° C, CD
was purified by applying the supernatant on a Ni2+ꢀchelating resin
(Qiagen). The column was then washed with five volumes of 20 mM
TrisꢀHCl pH 8, 500 mM NaCl, 20 mM imidazole (washing buffer)
and V. cholerae CD was finally eluted with a 20ꢀ300 mM imidazole
gradient in washing buffer. Fractions containing CD were identified
by 10% polyacrylamide gel electrophoresis analysis. They were
CD catalyzed deꢀNꢀacetylation of COs
Standard procedure for 1 mg of pure CO: COꢀIV, COꢀV or COꢀVI
was incubated with 50 ꢁg of CD in 0.5 mL of 10 mM ammonium
carbonate buffer pH 7.9 during 20 hours at 37 °C. The enzyme was
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1
heatꢀinactivated (10 min, 100 °C) and the reaction mixture was COꢀV(NV). H NMR (360 MHz, D2O) δ 5.20 (s, 0.5H, Hꢀ1αI), 4.88
centrifuged for 10 min at 21,100 g at 4 °C. The resulting supernatant (d, 1H, J1,2 = 8.2 Hz, Hꢀ1),4.84ꢀ4.69 (m, 0.5H, Hꢀ1βI), 4.65ꢀ4.54 (m,
was freezeꢀdried without further purification to give the 3H, Hꢀ1), 3.99ꢀ3.46 (m, 29H, Hꢀ2I,II,III,IV, Hꢀ3I,II,III,IV,V, Hꢀ4I,II,III,IV,V
,
corresponding CO(N) deꢀNꢀacetylated at the penultimate Hꢀ5I,II,III,IV,V, CH2ꢀ6I,II,III,IV,V), 3.15 (dd, 1H, J2,1 = J2,3 = 9.3 Hz, Hꢀ2V),
glucosaminyl residue from the reducing end, and characterized by 2.07 (s, 9H, 3N(C=O)CH3), 2.05 (s, 3H, N(C=O)CH3). HRESIMS:
low and high resolution MS, 1H NMR and HPSEC (ESI S1ꢀS3†).
m/z calcd for C38H65N5O25: 992.4047 [M+H]+, found 992.4030.
1
COꢀIV(NIII). H NMR (600 MHz, D2O) δ 5.16 (d, 0.6H, J1,2 = 2.8
Hz, Hꢀ1αI), 4.67 (d, 0.4H, J1,2 = 7.7 Hz, Hꢀ1βI), 4.59ꢀ4.50 (m, 3H, Hꢀ
Acylation of CO(N)s
1II,III,IV), 3.95ꢀ3.40 (m, 23H, Hꢀ2I,II,IV, Hꢀ3I,II,III,IV, Hꢀ4I,II,III,IV, Hꢀ Acylation reactions involved Nꢀhydroxysuccinimide activated esters,
5I,II,III,IV CH2ꢀ6I,II,III,IV), 2.74 (m, 1H, Hꢀ2III), 2.03 (s, 6H, which were obtained according to known literature procedures.18
2N(C=O)CH3), 2.01 (s, 3H, N(C=O)CH3). HRESIMS: m/z calcd for Triethylamine (1.6 equiv) was added to a solution of CO(N)s (5.4
C30H52N4O20: 789.3253 [M+H+]+, found 789.3265. mM, 1 equiv) and activated ester of oleic acid19 or palmitic acid20
,
COꢀV(NIV). 1H NMR (360 MHz, D2O) δ 5.19 (s, 0.5H, Hꢀ1αI), 4.72ꢀ (1.2 equiv) in dimethylsulfoxide in a polypropylene sealed tube. The
4.53 (m, 4.5H, Hꢀ1βI, Hꢀ1II,III,IV,V), 3.98ꢀ3.42 (m, 29H, Hꢀ2I,II,III,V, Hꢀ
resulting mixture was stirred at 60 °C for 6 days then concentrated
3I,II,III,IV,V, Hꢀ4I,II,III,IV,V, Hꢀ5I,II,III,IV,V, CH2ꢀ6I,II,III,IV,V), 3.01 (dd, 1H,
under vacuum. The residue was suspended in ethyl acetate/methanol
J2,1 = J2,3 = 9.3 Hz, Hꢀ2IV), 2.07 (s, 9H, 3N(C=O)CH3), 2.04 (s, 3H,
(9/1, 0.5 mL), sonicated for 10 min, centrifuged (7000 rpm, 10 min)
and the supernatant was removed. This operation was repeated four
times and the residue was reꢀsuspended in water (0.5 mL), freezeꢀ
dried to afford the desired LCO without further purification. The
resulting lipoꢀchitinoligosaccharides were characterized by 1H NMR,
and HRESIMS for new compounds (ESI S6ꢀS13†).
N(C=O)CH3). HRESIMS: m/z calcd for C38H64N5NaO25: 1014.3866
[M+Na]+, found 1014.3875.
COꢀVI(NV). MALDIꢀMS: m/z 1217 [M+Na].
Removal of the nonꢀreducing GlcNAc of COꢀV(NIV) and COꢀ
VI(NV)
1
LCOꢀIV (C16:0III). H NMR (600 MHz, DMSOꢀd6/D2O, 20:1) δ
I
I
4.87 (bs, 0.6H, Hꢀ1αI), 4.43 (d, J1β ,2 = 5.9 Hz, 0.4H, Hꢀ1βI), 4.38ꢀ
4.29 (m, 3H, Hꢀ1II,III,IV), 3.70 (app d, 1H, J6a, 6b = 11.1 Hz, Hꢀ6),
3.66ꢀ3.11 (m, 22H, Hꢀ2I,II,III,IV, Hꢀ3I,II,III,IV, 3Hꢀ4, Hꢀ5I,II,III,IV, 7Hꢀ6),
3.02 (dd, J3,4 = J4,5 = 9.2 Hz, Hꢀ4), 2.04 (t, 2H, J = 7.6 Hz,
NH(CO)CH2CH2), 1.86ꢀ1.76 (m, 9H, 3N(C=O)CH3), 1.50ꢀ1.41 (m,
2H, NH(CO)CH2CH2), 1.29ꢀ1.15 (m, 24H, 12CH2), 0.83 (t, 3H, J =
6.6 Hz, CH3). HRESIMS: m/z calcd for C48H82N4NaO21: 1049.5364
[M+Na]+, found 1049.5359.
Standard procedure for 1 mg of CO(N): Aspergillus oryzae βꢀ
galactosidase (0.1 mg, 0.8 IU, contaminated with βꢀNꢀ
acetylglucosaminidase activity)17 was solubilized in water and
dialyzed (Nanosep, Pall, 10 KDa MWCO) against water to remove
salts. The resulting enzyme solution was added to 1 mg of COꢀ
V(NIV) or COꢀVI(NV) in a final volume of 0.1 mL of water. The
reaction was carried out at 37°C during 20 hours. The reaction
mixture was dialyzed with a 10 KDa membrane (Nanosep, Pall) to
remove protein. The resulting CO(N) solution was precipitated in
water/acetone mixture (1:20) to remove liberated GlcNAc remaining
in the supernatant. Pellet constituted of CO(N) was reꢀdissolved in
LCOꢀIV (C18:1ꢁ9ZIII). 1H NMR (600 MHz, DMSOꢀd6/D2O, 20:1)
δ 5.38ꢀ5.27 (m, 2H, CH2CH=CHCH2), 4.88 (bs, 0.6H, Hꢀ1αI), 4.45
(m, 0.4H, Hꢀ1βI), 4.51ꢀ4.30 (m, 3H, Hꢀ1II,III,IV), 3.71 (app d, 1H, J6a,
water then freezeꢀdried, the resulting white solid was characterized 6b = 10.6 Hz, Hꢀ6), 3.68ꢀ3.09 (m, 22H, Hꢀ2I,II,III,IV, Hꢀ3I,II,III,IV, 3Hꢀ4,
by low and high resolution MS, and HPSEC (ESI S4, S5†).
Hꢀ5I,II,III,IV, 7Hꢀ6), 3.02 (dd, J3,4 = J4,5 = 9.0 Hz, Hꢀ4), 2.05 (t, 2H, J =
COꢀIV(NIV). 1H NMR (360 MHz, D2O) δ 5.21 (s, 0.6H, Hꢀ1αI), 6.3 Hz, NH(CO)CH2CH2), 2.01ꢀ1.95 (m, 4H, CH2CH=CHCH2),
4.85ꢀ4.69 (m, 0.4H, Hꢀ1βI), 4.65ꢀ4.56 (m, 2H, Hꢀ1II,III), 4.49 (d, 1H, 1.89ꢀ1.77 (m, 9H, 3N(C=O)CH3), 1.53ꢀ1.42 (m, 2H,
J1,2 = 8.3 Hz, Hꢀ1IV), 4.02ꢀ3.34 (m, 23H, Hꢀ2I,II,III, Hꢀ3I,II,III,IV, Hꢀ NH(CO)CH2CH2), 1.38ꢀ1.19 (m, 20H, 10CH2), 0.85 (t, 3H, J = 6.4
4I,II,III,IV, Hꢀ5I,II,III,IV, CH2ꢀ6I,II,III,IV), 2.69 (dd, 1H, J2,1 = J2,3 = 8.3 Hz, Hz, CH3). HRESIMS: m/z calcd for C48H84N4NaO21: 1075.5520
Hꢀ2IV), 2.09 (s, 6H, 2N(C=O)CH3), 2.06 (s, 3H, N(C=O)CH3). [M+Na]+, found 1075.5500.
HRESIMS: m/z calcd for C30H52N4O20: 789.3253 [M+H]+, found LCOꢀV (C16:0IV). 1H NMR (400 MHz, DMSOꢀd6/D2O, 20:1,
350K) δ 4.93 (s, 0.6H, Hꢀ1αI), 4.51ꢀ4.39 (m, 4.4H, Hꢀ1βI, Hꢀ
789.3250.
1II,III,IV,V), 3.77ꢀ3.05 (m, 30H, Hꢀ2I,II,III,IV,V, Hꢀ3I,II,III,IV,V, Hꢀ4I,II,III,IV,V
,
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Hꢀ5I,II,III,IV,V
,
CH2ꢀ6I,II,III,IV,V), 2.09 (t, 2H,
J
=
7.4 Hz, (Cꢀ6), 60.1 (Cꢀ6), 55.4 (Cꢀ2), 55.1 (Cꢀ2), 54.9 (Cꢀ2), 53.6 (Cꢀ2),
NH(CO)CH2CH2), 1.89ꢀ1.79 (m, 12H, 4N(C=O)CH3), 1.57ꢀ1.47 (m, 31.0 (CH2), 28.9 (CH2), 28.7 (CH2), 28.5 (CH2), 28.3 (CH2), 26.5
2H, NH(CO)CH2CH2), 1.34ꢀ1.20 (m, 24H, 12CH2), 0.86 (t, J = 6.5 (CH2CH=CH), 26.4 (CH2CH=CH), 24.7 (NH(CO)CH2CH2), 22.6
Hz, 3H, CH3). HRESIMS: m/z calcd for C54H95N5NaO26: 1252.6163 (N(C=O)CH3), 21.8 (N(C=O)CH3), 13.6 (CH3).
[M+Na]+, found 1252.6118.
MycꢀLCOꢀIV (C16:0).8 1H NMR (400 MHz, DMSOꢀd6/D2O, 20:1,
1
I I
LCOꢀV (C18:1ꢁ9ZIV). H NMR (400 MHz, DMSOꢀd6/D2O, 20:1, 355K) δ 4.93 (d, 0.6H, J1α ,2 = 2.7 Hz, Hꢀ1αI), 4.50ꢀ4.42 (m, 3.4H,
I
I
350K) δ 5.35ꢀ5.29 (m, 2H, CH2CH=CHCH2), 4.91 (d, 0.7H, J1α ,2
=
Hꢀ1βI, Hꢀ1II,III,IV), 3.73 (dd, 1H, J6a, 6b = 11.6 Hz, J6a, 5 = 2.2 Hz, Hꢀ6),
3.70ꢀ3.04 (m, 23H, Hꢀ2I,II,III,IV, Hꢀ3I,II,III,IV,, Hꢀ4I,II,III,IV, Hꢀ5I,II,III,IV
1.7 Hz, Hꢀ1αI), 4.48ꢀ4.35 (m, 4.3H, Hꢀ1βI, Hꢀ1II,III,IV,V), 3.76ꢀ3.03
(m, 30H, Hꢀ2I,II,III,IV,V, Hꢀ3I,II,III,IV,V, Hꢀ4I,II,III,IV,V, Hꢀ5I,II,III,IV,V, CH2ꢀ
6I,II,III,IV,V), 2.06 (t, 2H, J = 7.6 Hz, NH(CO)CH2CH2), 2.01ꢀ1.93 (m,
4H, CH2CH=CHCH2), 1.87ꢀ1.78 (m, 12H, 4N(C=O)CH3), 1.53ꢀ1.42
(m, 2H, NH(CO)CH2CH2), 1.34ꢀ1.18 (m, 20H, 10CH2), 0.84 (t, 3H,
,
7Hꢀ6), 2.09 (t, 2H, J = 7.6 Hz, NH(CO)CH2CH2), 1.88ꢀ1.89 (s, 9H,
3N(C=O)CH3), 1.55ꢀ1.46 (m, 2H, NH(CO)CH2CH2), 1.33ꢀ1.19 (m,
24H, 12CH2), 0.85 (t, 3H, J = 6.7 Hz, CH3); 13C NMR (100 MHz,
DMSO + εD2O, 355K) δ 172.3 (N(C=O)CH2), 169.3, 169.2, 169.1
J = 6.8 Hz, CH3). HRESIMS: m/z calcd for C56H97N5NaO26: (3N(C=O)CH3), 101.7 (Cꢀ1), 101.6 (Cꢀ1), 101.5 (Cꢀ1), 90.2 (Cꢀ1αI),
1278.6314 [M+Na]+, found 1278.6343.
81.5, 81.1, 81.0, 76.7, 74.8, 73.9, 72.3, 72.2, 70.6, 69.9, 68.8, 60.9
NodSmꢀIV (C18:1ꢁ9Z).8 1H NMR (400 MHz, DMSOꢀd6/D2O, (Cꢀ6), 60.1 (Cꢀ6), 59.9 (Cꢀ6), 55.4 (Cꢀ2), 54.8 (Cꢀ2), 53.6 (Cꢀ2),
20:1, 355K) δ 5.35ꢀ5.32 (m, 2H, CH2CH=CHCH2), 4.94 (d, 0.6H, 35.7 (NH(CO)CH2CH2), 31.1 (CH2), 28.9 (CH2), 28.8 (CH2), 28.7
J1α ,2 = 2.7 Hz, Hꢀ1αI), 4.52ꢀ4.41 (m, 3.4H, Hꢀ1βI, Hꢀ1II,III,IV), 3.74 (CH2), 28.5 (CH2), 24.8 (NH(CO)CH2CH2), 22.8, 22.7, 22.4
I
I
(dd, 1H, J6a, 6b = 11.6 Hz, J6a, 5 = 2.1 Hz, Hꢀ6), 3.69ꢀ3.07 (m, 23H, Hꢀ (3N(C=O)CH3), 21.9 (CH2), 13.7 (CH3)
2I,II,III,IV, Hꢀ3I,II,III,IV, Hꢀ4I,II,III,IV, Hꢀ5I,II,III,IV, 7Hꢀ6), 2.09 (t, 2H, J = MycꢀLCOꢀV (C16:0).8 1H NMR (400 MHz, DMSOꢀd6/D2O, 20:1,
7.6 Hz, NH(CO)CH2CH2), 2.01ꢀ1.97 (m, 4H, CH2CH=CHCH2), 355K) δ 4.92 (s, 0.6H, Hꢀ1αI), 4.50ꢀ4.38 (m, 4.4H, Hꢀ1βI, Hꢀ
1.87ꢀ1.81 (m, 9H, 3N(C=O)CH3), 1.54ꢀ1.48 (m, 2H,
NH(CO)CH2CH2), 1.34ꢀ1.24 (m, 20H, 10CH2), 0.86 (t, 3H, J = 7.0 Hꢀ5I,II,III,IV,V
1
II,III,IV,V), 3.75ꢀ3.06 (m, 30H, Hꢀ2I,II,III,IV,V, Hꢀ3I,II,III,IV,V, Hꢀ4I,II,III,IV,V
CH2ꢀ6I,II,III,IV,V), 2.09 (t, 2H,
7.8 Hz,
,
,
J
=
Hz, CH3); 13C NMR (100 MHz, DMSO + εD2O, 355K) δ 172.3
(N(C=O)CH2), 169.3 (N(C=O)CH3), 169.1 (N(C=O)CH3), 129.6
(CH2CH=CHCH2), 101.7 (Cꢀ1), 101.6 (Cꢀ1), 101.5 (Cꢀ1), 90.2 (Cꢀ
NH(CO)CH2CH2), 1.86ꢀ1.80 (m, 12H, 4N(C=O)CH3), 1.55ꢀ1.47 (m,
2H, NH(CO)CH2CH2), 1.26 (s, 24H, 12CH2), 0.87 (t, 3H, J = 6.5
Hz, CH3); 13C NMR (100 MHz, DMSO + εD2O, 355K) δ 172.2
1αI), 81.5, 81.1, 81.0, 76.7, 74.8, 73.9, 72.3, 72.2, 70.7, 69.9, 68.8, (N(C=O)CH2), 169.0 (N(C=O)CH3), 168.9 (N(C=O)CH3), 101.5 (Cꢀ
60.9 (Cꢀ6), 60.1 (Cꢀ6), 59.9 (Cꢀ6), 55.4 (Cꢀ2), 54.8 (Cꢀ2), 53.6 (Cꢀ 1), 101.4 (Cꢀ1), 101.3 (Cꢀ1), 90.1 (Cꢀ1αI), 81.3, 80.8, 76.6, 74.8,
2), 35.7 (NH(CO)CH2CH2), 31.1 (CH2), 29.1 (CH2), 29.0 (CH2), 74.7, 73.9, 72.1, 72.0, 70.6, 69.8, 68.6, 60.8 (Cꢀ6), 60.1 (Cꢀ6), 60.0
28.7 (CH2), 28.5 (CH2), 26.6 (CH2CH=CH), 26.5 (CH2CH=CH), (Cꢀ6), 59.9 (Cꢀ6), 55.4 (Cꢀ2), 55.0 (Cꢀ2), 54.9 (Cꢀ2), 54.8 (Cꢀ2),
24.8 (NH(CO)CH2CH2), 22.8 (N(C=O)CH3), 22.7 (N(C=O)CH3), 35.5 (NH(CO)CH2CH2), 31.0 (CH2), 28.7 (CH2), 28.6 (CH2), 28.3
22.4 (N(C=O)CH3), 21.9 (CH2), 13.7 (CH3)
(CH2), 24.7 (NH(CO)CH2CH2), 22.6 (N(C=O)CH3), 22.3
NodSmꢀV (C18:1ꢁ9Z).8 1H NMR (400 MHz, DMSOꢀd6/D2O, 20:1, (N(C=O)CH3), 21.7 (CH2), 13.5 (CH3).
355K) δ 5.35ꢀ5.33 (m, 2H, CH2CH=CHCH2), 4.93 (s, 0.6H, Hꢀ1αI),
4.51ꢀ4.41 (m, 4.4H, Hꢀ1βI, Hꢀ1II,III,IV,V), 3.76ꢀ3.08 (m, 30H, Hꢀ
2I,II,III,IV,V, Hꢀ3I,II,III,IV,V, Hꢀ4I,II,III,IV,V, Hꢀ5I,II,III,IV,V, CH2ꢀ6I,II,III,IV,V),
Results and discussion
2.10 (t, 2H, J = 7.6 Hz, NH(CO)CH2CH2), 2.02ꢀ1.98 (m, 4H,
CH2CH=CHCH2), 1.86ꢀ1.82 (m, 12H, 4N(C=O)CH3), 1.55ꢀ1.48 (m,
Expression, purification and characterization of V. cholerae CD
2H, NH(CO)CH2CH2), 1.33ꢀ1.25 (m, 20H, 10CH2), 0.87 (t, 3H, J = V. cholerae CD was obtained from E. coli BL21 harbouring the
7.0 Hz, CH3); 13C NMR (100 MHz, DMSO + εD2O, 355K) δ 172.3
overexpression vector pET26bꢀVC1280. In this plasmid, the cd
gene is fused to an Nꢀterminal pelB sequence for a periplasmic
localization and to a Cꢀterminal 6ꢀHis Tag sequence to allow
the enzyme purification by Immobilized Metal ion Affinity
Chromatography (IMAC) methodology. The purification
(N(C=O)CH2), 169.7 (N(C=O)CH3), 169.6 (N(C=O)CH3), 169.3
(N(C=O)CH3), 169.1 (N(C=O)CH3), 129.5 (CH2CH=CHCH2), 101.6
(Cꢀ1), 101.5 (Cꢀ1), 101.4 (Cꢀ1), 90.2 (Cꢀ1αI), 81.3, 81.1, 80.9, 80.8,
76.7, 74.8, 74.6, 73.9, 72.4, 72.2, 70.7, 69.8, 68.8, 60.9 (Cꢀ6), 60.3
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

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VJieowuArrtnicalelONnlainme e
ARTICLE
procedure gave 120 mg of pure protein per litre of culture, as V(NIV) and COꢀVI(NV) regioselectively deꢀ
judged by SDSꢀPAGE (data not shown). Determination of the penultimate GlcNAc unit from the reducing end in quantitative
DOI: 10.1039/C5GC00623F
N
ꢀacetylated at the
kinetic parameters for deꢀ ꢀacetylation of COꢀII, the best yield after protein removal as judged by MALDIꢀMS, TLC and
N
substrate, gave the following values: KM = 3.75 ± 0.75 mM; kcat HPSEC analysis (ESI S1ꢀS3†). Ammonium carbonate buffer
= 0.45 ± 0.06 sꢀ1. VC1280 is present in different oligomeric was used because of its ease of elimination by concentration
forms in solution, the monomeric form being the most active.21 followed by freeze drying without remaining inorganic salts
For our synthesis purpose, we chose to work with the mixture contaminating the reaction product. Isolated yields were 97 %,
of monomeric and oligomeric forms, and this can explain the 91 % and 79 % for COꢀIV(NIII), COꢀV(NIV) and COꢀVI(NV)
high enzyme/substrate ratio used in the deꢀ
reactions.
N
ꢀacetylation respectively, precursors of LCO analogues bearing a fatty acid
chain at the penultimate residue from the reducing unit (Table
2).
Enzymatic conversion of COs to LCOs precursors
The synthetic strategy adopted for the synthesis of LCOs, its Table 2 Enzymatic deꢀNꢀacetylation of COs
precursors and analogues is summarized in figure 1.
Chitin oligomers can be prepared through colloidal chitin
chemical or enzymatic depolymerization. However this
approach is tedious and cannot afford chitintetraose to
chitinhexaose (COꢀIV to COꢀVI) in large quantities. We found
more appropriate to start from a commercial mixture of
Starting
Quantity
(mg)
Quantity Isolated yield
Product11
compound
(mg)
(%)
COꢀIV
COꢀV
COꢀVI
18.7
19.1
5.1
COꢀIV(NIII)
COꢀV(NIV)
COꢀVI(NV)
17.3
16.6
3.9
97
91
79
chitosan oligomers which were reꢀNꢀacetylated and purified by
ion exchange chromatography followed by gel filtration to give
individual oligomers (COꢀIV, COꢀV and COꢀVI) in pure form
(Table 1).
Aspergillus oryzae βꢀNꢀacetylglucosaminidase (GlcNAcase) is
an exoꢀacting glycoside hydrolase liberating GlcNAc units
from the nonꢀreducing end of chitinoligomers. It is found as a
contaminant in commercial preparations of
β
ꢀgalactosidase.17
Table 1 Quantities of isolated COs starting from 1 g of COs
This enzyme is inactive on glucosaminyl residue and is
appropriate to release only the nonꢀreducing GlcNAc residue
mixture:
Chitin
Quantity Isolated yield
from chitinoligomers deꢀ
GlcNAc unit from the reducing end (Figure 1). COꢀV(NIV) and
COꢀVI(NV) where incubated with A. oryzae
Nꢀacetylated at the penultimate
oligomer
(mg)
(%)
βꢀNꢀ
COꢀII
COꢀIII
COꢀIV
COꢀV
COꢀVI
86.4
157.2
196.4
148
8.6
15.7
19.6
14.8
7.8
acetylglucosaminidase in water, followed by dialysis and
precipitation in water/acetone mixture to remove protein and
liberated GlcNAc remaining in the supernatant. Isolated yields
were 66 % and 43 % for COꢀIV(NIV) and COꢀV(NV)
respectively, precursors of LCOꢀIV and LCOꢀV (Figure 1, ESI
S4ꢀS5†).
78.4
Each oligomer was treated with V. cholerae CD in ammonium
carbonate buffer to give the corresponding COꢀIV(NIII), COꢀ
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5GCA00R6T2I3CFLE
Fig. 1 Access to LCOs and analogues from COs.
Table 3 Acylation of CO(N)s
Acylation of CO(N)s
Isolated
Starting
Quantity
(mg)
Quantity
yield
Product22
The combined Vibrio cholerae CD and Aspergillus oryzae
GlcNAcase approach led selectively to chitinoligosaccharides
with the free amine at the nonꢀreducing end, which are
advanced precursors of lipoꢀchitinoligosaccharides such as Nod
and MycꢀLCOs. The synthesis of these plant growth promotors
compound
(mg)
(%)
COꢀIV(NIII)
COꢀIV(NIII)
COꢀIV(NIV)
COꢀIV(NIV)
COꢀV(NIV)
COꢀV(NIV)
COꢀV(NV)
COꢀV(NV)
3.1
3.1
LCOꢀIV(C16:0 III
)
4.0
4.2
100
100
100
100
85
LCOꢀIV(C18:1ꢁ9ZIII)
MycꢀLCOꢀIV(C16:0)
NodSmꢀIV(C18:1ꢁ9Z)
LCOꢀV(C16:0IV)
2.1
2.8
requires a
Nꢀacylation reaction, which was performed with the
2.7
3.6
N
ꢀhydroxysuccinimide ester18 of oleic acid19 (C18:1
ꢁ
9Z) or of
11.0
6.0
11.6
7.3
palmitic acid20 (C16:0) in DMSO at 60 °C in the presence of an
excess of triethylamine. The expected Nod and Myc LCOs1,4,8
were isolated in 68 % to quantitative yields (Figure 2).
LCOꢀV(C18:1ꢁ9ZIV)
MycꢀLCOꢀV(C16:0)
NodSmꢀV(C18:1ꢁ9Z)
96
3.3
2.8
68
13.0
14.6
89
Applying the same procedure to COꢀIV(NIII) and COꢀV(NIV)
provided LCOs analogues, which were isolated in 85% to
quantitative yields, attesting the efficiency of the acylation
reaction (Table 3, ESI S6ꢀS13†).
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DOI: 10.1039/C5GC00623F
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RSCPublishing
ARTICLE
Fig 2. Acylation of COs
degradation using A. oryzae
Efficient acylation procedure using
acid ester led to natural Nod and MycꢀLCOs. Alternatively,
omitting the ꢀacetylglucosaminidase treatment gave access
β
ꢀ
N
ꢀacetylglucosaminidase.
N
ꢀhydroxysuccinimide fatty
Conclusion
The present work reports an efficient and straightforward in
vitro chemoꢀenzymatic access to natural LCO fertilizers and
analogues from biomass. Commercial source of chitosan
oligomers was found to be convenient for the preparation of
βꢀN
to LCO analogues bearing the fatty acid chain at the
penultimate residue from the reducing end. They may play also
a role in the plant signaling events.
pure COs by
exclusion chromatography.
Selective ꢀdeacetylation of theses COs was achieved by
Nꢀacetylation and subsequent purification by sizeꢀ
This methodology opened the way for the largeꢀscale
production of lipoꢀchitinoligosaccharides, high value molecules
of major agrochemical interest, starting from chitin an abundant
renewable resource.
N
VC1280 CD which was produced in high yield (120 mg.Lꢀ1) by
periplasmic expression in E. coli. The enzyme was active in
carbonate buffer, allowing a straightforward route to COs
specifically deꢀNꢀacetylated at the penultimate residue from the
Acknowledgements
reducing end. Very high yield could be achieved after a simple
purification procedure consisting of protein thermal
denaturation and centrifugation followed by freeze drying,
leading to pure CO(N). These CO(N) could be transformed in
Nod and MycꢀLCO precursors by controlled enzymatic
Authors are indebted to late Prof. S. Roseman for providing
VC1280 CD gene and are grateful to S. Pradeau, M. Touvrey
and P. Chaud for skillful assistance. Authors acknowledge
support from ICMG FR 2607, LabEx ARCANE (ANRꢀ11ꢀ
LABXꢀ0003ꢀ01), PolyNat Carnot Institute, French Agence
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ARTICLE
DOI: 10.1039/C5GC00623F
Nationale de la Recherche (ANRꢀ10ꢀCD2Iꢀ0008, BioCOS 8. G. Despras, A. Alix, D. Urban, B. Vauzeilles and J.ꢀM. Beau,
2010ꢀ2014), and Bill & Melinda Gates Foundation (ENSA Angew. Chem. Int. Ed., 2014, 53, 11912–11916.
2012ꢀ2017).
9. E. Samain, V. Chazalet and R. A. Geremia, J. Biotechnol., 1999,
72, 33ꢀ47.
10. S. K. Kim and N. Rajapakse, Carbohydr. Polym., 2005, 62, 357ꢀ
368.
Abbreviations
Amp, ampicillin; CO, chitinoligosaccharide; CO(N), deꢀNꢀacetylated
11. We followed the nomenclature already adopted for LCOs and
derivatives (W. D'Haeze and M. Holsters, Glycobiology, 2002, 12,
79Rꢀ105R). Concerning COs deꢀNꢀacetylated at a specific position
(CO(Nx)), the N letter indicates a free amino group at Cꢀ2 of the
glycosyl unit, and the roman upper case number specifies the unit
position starting from the reducing end. i.e. COꢀII (NI) for
chitinbiose deꢀNꢀacetylated at the reducing unit).
chitinoligosaccharide; CD, chitin deacetylase; COꢀIV, chitintetraose;
COꢀV, chitinpentaose; COꢀVI, chitinhexaose; GlcN, Dꢀglucosamine;
GlcNAc,
Nꢀacetylꢀ
D
ꢀglucosamine;
Kan,
IPTG,
isopropyl
LCO,
βꢀDꢀ
thiogalactopyranoside;
kanamycin;
lipoꢀ
chitinoligosaccharide; LB, Luria Broth; NodSm, Nod factors
produced by Sinorhizobium meliloti; SDSꢀPAGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis; TLC, thin layer
chromatography
12. X. B. Li, L. X. Wang, X. S. Wang and S. Roseman,
Glycobiology, 2007, 17, 1377ꢀ1387.
13. J. W. Walkley and J. Tillman, J. Chromatogr., 1977, 132, 172ꢀ
174.
Notes and references
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CNRS, CERMAV, Fꢀ38000 Grenoble, France.
bCNRS and Université ParisꢀSud, Glycochimie Moléculaire et
Macromoléculaire, ICMMO, UMR 8182, 91405 Orsay, France.
cCNRS, ICSN, Centre de Recherche de Gif, 1 avenue de la Terrasse, Fꢀ
91198 GifꢀsurꢀYvette, France
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Enzym.,1999, 6, 483–494.
† Electronic Supplementary Information (ESI) available: [Low
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1
resolution MALDIꢀToF MS, HRESIMS, H NMR and HPSEC
analyses for purified compounds]. See DOI: 10.1039/b000000x/
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22. We propose a specific nomenclature for LCOs analogues where
the roman upper case number specifies the position starting from the
reducing end (i.e. LCOꢀIV(C16:0III) for chitintetraose Nꢀ
palmitoylated at the penultimate residue from the reducing unit).
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lipo-chitinoligosaccharides (LCOs), keys molecules in plant-symbiotic microorganisms
DOI: 10.1039/C5GC00623F
communication, are readily obtained via chemoenzymatic synthesis from chitin, a renewable
abundant biopolymer.