SUPPLEMENTARY MATERIAL
Biotechnology and Bioengineering
Enzymatic degradation of lignin-carbohydrate complexes (LCCs): Model studies using a
fungal glucuronoyl esterase from Cerrena unicolor
Clotilde d’Errico,a Jonas O. Jørgensen,a Kristian B. R. M. Krogh,b Nikolaj Spodsberg,b Robert Madsen,a Rune
Nygaard Monradb,*
a
Department of Chemistry, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark; bNovozymes A/S,
Krogshøjvej 36, 2880 Bagsværd, Denmark, rnmo@novozymes.com
Table of contents
Isolation and sequencing of the Cerrena unicolor strain .................................................................................................. S2
Glycosylation pattern of CuGE......................................................................................................................................... S5
Synthesis of methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside (11) .................................................................................. S6
1
H and 13C NMR of benzyl (methyl 4-O-methyl-α-D-glucopyranoside) uronate (1) ........................................................ S7
H and 13C NMR of benzyl (methyl α-D-glucopyranoside) uronate (2)............................................................................ S8
1
1
H and 13C NMR of phenylpropyl (methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside) uronate (13) ................................. S9
H and 13C NMR of phenylpropyl (methyl α-D-glucopyranoside) uronate (3) ............................................................... S10
1
1
H and 13C NMR of phenyl (methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside) uronate (14) ......................................... S11
H and 13C NMR of phenyl (methyl α-D-glucopyranoside) uronate (4) ......................................................................... S12
1
13
C NMR of ethyl α-D-glucuronate ................................................................................................................................. S13
Kinetic graphs of degradation of benzyl (methyl 4-O-methyl-α-D-glucopyranoside) uronate (1) ................................. S14
Kinetic graphs of degradation of benzyl (methyl α-D-glucopyranoside) uronate (2)...................................................... S15
Kinetic graphs of degradation of phenylpropyl (methyl α-D-glucopyranoside) uronate (3) ........................................... S16
Kinetic graphs of degradation of phenyl (methyl α-D-glucopyranoside) uronate (4) ..................................................... S17
References ...................................................................................................................................................................... S18
S1
Isolation and sequencing of the Cerrena unicolor strain
The Cerrena unicolor strain MS01356 was isolated by spore isolation from fruit-body of a specimen collected in the
summer of 1997 in Kamchatka, Russia. MS01356 was cultured in 100 ml PDL medium and incubated at 20 ºC, 85 rpm
for 11 days. The mycelium was harvested by pouring the culture through Miracloth (Millipore).
DNA and RNA extraction
Mycelia from a culture of Cerrena unicolor MS01356 were collected and frozen in liquid nitrogen stored in a -80 ºC
freezer until use. The frozen mycelia were transferred into a liquid nitrogen pre-chilled mortar and pestle and ground to
a fine powder with a small amount of baked quartz sand. Total RNA was prepared from the powdered mycelia by
extraction with guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion (Chirgwin et al.
1979). The polyA enriched RNA was isolated by oligo (dT)-cellulose affinity chromatography (Aviv and Leder 1972).
Double stranded cDNA was synthesised according to the general methods using a Not I-(dT)18 primer (GE Healthcare)
(Gubler and Hoffman 1983; Kofod et al. 1994; Sambrook et al. 1989). After synthesis, the cDNA was treated with
mung bean nuclease, blunt ended with T4 DNA polymerase, and ligated to an Eco RI adaptor (GE Healthcare). The
cDNA was cleaved with Not I and the cDNA was size fractionated by 0.8% agarose gel electrophoresis using 44 mM
Tris base, 44 mM boric acid, 0.5 mM EDTA (ethylenediaminetetraacetic acid) (TBE) buffer. The fraction of cDNA of
700 bp and larger was excised from the gel and purified using a GFX PCR DNA and Gel Band Purification Kit (GE
Healthcare) according to the manufacturer’s instructions.
Construction of cDNA library
The prepared cDNA was then directionally cloned by ligation into Eco RI-Not I cleaved pMHas5 (Patent
WO2004013350) using T4 ligase (Promega) according to the manufacturer’s instructions. The ligation mixture was
electroporated into E. coli DH10B cells (Boehringer Mannheim) using a Gene Pulser and Pulse Controller (Bio-Rad) at
200 ohms, 25 mF, 1.8 kV with a 2 mm gap cuvette according to the manufacturer’s procedure. The electroporated cells
were plated onto LB plates supplemented with 100 µg of ampicillin per ml. A cDNA plasmid pool was prepared from
34,000 total transformants of the original pMHas5 vector ligation. Plasmid DNA was prepared directly from the pool of
colonies using a Qiagen-tip 100 (Qiagen).
Construction of SigA4 transposon containing the beta-lactamase reporter gene
A transposon containing plasmid designated pSigA4 was constructed from the pSigA2 transposon containing plasmid in
order to create an improved version of the signal trapping transposon of pSigA2 with decreased selection background
(Patent WO200177315). The pSigA2 transposon contains a signal-less beta-lactamase construct encoded on the
transposon itself. PCR was used to create a deletion of the intact beta-lactamase gene found on the plasmid backbone
using a proofreading Proofstart DNA polymerase (Qiagen) and the 5’ phosphorylated primers SigA2NotU-P and
SigA2NotD-P (TAG Copenhagen) (Table SI). The amplification reaction was composed of 1 µl of pSigA2 (10 ng/µl), 5
S2
µl of 10X Proofstart Buffer (Qiagen), 2.5 µl of dNTP mix (20 mM), 0.5 µl of SigA2NotU-P (10 mM), 0.5 µl of
SigA2NotD-P (10 mM), 10 µl of Q solution (Qiagen), and 31.3 µl of deionized water. The amplification reaction was
incubated in a PTC-200 DNA Engine Thermal Cycler (MJ Research) programmed for 1 cycle at 95 °C for 5 minutes;
and 20 cycles each at 94 °C for 30 seconds, 62 °C for 30 seconds, and 72 °C for 4 minutes. A 3.9 kb PCR reaction
product was isolated by 0.8% agarose gel electrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mM
disodium EDTA (TAE) buffer, and 0.1 µg of ethidium bromide per ml. The DNA band was visualized with the aid of
an Eagle Eye Imaging System (Stratagene) at 360 nm. The 3.9 kb DNA band was excised from the gel and purified
using a GFX PCR DNA and Gel Band Purification Kit according to the manufacturer’s instructions. The 3.9 kb
fragment was self-ligated at 16 ºC overnight with 10 units of T4 DNA ligase (New England Biolabs), 9 µl of the 3.9 kb
PCR fragment, and 1 µl of 10X ligation buffer (New England Biolabs). The ligation reaction was heat inactivated for 10
minutes at 65 ºC and then digested with Dpn I at 37 ºC for 2 hours. After incubation, the digestion was purified using a
GFX PCR DNA and Gel Band Purification Kit. The purified material was then transformed into E. coli Top10
competent cells (Invitrogen) according to the manufacturer’s instructions. The transformation mixture was plated onto
LB plates supplemented with 25 µg of chloramphenicol per ml. Plasmid mini-preps were prepared from several
transformants and digested with Bgl II. One plasmid with the correct construction was chosen. The plasmid was
designated pSigA4. Plasmid pSigA4 contains the Bgl II flanked transposon SigA2 identical to that disclosed in literature
(Patent WO200177315). A 60 µl sample of plasmid pSigA4 DNA (0.3 µg/µl) was digested with Bgl II and separated by
0.8% agarose gel electrophoresis using TBE buffer. A SigA2 transposon DNA band of 2 kb was eluted with 200 µl of
EB buffer (Qiagen) and purified using a GFX PCR DNA and Gel Band Purification Kit according to the manufacturer’s
instructions and eluted in 200 µl of EB buffer. SigA2 was used for transposon assisted signal trapping.
Transposon Assisted Signal Trapping of Cerrena unicolor MS01356 cDNA
A cDNA plasmid pool was prepared from 34,000 total transformants of the original cDNA-pMHas5 vector ligation.
Plasmid DNA was prepared directly from a pool of colonies recovered from solid LB selective medium using a Qiaprep
Spin Midi/Maxiprep Kit (Qiagen). The plasmid pool was treated with transposon SigA2 and MuA transposase
(Finnzymes) according to the manufacturer’s instructions. For in vitro transposon tagging of the Cerrena unicolor
MS01356 cDNA library, 4 µl of SigA2 transposon containing approximately 100 ng of DNA were mixed with 5 µl of
the plasmid DNA pool of the Cerrena unicolor MS01356 cDNA library containing 3 µg of DNA, 1 µl of MuA
transposase (0.22 µg/µl), and 4 µl of 5X buffer (Finnzymes) in a total volume of 20 µl and incubated at 37 °C for 2.5
hours followed by heat inactivation at 70 °C for 10 minutes. The DNA was precipitated by addition of 2 µl of 3 M
sodium acetate pH 5 and 55 µl of 96% ethanol and centrifuged for 30 minutes at 10,000 x g, 4 °C. The pellet was
washed in 70% ethanol, air dried at room temperature, and resuspended in 7 µl of deionized water. A 1.5 µl volume of
the transposon tagged plasmid pool was electroporated into 40 µl of E. coli ElectroMAX DH10B cells (Invitrogen)
according to the manufacturer’s instructions using a Gene Pulser and Pulse Controller (Bio-Rad) at 25 uF, 25 mAmp,
2.5 kV with a 2 mm gap cuvette according to the manufacturer’s procedure. The electroporated cells were incubated in
SOC medium with shaking at 225 rpm for 1 hour at 37 C before being plated on the following selective media: LB
medium supplemented with 50 µg of kanamycin per ml; LB medium supplemented with 50 µg of kanamycin per ml
and 15 µg of chloramphenicol per ml; and LB medium supplemented with 50 µg of kanamycin per ml, 15 µg of
S3
chloramphenicol per mL, and 15 µg of ampicillin per ml. From plating of the electroporation onto LB medium
supplemented with 50 µg of kanamycin per ml, 15 µg of chloramphenicol per ml, and 15 µg of ampicillin per ml,
approximately 180 colonies were observed after 3 days at 30 C. All colonies were replica plated onto LB plates
supplemented with kanamycin, chloramphenicol with 50 µg/ml ampicillin. Further electroporation and plating
experiments were performed until 500 total colonies were recovered under triple selection. The colonies were miniprepped using a Qiaprep 96 Turbo Miniprep Kit (Qiagen). Plasmids were sequenced with the transposon forward and
reverse primers (primers A and B, Table S1).
Sequence assembly and annotation
DNA sequences were obtained for the reactions on a MegaBACE 500 DNA Analysis System (GE Healthcare). Primer
A and primer B sequence reads for each plasmid were trimmed to remove vector and transposon sequence. This resulted
in roughly 200 assembled sequences which were grouped into 95 contigs by using the program PhredPhrap (Ewing et
al. 1998; Ewing and Green 1998). All 95 contigs were subsequently compared to sequences available in standard public
DNA and protein sequences databases (TrEMBL, SWALL, PDB, EnsemblPep, GeneSeqP) by using the program
BlastX 2.0a19MP-WashU [14-Jul-1998] [Build linux-x86 18:51:44 30-Jul-1998] (Gish and States 1993). The family
CE15 candidate was identified directly by analysis of the BlastX results (accession number: GENESEQP:BAY14951).
Table SI Primers used
Primer
Sequence
SigA2NotU-P
5’-TCGCGATCCGTTTTCGCATTTATCGTGAAACGCT-3’
SigA2NotD-P
5’-CCGCAAACGCTGGTGAAAGTAAAAGATGCTGAA-3’
Primer A
5’-AGCGTTTGCGGCCGCGATCC-3’
Primer B
5’-TTATTCGGTCGAAAAGGATC C-3’
S4
Glycosylation pattern of CuGE as observed by MS analysis
Intens.
Glc - 2.0943
Glc - 0.1447
Glc - 4.0900
1000
Glc + 0.3033
154.2395
Glc + 3.4539
Glc + 0.5863
Glc + 2.5672
Glc - 0.9446
2000
Glc - 0.2504
Glc - 1.5797
Glc - 1.6192
Glc + 2.2925
Glc - 0.8775
Glc + 1.3350
51799.2346
Glc + 1.9833
Glc + 1.2360
Glc + 0.2975
Glc + 0.9898
Glc - 0.3325
Glc - 0.2673
Glc + 1.3707
Glc + 0.1453
Glc - 0.0475
Glc - 0.2038
Glc - 1.0686
51474.1603
Glc - 1.1422
3000
Glc - 0.3980
Glc + 1.8399
4000
Glc - 0.2139
+MS, 5.1-5.4min #(211-224), Deconvoluted (maximum entropy)
57712.0588
55546.1907
56652.2674
48702.2855
0
48000
49000
50000
51000
52000
53000
54000
55000
56000
57000
m/z
Fig. S1 Full length Mw by LC-MS. A clear glycosylation pattern with 162 Da spacing corresponding to a hexose unit is observed
around 51 kDa
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Synthesis of Methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside (11)
A solution of methyl α-D-glucopyranoside 9 (15.0 g, 77.3 mmol) and trityl chloride (23.6 g, 92.8 mmol) in dry pyridine
(200 ml) was stirred at 90 °C until disappearance of the starting material. After 4 hours, the solvent was evaporated
under reduced pressure, the reaction mixture diluted with CH2Cl2 and then washed twice with water. The combined
organic layers were dried over MgSO 4 and concentrated under reduced pressure. The tritylated compound was
crystallized from toluene and rinsed with heptane (28.9 g, 86%). The compound (5.02 g, 11.4 mmol) was then dissolved
in dry DMF with NaH (2.31 g, 57.0 mmol). After stirring for 20 minutes benzyl bromide (7.0 ml, 57.0 mmol) and
tetrabutylammonium iodide (0.34 g, 0.912 mmol) were added at 0 °C. The reaction mixture was stirred at room
temperature for 18 hours then diluted with ethyl acetate and washed with brine. The combined organic layers were dried
with MgSO4 and concentrated under reduced pressure. The fully protected crude compound was dissolved in methanol
with 1% of H2SO4 and stirred at room temperature for 1 hour until complete cleavage of the trityl group had occurred
according to TLC. The reaction mixture was treated with Na2CO3 (7.30 g) until neutrality (pH 7). After 1 hour the
mixture was filtered and concentrated then diluted with CH2Cl2 and washed twice with brine. The organic layers were
dried over MgSO4 and evaporated under reduced pressure. The crude residue was purified by silica gel column
chromatography (heptane/ethyl acetate, 7:3) to give 11 as a white solid (4.20 g, 80%). (Bernet and Vasella 1979). 1H
NMR (400 MHz, CDCl3): δ 7.41 – 7.25 (m, 15H, ArH), 5.01 (d, J = 10.9 Hz, 1H, OCH2Ph), 4.91 (d, J = 11.0 Hz, 1H,
OCH2Ph), 4.86 (d, J = 10.9 Hz, 1H, OCH2Ph), 4.82 (d, J = 12.1 Hz, 1H, OCH2Ph), 4.68 (d, J = 12.0 Hz, 1H, OCH2Ph),
4.66 (d, J = 11.0 Hz, 1H, OCH2Ph), 4.59 (d, J = 3.6 Hz, 1H, H-1), 4.03 (t, J = 9.3 Hz, 1H, H-3), 3.79 (dd, J = 11.5, 2.4
Hz, 1H, H-6a), 3.71 (dd, J = 11.7, 3.8 Hz, 1H, H-6b), 3.70 – 3.61 (m, 1H, H-5), 3.55 (t, J = 9.3 Hz, 1H, H-4), 3.52 (dd, J
= 9.6, 3.6 Hz, 1H, H-2), 3.38 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3): δ 138.8, 138.2, 138.2, 138.0, 128.5, 128.5,
128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 127.9, 127.7, 98.2, 82.0, 80.0, 77.5, 75.8, 75.8, 73.5, 70.7, 61.9, 55.2. Spectral
data are in agreement with data reported in literature (Bernet and Vasella 1979; Dorgeret et al. 2011).
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S7
S8
S9
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S11
S12
The compound is contaminated with small amounts of D-glucofuranurono-6,3-lactone.
S13
S14
S15
S16
S17
References
Aviv H, Leder P (1972) Purification of biologically active globin messenger RNA by chromatography on
oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A 69:1408-1412
Bernet B, Vasella A (1979) Carbocyclic compounds from monosaccharides. 1.Transformations in the glucose series.
Helv Chim Acta 62:1990-2016
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from
sources enriched in ribonuclease. Biochemistry 18:5294-5299
Dorgeret B, Khemtémourian L, Correia I, Soulier J-L, Lequin O, Ongeri S (2011) Sugar-based peptidomimetics inhibit
amyloid β-peptide aggregation. Eur J Med Chem 46:5959-5969
Ewing B, Green P (1998) 1. Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome
Res 8:186-194
Ewing B, Hillier LD, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using Phred. I. Accuracy
assessment. Genome Res 8:175-185
Gish W, States DJ (1993) Identification of protein coding regions by database similarity search. Nat Genet 3:266-272
Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263-269
Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, Dörreich K, Dalboege H (1994) Cloning and
characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus.
J Biol Chem 269:29182-29189
Sambrook J, Fritsch EF, Maniantis T (1989) Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York
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