Supplementary Information
The novel catabolic pathway of 3,6-anhydro-L-galactose, the main component of red
macroalgae, in a marine bacterium
Eun Ju Yun1, Saeyoung Lee1, Hee Taek Kim1, Jeffrey G. Pelton2, Sooah Kim1, Hyeok-Jin Ko1,
In-Geol Choi1* and Kyoung Heon Kim1*
1
Department of Biotechnology, Korea University Graduate School, Seoul 136-713, Korea;
2
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley,
California 94720, USA.
*Corresponding authors.
E-mail address: khekim@korea.ac.kr (K.H. Kim), igchoi@korea.ac.kr (I.-G. Choi),
Telephone number: +82 2 3290 3028 (K.H. Kim), +82 2 3290 3152 (I.-G. Choi)
1
Fig. S1. Conversion of AHG using crude enzyme of EJY3. Enzymatic reactions were
performed using 200 μl of reaction mixtures containing 74 μg crude enzyme and 10 mM
AHG with 1.5 mM NADPH or NAD+ in 20 mM Tris-HCl buffer (pH 7.4) at 30°C for 12 h.
Control, thermally inactivated crude enzyme with AHG; Crude, crude enzyme with AHG;
NADPH, crude enzyme with AHG and NADPH; NAD+, crude enzyme with AHG and NAD+.
2
Fig. S2. (A) TLC of AHGA purified from the in vitro reaction product of AHG with
VejAHGD. (B) TLC of KDGal purified from the in vitro reaction product of AHG with
VejAHGD and VejACI. Purification was performed by fast protein liquid chromatography
(AKTA Prime; GE Healthcare, Piscataway, NJ) by using a Sephadex G-10 column. Gal and
KDGlc were used as markers.
3
Fig. S3. (A)
13
C NMR spectrum, (B) 1H-13C HSQC NMR spectrum and (C) 1H-13C HMBC
NMR spectrum of KDGal purified from the in vitro reaction product of AHG with AHG
dehydrogenase and 3,6-anhydrogalactonate cycloisomerase. Chemical shifts were referenced
to those of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid.
4
Table S1. Comparison of the 1H- and 13C chemical shifts of AHGA purified in this study with
previously published values for AHG (Yun et al. 2013).
1
13
H Chemical shift (ppm)
C Chemical shift (ppm)
AHGA
AHGa
Difference
AHGA
AHGa
Difference
H-1
NAb
5.00
NAb
181.5
92.1
89.4
H-2
4.19
3.61
0.58
74.6
75.0
–0.4
H-3
4.07
3.94
0.13
89.0
86.0
3.0
H-4
4.29
4.21
0.08
80.8
78.8
2.0
H-5
4.28
4.25
0.03
79.6
80.2
–0.6
H-6
3.87
3.84
0.03
75.6
74.9
0.7
H-6’
a 1
4.04
4.01
0.03
H and 13C chemical shifts of AHG in comparison with previously published values (Yun et
al. 2013)
b
Not applicable
5
Table S2. Comparison of the 1H- and
13
C chemical shifts of KDGal and AHGA purified in
this study.
1
13
H Chemical shift (ppm)
C Chemical shift (ppm)
KDGal
AHGA
Difference
KDGal
AHGA
Difference
H-1
NAa
NAa
NAa
179.0
181.5
-2.5
H-2
NAa
4.19
NAa
99.3
74.6
24.7
4.07
NAa
42.0
89.0
–47.0
2.20
H-3
1.85
H-4
3.91
4.29
–0.38
71.8
80.8
–9.0
H-5
3.64
4.28
–0.64
73.6
79.6
–6.0
H-6
3.66
3.87
–0.21
66.0
75.6
–9.6
H-6’
a
3.85
4.04
–0.19
Not applicable
6
Table S3. Strains and plasmids used in this study.
Name
Description
Reference
Strains
EJY3
Vibrio sp., an agarolytic marine bacterium metabolizing 3,6-
(Roh et al., 2012)
anhydro-L-galactose
DH5α
F-, endA1, supE44, thi-1, recA1, relA1, gyrA96, deoR, nupG,
(Grant et al., 1990)
Φ80dlacZΔM15,
Δ (lacZYA-argF) U169, hsdR17 (rK-, mK+) and λ–
DH5α_ Vejahgd
DH5α harbouring Vejahgd in pBAD/myc His
In this study
DH5α_ Vejaci
DH5α harbouring Vejaci in pET21α
In this study
BL21(DE3)
F–, ompT, gal, dcm, lon, hsdSB (rB- mB-) and λ (DE3 [lacI, lacUV5-
(Studier & Moffatt, 1986)
T7 gene 1, ind1, sam7 and nin5])
BL21(DE3)_ Vejahgd
BL21(DE3) harbouring Vejahgd in pBAD/myc His
In this study
BL21(DE3)_ Vejaci
BL21(DE3) harbouring Vejaci in pET21α
In this study
MG1655
F-, λ-, ilvG, rfb50 and rph-1
(Heath et al., 1992)
7
MG1655_empty vector
MG1655 harbouring an empty vector, pBAD/myc His
In this study
MG1655_Vejahgd+Vejaci
MG1655 harbouring Vejahgd and Vejaci genes in pBAD/myc His
In this study
KO11
Hyperexpressive for pdc and adhB from Z. mobilis, high Cmr (600
(Ohta et al., 1991)
ug ml-1) and frd
KO11_ empty vector
KO11 harbouring an empty vector, pBAD/myc His
In this study
KO11_ Vejahgd+Vejaci
KO11 harbouring Vejahgd and Vejaci genes in pBAD/myc His
In this study
pBAD/myc His
araBAD promoter, rrnB terminator, pBR322 origin and Ampr
(Lee et al., 1981)
pBAD/myc His_ Vejahgd
pBAD cloned Vejahgd from EJY3
In this study
pBAD/myc His_Vejahgd+Vejaci
pBAD cloned Vejahgd and Vejaci from EJY3
In this study
pET21α
T7 lac promoter, T7 terminator, bacterial origin (ori) and Ampr
pET21α_ Vejaci
pET21α cloned Vejaci from EJY3
Plasmids
8
(Pan & Malcolm, 2000)
In this study
Table S4. Primers used in this study.
Target DNA name
Sequence
Vejahgd
F_ahgd
GAAGGAGATATAAGGATGAAACGTTACCAAATGTACGTTG
R_ahgd
ATGATGGTGATGGTGGTCGAAATTCACATAGAATGTCTT
Vejaci
F_aci
GAAGGAGATATAAGGATGAAAACAACAATCAAAGACATCAAAA
R_aci
ATGATGGTGATGGTGCACTTCGTACTGAGCAATTTTGT
Vejahgd + Vejaci
F_ahgd
GCGCTCGAGATGAAACGTTACCAAATGTACGTTG
R_ahgd
GCGTCTAGATTAGTCGAAATTCACATAGAATGTCT
F_aci
GCGTCTAGAATGAAAACAACAATCAAAGACATCAAAAC
R_aci
GCGTACGTACACTTCGTACTGAGCAATTTTGTC
9
Supplementary Methods
Supplementary Method 1: Preparation of agarose hydrolysate
1) Chemical liquefaction of agarose using acetic acid. Enzymatic hydrolysates were obtained
by combining chemical liquefaction and enzymatic hydrolysis using β-agarase and
neoagarobiose hydrolase as previously described (Kim et al., 2012; Yun et al., 2012). Briefly,
5 g of agarose was prehydrolysed in 100 mL of 27.4% (w/v) acetic acid at 80°C for 3 h at 150
rpm in a shaking water bath. The reaction product was dried using a rotary vacuum
evaporator at 65°C for 2 h. Agarooligosaccharides were then precipitated with 1.5 L of 94.0%
(v/v) ethanol (Daejung, Siheung, Korea) and filtered through a membrane filter (pore size,
0.45 μm; Whatman, Dassel, Germany).
2) Enzymatic hydrolysis of agarooligosaccharides to 3,6-anhydro-L-galactose (AHG) and
galactose. To obtain AHG and galactose from agarooligosaccharides, two enzymes, an exotype β-agarase (Aga50D) and a neoagarobiose hydrolase (SdNABH (Lee et al., 2009; Kim et
al., 2010; Ha et al., 2012)), were used. Expression and purification of Aga50D and SdNABH
were performed as previously described (Lee et al., 2009; Kim et al., 2010; Ha et al., 2012).
For enzymatic reactions, agarooligosaccharides (5% [w/v]) were used as substrate, which
were hydrolysed to neoagarobiose by Aga50D at 30°C for 72 h in 50 mM Tris–HCl buffer
(pH 7.4). Neoagarobiose produced by the enzymatic reaction with Aga50D was hydrolysed to
AHG and galactose by an enzymatic reaction using SdNABH at the same conditions (Yun et
al., 2012).
Supplementary Methods 2: Purification of AHG from the enzymatic hydrolysate of
agarose
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1) Silica gel chromatography. To obtain purified AHG, the concentrated enzymatic reaction
product of agarose was mixed with Celite powder for loading onto the silica gel column (I.D.
4 × 100 cm) packed with silica gel 60 (70-230 mesh ASTM; Merck, Darmstadt, Germany).
After loading the sample onto the column, a solvent mixture of chloroform/methanol/H2O
(78:20:2, v/v/v) was used as an eluent, and the fraction containing AHG was collected and
dried using a rotary vacuum evaporator at 35°C for 2 h (Yun et al., 2012).
2) Bio-Gel P-2 chromatography. The concentrated sample obtained by silica gel
chromatography of the agarose hydrolysate was loaded onto a Bio-Gel P-2 column (I.D. 2.1 ×
35.8 cm, Amersham Biosciences, Piscataway, NJ) for further purification of AHG. Water
was used as mobile phase, and only the sample fractions containing AHG were collected
(Yun et al., 2012).
Supplementary Method 3: Quantitative analysis of AHG and galactose by gas
chromatography/mass spectrometry (GC/MS)
AHG and galactose produced from agarose were quantified by GC-MS analysis as previously
described (Yun et al., 2011). Briefly, 20 µl of the reaction product, which contained mainly
AHG and galactose, was dried in a speed vacuum concentrator (Labconco, Kansas City, MO).
The dried sample was derivatized by adding 50 μl of 2% (w/v) of methoxyamine
hydrochloride in pyridine (Sigma, St. Louis, MO) at 75°C for 30 min. For the
trimethylsilylation of the sample, 80 μl of N-methyl-N-(trimethylsilyl)-trifluoroacetamide
(MSTFA; Fluka, St. Louis, MO) was added and the mixture was incubated at 40°C for 30
min. For GC-MS analysis, the Agilent 7890A GC/5975 C MSD system (Agilent
Technologies, Wilmington, DE) equipped with a DB-5ms column (I.D. 30 m × 0.25 mm,
0.25 μm film thickness; Agilent Technologies) was used. The oven temperature was initially
11
set at 100°C for 3.5 min and increased to 160°C at 15°C min-1, which was held for 20 min,
and then increased to 200°C at 20°C min-1, which was held for 15 min. Finally, the
temperature was increased to 280°C at 20°C min-1 and maintained for 5 min. Mass spectra
were acquired in a scan range of 50–700 m/z. The concentrations of AHG and galactose were
determined using the GC-MS calibration curve obtained using the standards of D-AHG
(Dextra Laboratories, Berkshire, UK) and D-galactose.
Supplementary Method 4: Quantitative analysis of ethanol by high performance liquid
chromatography (HPLC)
The ethanol concentration was quantified by HPLC (1200 Series, Agilent Technologies)
using a H+ column (Rezex ROA-Organic Acid, Phenomenex, Torrance, CA) and a refractive
index (RI) detector. We used 0.005N H2SO4 as mobile phase at a flow rate of 0.6 ml min-1;
column and RI detector temperatures were set at 50°C (Ha et al., 2010).
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