electronic supplementary information · were used in subsequent experiments (2,3-butanediol and...
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Electronic Supplementary Information
Artificial synthetic pathway for acetoin, 2,3-butanediol, and 2-butanol
production from ethanol using cell free multi-enzyme catalysis
Liaoyuan Zhang*,a,b,† Raushan Singh,b,† Sivakumar D,b Zewang Guo,a Jiahuan Li,a
Fanbin Chen,a Yuanzhi He,a Xiong Guan,a Yun Chan Kang,*,c and Jung-Kul Lee*,b
aKey Laboratory of Biopesticide and Chemical Biology, Ministry of Education,
College of Life Sciences, Gutian Edible Fungi Research Institute, Fujian Agriculture
and Forestry University, Fuzhou, Fujian province, 350002, PR ChinabDepartment of Chemical Engineering, Konkuk University, Seoul 143-701, Republic
of KoreacDepartment of Materials Science and Engineering, Korea University, Seoul 02841,
Republic of Korea
†: These authors contributed equally to this work.
*Corresponding author: Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Ministry of Education, FuZhou, Fujian Province, 350002, PR China Tel.: +86-591-83789492; Fax: +86-591-83789121; E-mail: [email protected]*Corresponding author: Department of Materials Science and Engineering, Korea University, Seoul 143-701, Republic of Korea Tel.: +82-2-4503505; Fax: +82-2-4583504;E-mail: [email protected]*Corresponding author: Department of Chemical Engineering, Konkuk University, Seoul 05029, Republic of Korea Tel.: +82-2-4503505; Fax: +82-2-4583504;E-mail: [email protected]
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Electronic Supplementary Material (ESI) for Green Chemistry.This journal is © The Royal Society of Chemistry 2017
Voges-proskauer (VP) reaction
Samples in the reaction solution were centrifuged at 10,000 ×g for 5 min at 4°C.
The concentration of acetoin in the samples was analyzed and quantified in the Voges-
proskauer (VP) reaction. Briefly, 0.3 mL of diluted sample, 0.3 mL of 0.5% creatine,
0.3 mL of 5% alpha-naphthol, and 0.3 mL of 5% NaOH were added to a 10-mL tube,
which was shaken gently for 30 min at 30°C. The optical density of the reaction
solution was determined at 520 nm using a spectrophotometer (UV-1800, MAPADA)
and the acetoin concentration was calculated from the calibration curve. The
calibration graph was plotted between standard acetoin concentration and the
corresponding optical density at 520 nm after the VP reaction in the range of 0.04–0.4
mM.
Optimization of reaction conditions
To obtain higher acetoin yield from ethanol, four factors including pH,
temperature, coenzyme (NAD+ and TPP), and metal ions were optimized in the
artificial cascade reaction using single-factor experiments. As shown in Figure S6, the
reaction mixtures containing 0.1 mg mL-1 EtDH, 0.2 mg mL-1 FLS, 0.1 mg mL-1 NOX,
4 mM NAD+, 0.1 mM TPP, 1 mM Mg2+, 1 mM DTT, 20% DMSO, and 100 mM
ethanol were evaluated at different pH values (6.0–9.0) at 30°C for 6 h. The results
showed that the maximum acetoin yield of 18.68 mM was produced at pH 8.0. Thus,
the optimized pH value of 8.0 was used in subsequent experiments. The effect of
temperature on acetoin production was evaluated in the range of 20–42°C for 6 h. The
results indicated that acetoin yield gradually increased with increasing temperature
from 20 to 42°C. Considering the enzyme stability, a temperature of 30°C was
employed for the cascade reaction. During the cascade reaction, coenzymes NAD+
and TPP were required for the activity of EtDH and FLS. Thus, their concentrations
were optimized in the range of 1–8 mM and 0–0.5 mM respectively. Similar results
were observed as shown in Figure S6 when the NAD+ concentration was 1, 2, and 4
mM, whereas NAD+ concentrations of 6 and 8 mM led to clear decreases in acetoin
production. Coenzyme TPP improved acetoin production as shown in Figure S6.
However, increasing the TPP concentration only minimally affected the cascade
reaction. Considering the reaction cost, 1 mM NAD+ and 0.1 mM TPP were used to
carry out the cascade reaction in subsequent experiments. Metal ions often activate
and improve enzyme catalytic efficiency. In the cascade reaction, Mg2+, Mn2+, Ca2+,
2
Fe2+, Ni2+, Cu2+, and Zn2+ at 1 mM were used to investigate the effects on acetoin
production. The results shown in Figure S6 revealed that metal ions have important
effects on acetoin production in the current cascade reaction. Only 2.15 mM acetoin
was produced in the absence of metal ions. All metal ions listed, particularly Mg2+
and Mn2+, significantly enhanced acetoin yield. Ultimately, the relatively optimal
reaction conditions of pH (8.0), NAD+ (1 mM), TPP (0.1 mM), and Mg2+ (1 mM)
were used in subsequent experiments (2,3-butanediol and 2-butanol production).
Quantification of substrate and products
The concentrations of ethanol, acetaldehyde, acetoin, 2,3-butanediol, butanone,
and 2-butanol in the artificial cascade reaction were determined with the addition of
isoamylol (50 mM) as an internal standard and quantified using a gas chromatograph
system (Agilent GC9860) equipped with a chiral column (Supelco β-DEX™ 120, 30-
m length, 0.25-mm inner diameter). The standard samples of ethanol (5–100 mM),
acetaldehyde (5–100 mM), acetoin (5–150 mM), 2,3-butanediol (5–150 mM),
butanone (5–150 mM), and 2-butanol (2–20 mM) at different concentrations were
prepared using ethyl acetate as a solvent. Each standard sample mixed with an
equivalent volume of isoamylol solution (50 mM) was analyzed using the GC system.
The calibration curves for each standard were constructed by plotting the area ratio
(standard area/isoamylol area) against the standard concentration. The concentrations
of substrate and products in the cascade reaction were calculated from the calibration
curve of each standard.
GC-MS analysis of product from acetaldehyde by FLS
The product from acetaldehyde by whole-cell biocatalysis was analyzed with an
Agilent GC 7890B coupled to a 5977A MSD using a chiral column (Supelco β-
DEX™ 120, 30-m length, 0.25-mm inner diameter) with the following temperature
program: 1.5-min hold at an initial temperature of 50°C followed by a 8°C min-1 ramp
to 110°C, with a final hold time of 15 min. Helium was used as the carrier gas at a
flow rate of 16.2 mL min-1.
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Figure S1 Verification of conversion from acetaldehyde to acetoin by FLS enzyme using the VP reaction. A, 50 mM acetaldehyde at 0 h; B, 100 mM acetaldehyde at 0 h; C, 50 mM acetaldehyde at 6 h; D, 100 mM acetaldehyde at 6 h.
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A B C D
Figure S2 SDS-PAGE analysis of purified EtDH, NOX, FLS, BDH, and their variants.
5
KDa120100
70
50
40
30
25
14
1 2 3 M 4 5 6
dhaB1
dhaB2
dhaB3
dhaR1
dhaR2
Figure S3 SDS-PAGE analysis of purified DDH:Q337A/F375I and purified dhaR. M, marker proteins; Lane 1, purified DDH:Q337A/F375I; Lane 2, crude enzyme of DDH:Q337A/F375I; Lanes 3 and 4, E. coli BL21(DE3) harboring pET28a; Lane 5, crude enzyme of dhaR protein; Lane 6, purified dhaR protein.
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Figure S4 Analysis of substrate stereoselectivity of DDH through whole-cell biocatalysis using meso-2,3-butanediol, (2R,3R)-butanediol and (2S,3S)-butanediol as substrates.
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Figure S5 Thermostability of the purified enzymes shown by residual activities. The activities were determined after the enzyme solutions were incubated at 30, 37, and 45°C for 6 h. 1, EtDH (ethanol with NAD+); 2, EtDH:D46G (ethanol with NAD+); 3, EtDH:D46G (ethanol with NADP+); 4, FLS (acetaldehyde with TPP); 5, FLS:L482S (acetaldehyde with TPP); 6, BDH:S199A (acetoin with NADPH); 7, BDH:S199A (butanone with NADPH); 8, DDH:Q337A F375 with dhaR (meso-2,3-butanediol with B12); 9, NOX (O2 with NADH).
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Figure S6 Effects of pH, temperature, NAD+, TPP, and metal ions on acetoin production from ethanol in the reaction system containing EtDH, FLS, and NOX enzymes.
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Figure S7 Time-course of acetoin production from 100 mM ethanol in the reaction system containing EtDH, FLS, and NOX enzymes under optimized conditions. Results are the means ± SD of three parallel replicates.
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Figure S8 Hot spots prediction was performed using the HotSpot Wizard 2.0 server. A, Results of hot spot prediction; B, structure model of FLS enzyme (blue: substrate tunnel pocket; golden, active site pocket; red: high mutable residues in the FLS enzyme; yellow: moderate mutable residues in the FLS enzyme).
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A B
Figure S9 Molecular interaction of the active site residue W480 with substrate (acetaldehyde) in A) wild-type FLS and B) mutant FLS:L482S. 100-ns molecular dynamics simulation results showing interaction fraction of key residues (given in X-axis) with the substrate in C) wild-type FLS and D) mutant FLS:L482S.
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NAD+
NAD+/NADP+
Figure S10 Alignment of amino acid sequence from the EtDH (CnMDH) enzyme with those of other dehydrogenases from PDB database. Red box shows that the enzymes only used NAD+ as a coenzyme; Blue box shows that the enzymes can simultaneously utilize NAD+ and NADP+ as coenzymes.
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Figure S11 Effects of NAD+ and NADP+ on 2,3-butanediol production from ethanol in the cascade reaction.
14
KDa120100
70
50
40
30
25
14
dhaR1dhaB1
dhaB2
dhaB3
M 1 2
dhaR2
Figure S12 Expression analysis of DDH:Q337A/F375I and its activator dhaR in E. coli BL21(DE3). M, marker proteins; 1, E. coli BL21(DE3) harboring pET28a; 2, crude enzyme of DDH:Q337A/F375I enzyme and its activator dhaR protein.
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Figure S13 Molecular interaction of an active site residue E171 with substrate (2,3-butanediol) in A) wild-type DDH and B) double mutant (Q337A/F375I) of DDH. 100 ns molecular dynamics simulation results showing interaction fraction of key residues (given in X-axis) with the substrate in C) wild-type DDH and D) double mutant (Q337A/F375I) of DDH.
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Acetaldehyde
Ethanol
2-Butanol
Butanone
(3R)-Acetoin (3S)-Acetoin
(2R,3R)-Butanediol
meso-2,3-Butanediol
Acetaldehyde
Ethanol
(3R)-Acetoin
(3S)-Acetoin
Acetaldehyde
Ethanol
(3R)-Acetoin (3S)-Acetoin
(2R,3R)-Butanediol
meso-2,3-Butanediol
Acetaldehyde
Ethanol
2-Butanol
Butanone
(3R)-A
cetoin (3S)-Acetoin
(2R,3R)-Butanediolmeso-2,3-Butanediol
A
B
C
D
Figure S14 Chiral-column GC analysis of the substrate and products in the reaction solutions. A, profile of mixture of standard chemicals; B, reaction from ethanol to acetoin via the intermediate acetaldehyde; C, reaction from ethanol to 2,3-butanediol via the intermediate acetaldehyde and acetoin; D, reaction from ethanol to 2-butanol via the intermediate acetaldehyde, acetoin, 2,3-butanediol, and butanone.
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18
Figure S15 GC-MS analysis of the product from acetaldehyde by FLS enzyme.
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Figure S16 GC analysis of 2-butanol enantiomers produced from 2-butanone using the whole-cell biocatalytic system including 50 mM HEPES buffer (pH 8.0), 40 g L-1 induced E. coli/pET28a-BDH:S199A cells (wet cell weight), 25 mM butanone at 30 oC for 6 h. 2-Butanol was analyzed using a gas chromatograph system (Agilent GC9860) with equipped with a chiral column (Supelco β-DEX™ 120, 30-m length, 0.25-mm inner diameter). The operation conditions were as follows: N2 was used as the carrier gas at flow rate of 1.2 mL min-1; injector temperature and detector temperature were 215 and 245°C, respectively; and column temperature was maintained at 50°C for 1.5 min, and then increased to 60 °C at a rate of 2 °C min-1, ultimately increased to 180 °C at a rate of 8 °C min-1.
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R
S2-Butanol
2-Butanone
Table S1 Primers used in this study.Primers Sequence (5′-3′) Mutation siteEtDH1 GATTGTTACCGGTGCTGGCCTGCATAAAATG D46GEtDH2 CATTTTATGCAGGCCAGCACCGGTAACAATC D46GFLS1 GGTAGCGGATGGTGGCCTGNNNTATCTCTGGCTGTCC T396FLS2 GGACAGCCAGAGATANNNCAGGCCACCATCCGCTACC T396FLS3 CCGCCGCACGATCCTTGTGNNNGGCGATGGCTCGGTG T446FLS4 CACCGAGCCATCGCCNNNCACAAGGATCGTGCGGCGG T446FLS5 GCCGCTGATCGTCATCATCNNNAACAACCAAAGCTGG M473FLS6 CCAGCTTTGGTTGTTNNNGATGATGACGATCAGCGGC M473FLS7 CATCATCATGAACAACCAANNNTGGGGGTGGACATTG S477FLS8 CAATGTCCACCCCCANNNTTGGTTGTTCATGATGATG S477FLS9 CCAAAGCTGGGGGTGGACANNNCATTTCCAGCAATTG L482FLS10 CAATTGCTGGAAATGNNNTGTCCACCCCCAGCTTTGG L482FLS11 TCGCGTGACGGGCACCCGTNNNGAAAATGGCTCCTAT L499FLS12 ATAGGAGCCATTTTCNNNACGGGTGCCCGTCACGCGA L499BDH1 GAATTATCGGTGTTGGAGCCAGACCTGTTTGTGTTG S199ABDH2 CAACACAAACAGGTCTGGCTCCAACACCGATAATTC S199ADDH1 CTCCAAAACGGTGGGGTTGCTTGTATTGGGATTCCAG S302ADDH2 CTGGAATCCCAATACAAGCAACCCCACCGTTTTGGAG S302ADDH3 ATGTGCGTCTGCTAATGACGCAGCGTTCTCCCATTCTG Q337ADDH4 CAGAATGGGAGAACGCTGCGTCATTAGCAGACGCACAT Q337ADDH5 CACCTAACTATGACAACACGATTGCGGGGTCAAACACCG F375IDDH6 CGGTGTTTGACCCCGCAATCGTGTTGTCATAGTTAGGTG F375I
Table S2 The specific activities of five enzymes in the artificial reaction pathway.Enzyme Substrate/coenzyme Specific activity (U mg-1)EtDH Ethanol/NAD+ 10.64 ± 0.15
Ethanol/NAD+ 8.81 ± 0.13EtDH:D46G
Ethanol/NADP+ 1.35 ± 0.01FLS:L482S Acetaldehyde/TPP 0.26 ± 0.01
Acetoin/NADPH 51.13 ± 3.74BDH:S199A
Butanone/NADPH 57.55 ± 2.65DDH:Q337A/F375I with dhaR meso-2,3-Butanediol/B12 0.05 ± 0.01NOX O2/NADH 9.83 ± 0.48
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Table S3 Effects of coenzyme B12, ATP, Mg2+, and dhaR on butanone production from meso-2,3-butanediol by wild-type DDH enzyme.
Reaction DDH Coenzyme B12 ATP Mg2+ dhaR ButanoneA 0.2 mg ml-1 0 mM 0 mM 0 mM 0 mg ml-1 0 ± 0 mMB 0.2 mg ml-1 1 mM 0 mM 0 mM 0 mg ml-1 0 ± 0 mMC 0.2 mg ml-1 1 mM 100 mM 0 mM 0 mg ml-1 1.38 ± 0.12 mMD 0.2 mg ml-1 1 mM 100 mM 1 mM 0 mg ml-1 2.48 ± 0.16 mME 0.2 mg ml-1 1 mM 100 mM 1 mM 0.2 mg ml-1 5.69 ± 0.21 mM
The results indicate that the coenzyme B12 and ATP are required for the catalytic reaction, whereas the reactivating factor dhaR and Mg2+ efficiently enhanced butanone production from meso-2,3-butanediol.
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Full-length EtDH and its variantEtDHATGACCCATCTGAATATTGCTAATCGCGTGGATAGTTTTTTTATTCCGTGTGTTACCCTGTTTGGCCCG
GGTTGCGCACGTGAAACCGGCGCCCGTGCACGTAGTCTGGGCGCACGTAAAGCCCTGATTGTTACCGAT
GCTGGCCTGCATAAAATGGGCCTGTCAGAAGTTGTTGCAGGTCATATTCGTGAAGCTGGTCTGCAAGCA
GTGATTTTTCCGGGCGCTGAACCGAATCCGACCGATGTTAATGTGCATGATGGTGTGAAACTGTTTGAA
CGTGAAGAATGTGATTTTATTGTGTCTCTGGGTGGCGGTAGCAGCCATGATTGCGCTAAAGGCATTGGC
CTGGTTACCGCGGGTGGCGGCCATATTCGCGATTATGAAGGTATTGATAAAAGTACCGTGCCGATGACC
CCGCTGATTTCAATTAATACCACCGCTGGTACAGCCGCCGAAATGACCCGCTTTTGTATTATTACCAAT
TCTAGTAATCATGTTAAAATGGCAATTGTTGATTGGCGTTGCACCCCGCTGATTGCGATTGATGACCCT
AGTCTGATGGTGGCAATGCCGCCGGCGCTGACCGCGGCAACCGGCATGGATGCTCTGACCCATGCAATT
GAAGCGTATGTGTCAACCGCAGCCACCCCGATTACCGATGCGTGTGCAGAAAAAGCGATTGTGCTGATT
GCGGAATGGCTGCCGAAAGCGGTTGCGAATGGCGATAGCATGGAAGCCCGTGCGGCTATGTGCTATGCA
CAGTATCTGGCAGGCATGGCGTTTAATAATGCATCACTGGGTTATGTGCACGCTATGGCACATCAGCTG
GGCGGCTTTTATAATCTGCCGCATGGTGTGTGCAATGCAATTCTGCTGCCGCATGTGTCAGAATTTAAT
CTGATTGCCGCACCGGAACGCTATGCACGCATTGCCGAACTGCTGGGTGAAAATATTGGTGGTCTGTCT
GCTCATGATGCGGCGAAAGCTGCTGTTAGCGCGATTCGCACCCTGTCTACCTCTATTGGTATTCCGGCT
GGTCTGGCGGGTCTGGGTGTTAAAGCCGATGACCATGAAGTTATGGCCAGCAATGCTCAGAAAGATGCT
TGTATGCTGACCAATCCGCGCAAAGCCACCCTGGCCCAGGTTATGGCGATTTTTGCTGCCGCCATGTAA
EtDH:D46GATGACCCATCTGAATATTGCTAATCGCGTGGATAGTTTTTTTATTCCGTGTGTTACCCTGTTTGGCCCG
GGTTGCGCACGTGAAACCGGCGCCCGTGCACGTAGTCTGGGCGCACGTAAAGCCCTGATTGTTACCGGT
GCTGGCCTGCATAAAATGGGCCTGTCAGAAGTTGTTGCAGGTCATATTCGTGAAGCTGGTCTGCAAGCA
GTGATTTTTCCGGGCGCTGAACCGAATCCGACCGATGTTAATGTGCATGATGGTGTGAAACTGTTTGAA
CGTGAAGAATGTGATTTTATTGTGTCTCTGGGTGGCGGTAGCAGCCATGATTGCGCTAAAGGCATTGGC
CTGGTTACCGCGGGTGGCGGCCATATTCGCGATTATGAAGGTATTGATAAAAGTACCGTGCCGATGACC
CCGCTGATTTCAATTAATACCACCGCTGGTACAGCCGCCGAAATGACCCGCTTTTGTATTATTACCAAT
TCTAGTAATCATGTTAAAATGGCAATTGTTGATTGGCGTTGCACCCCGCTGATTGCGATTGATGACCCT
AGTCTGATGGTGGCAATGCCGCCGGCGCTGACCGCGGCAACCGGCATGGATGCTCTGACCCATGCAATT
GAAGCGTATGTGTCAACCGCAGCCACCCCGATTACCGATGCGTGTGCAGAAAAAGCGATTGTGCTGATT
GCGGAATGGCTGCCGAAAGCGGTTGCGAATGGCGATAGCATGGAAGCCCGTGCGGCTATGTGCTATGCA
CAGTATCTGGCAGGCATGGCGTTTAATAATGCATCACTGGGTTATGTGCACGCTATGGCACATCAGCTG
GGCGGCTTTTATAATCTGCCGCATGGTGTGTGCAATGCAATTCTGCTGCCGCATGTGTCAGAATTTAAT
CTGATTGCCGCACCGGAACGCTATGCACGCATTGCCGAACTGCTGGGTGAAAATATTGGTGGTCTGTCT
GCTCATGATGCGGCGAAAGCTGCTGTTAGCGCGATTCGCACCCTGTCTACCTCTATTGGTATTCCGGCT
GGTCTGGCGGGTCTGGGTGTTAAAGCCGATGACCATGAAGTTATGGCCAGCAATGCTCAGAAAGATGCT
TGTATGCTGACCAATCCGCGCAAAGCCACCCTGGCCCAGGTTATGGCGATTTTTGCTGCCGCCATGTAA
Full-length FLS and its variantFLSATGGCGATGATTACAGGCGGCGAACTGGTTGTTCGCACCCTAATAAAGGCTGGGGTCGAACATCTGTTC
GGCCTGCACGGCATTCATATCGATACGATTTTTCAAGCCTGTCTCGATCATGATGTGCCGATCATCGAC
ACCCGCCATGAGGCCGCCGCAGGGCATGCGGCCGAGGGCTATGCCCGCGCTGGCGCCAAGCTGGGCGTG
GCGCTGGTCACGGCGGGCGGGGGATTTACCAATGCGGTCACGCCCATTGCCAACGCTCGTACCGATCGC
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ACGCCGGTGCTCTTCCTCACCGGATCGGGCGCGCTGCGTGATGATGAAACCAACACGTTGCAGGCGGGG
ATTGATCAGGTCGCCATGGCGGCGCCCATTACCAAATGGGCGCATCGGGTGATGGCAACCGAGCATATC
CCACGGCTGGTGATGCAGGCGATCCGCGCCGCGTTGAGCGCGCCACGCGGGCCGGTGTTGCTGGATCTG
CCGTGGGATATTCTGATGAACCAGATTGATGAGGATAGCGTCATTATCCCCGATCTGGTCTTGTCCGCG
CATGGGGCCCATCCCGACCCTGCCGATCTGGATCAGGCTCTCGCGCTTTTGCGCAAGGCGGAGCGGCCG
GTCATCGTGCTCGGCTCAGAAGCCTCGCGGACAGCGCGCAAGACGGCGCTTAGCGCCTTCGTGGCGGCG
ACTGGCGTGCCGGTGTTTGCCGATTATGAAGGGCTAAGCATGCTCTCGGGGCTGCCCGATGCTATGCGG
GGCGGGCTGGTGCAAAACCTCTATTCTTTTGCCAAAGCCGATGCCGCGCCAGATCTCGTGCTGATGCTG
GGGGCGCGCTTTGGCCTTAACACCGGGCATGGATCTGGGCAGTTGATCCCCCATAGCGCGCAGGTCATT
CAGGTCGACCCTGATGCCTGCGAGCTGGGACGCCTGCAGGGCATCGCTCTGGGCATTGTGGCCGATGTG
GGTGGGACCATCGAGGCTTTGGCGCAGGCCACCGCGCAAGATGCGGCTTGGCCGGATCGCGGCGACTGG
TGCGCCAAAGTGACGGATCTGGCGCAAGAGCGCTATGCCAGCATCGCTGCGAAATCGAGCAGCGAGCAT
GCGCTCCACCCCTTTCACGCCTCGCAGGTCATTGCCAAACACGTCGATGCAGGGGTGACGGTGGTAGCG
GATGGTGGCCTGACCTATCTCTGGCTGTCCGAAGTGATGAGCCGCGTGAAACCCGGCGGTTTTCTCTGC
CACGGCTATCTAAACTCGATGGGCGTGGGCTTCGGCACGGCGCTGGGCGCGCAAGTGGCCGATCTTGAA
GCAGGCCGCCGCACGATCCTTGTGACCGGCGATGGCTCGGTGGGCTATAGCATCGGTGAATTTGATACG
CTGGTGCGCAAACAATTGCCGCTGATCGTCATCATCATGAACAACCAAAGCTGGGGGTGGACATTGCAT
TTCCAGCAATTGGCCGTCGGCCCCAATCGCGTGACGGGCACCCGTTTGGAAAATGGCTCCTATCACGGG
GTGGCCGCCGCCTTTGGCGCGGATGGCTATCATGTCGACAGTGTGGAGAGCTTTTCTGCGGCTCTGGCC
CAAGCGCTCGCCCATAATCGCCCCGCCTGCATCAATGTCGCGGTCGCGCTCGATCCGATCCCGCCCGAA
GAACTCATTCTGATCGGCATGGACCCCTTCGCATGA
FLS:L482SATGGCGATGATTACAGGCGGCGAACTGGTTGTTCGCACCCTAATAAAGGCTGGGGTCGAACATCTGTTC
GGCCTGCACGGCATTCATATCGATACGATTTTTCAAGCCTGTCTCGATCATGATGTGCCGATCATCGAC
ACCCGCCATGAGGCCGCCGCAGGGCATGCGGCCGAGGGCTATGCCCGCGCTGGCGCCAAGCTGGGCGTG
GCGCTGGTCACGGCGGGCGGGGGATTTACCAATGCGGTCACGCCCATTGCCAACGCTCGTACCGATCGC
ACGCCGGTGCTCTTCCTCACCGGATCGGGCGCGCTGCGTGATGATGAAACCAACACGTTGCAGGCGGGG
ATTGATCAGGTCGCCATGGCGGCGCCCATTACCAAATGGGCGCATCGGGTGATGGCAACCGAGCATATC
CCACGGCTGGTGATGCAGGCGATCCGCGCCGCGTTGAGCGCGCCACGCGGGCCGGTGTTGCTGGATCTG
CCGTGGGATATTCTGATGAACCAGATTGATGAGGATAGCGTCATTATCCCCGATCTGGTCTTGTCCGCG
CATGGGGCCCATCCCGACCCTGCCGATCTGGATCAGGCTCTCGCGCTTTTGCGCAAGGCGGAGCGGCCG
GTCATCGTGCTCGGCTCAGAAGCCTCGCGGACAGCGCGCAAGACGGCGCTTAGCGCCTTCGTGGCGGCG
ACTGGCGTGCCGGTGTTTGCCGATTATGAAGGGCTAAGCATGCTCTCGGGGCTGCCCGATGCTATGCGG
GGCGGGCTGGTGCAAAACCTCTATTCTTTTGCCAAAGCCGATGCCGCGCCAGATCTCGTGCTGATGCTG
GGGGCGCGCTTTGGCCTTAACACCGGGCATGGATCTGGGCAGTTGATCCCCCATAGCGCGCAGGTCATT
CAGGTCGACCCTGATGCCTGCGAGCTGGGACGCCTGCAGGGCATCGCTCTGGGCATTGTGGCCGATGTG
GGTGGGACCATCGAGGCTTTGGCGCAGGCCACCGCGCAAGATGCGGCTTGGCCGGATCGCGGCGACTGG
TGCGCCAAAGTGACGGATCTGGCGCAAGAGCGCTATGCCAGCATCGCTGCGAAATCGAGCAGCGAGCAT
GCGCTCCACCCCTTTCACGCCTCGCAGGTCATTGCCAAACACGTCGATGCAGGGGTGACGGTGGTAGCG
GATGGTGGCCTGACCTATCTCTGGCTGTCCGAAGTGATGAGCCGCGTGAAACCCGGCGGTTTTCTCTGC
CACGGCTATCTAAACTCGATGGGCGTGGGCTTCGGCACGGCGCTGGGCGCGCAAGTGGCCGATCTTGAA
GCAGGCCGCCGCACGATCCTTGTGACCGGCGATGGCTCGGTGGGCTATAGCATCGGTGAATTTGATACG
CTGGTGCGCAAACAATTGCCGCTGATCGTCATCATCATGAACAACCAAAGCTGGGGGTGGACAAGTCAT
24
TTCCAGCAATTGGCCGTCGGCCCCAATCGCGTGACGGGCACCCGTTTGGAAAATGGCTCCTATCACGGG
GTGGCCGCCGCCTTTGGCGCGGATGGCTATCATGTCGACAGTGTGGAGAGCTTTTCTGCGGCTCTGGCC
CAAGCGCTCGCCCATAATCGCCCCGCCTGCATCAATGTCGCGGTCGCGCTCGATCCGATCCCGCCCGAA
GAACTCATTCTGATCGGCATGGACCCCTTCGCATGA
Full-length BDH and its variantBDHATGAAAGGTTTTGCAATGTTAGGTATTAACAAATTAGGATGGATTGAAAAGAAAAACCCAGTGCCAGGT
CCTTATGATGCGATTGTACATCCTCTAGCTGTATCCCCATGTACATCAGATATACATACGGTTTTTGAA
GGAGCACTTGGTAATAGGGAAAATATGATTTTAGGCCATGAAGCTGTAGGTGAAATAGCCGAAGTTGGC
AGCGAAGTTAAAGATTTTAAAGTTGGCGATAGAGTTATCGTACCATGCACAACACCTGACTGGAGATCT
TTAGAAGTCCAAGCTGGTTTTCAGCAGCATTCAAACGGTATGCTTGCAGGATGGAAGTTTTCCAATTTT
AAAGATGGTGTATTTGCAGATTACTTTCATGTAAACGATGCAGATATGAATCTTGCCATACTCCCAGAT
GAAATACCTTTAGAAAGTGCAGTTATGATGACAGACATGATGACTACTGGTTTTCATGGAGCAGAACTT
GCAGACATAAAAATGGGCTCCAGCGTTGTAGTAATTGGTATAGGAGCTGTTGGATTAATGGGAATAGCC
GGTTCCAAACTTCGAGGAGCAGGCAGAATTATCGGTGTTGGAAGCAGACCTGTTTGTGTTGAAACAGCT
AAATTTTATGGAGCAACTGATATTGTAAATTATAAAAATGGTGATATAGTTGAACAAATCATGGACTTA
ACTCATGGTAAAGGTGTAGACCGTGTAATCATGGCAGGCGGTGGTGCTGAAACACTAGCACAAGCAGTA
ACTATGGTTAAACCTGGCGGCGTAATTTCTAACATCAACTACCATGGAAGCGGTGATACTTTACCAATA
CCTCGTGTTCAATGGGGCTGCGGCATGGCTCACAAAACTATAAGAGGAGGATTATGCCCCGGCGGACGT
CTTAGAATGGAAATGCTAAGAGATCTTGTTCTATATAAACGTGTTGATTTGAGTAAACTTGTTACTCAT
GTATTTGATGGTGCAGAAAATATTGAAAAGGCCCTTTTGCTTATGAAAAATAAGCCAAAAGATTTAATT
AAATCAGTAGTTACATTCTAA
BDH:S199AATGAAAGGTTTTGCAATGTTAGGTATTAACAAATTAGGATGGATTGAAAAGAAAAACCCAGTGCCAGGT
CCTTATGATGCGATTGTACATCCTCTAGCTGTATCCCCATGTACATCAGATATACATACGGTTTTTGAA
GGAGCACTTGGTAATAGGGAAAATATGATTTTAGGCCATGAAGCTGTAGGTGAAATAGCCGAAGTTGGC
AGCGAAGTTAAAGATTTTAAAGTTGGCGATAGAGTTATCGTACCATGCACAACACCTGACTGGAGATCT
TTAGAAGTCCAAGCTGGTTTTCAGCAGCATTCAAACGGTATGCTTGCAGGATGGAAGTTTTCCAATTTT
AAAGATGGTGTATTTGCAGATTACTTTCATGTAAACGATGCAGATATGAATCTTGCCATACTCCCAGAT
GAAATACCTTTAGAAAGTGCAGTTATGATGACAGACATGATGACTACTGGTTTTCATGGAGCAGAACTT
GCAGACATAAAAATGGGCTCCAGCGTTGTAGTAATTGGTATAGGAGCTGTTGGATTAATGGGAATAGCC
GGTTCCAAACTTCGAGGAGCAGGCAGAATTATCGGTGTTGGAGCCAGACCTGTTTGTGTTGAAACAGCT
AAATTTTATGGAGCAACTGATATTGTAAATTATAAAAATGGTGATATAGTTGAACAAATCATGGACTTA
ACTCATGGTAAAGGTGTAGACCGTGTAATCATGGCAGGCGGTGGTGCTGAAACACTAGCACAAGCAGTA
ACTATGGTTAAACCTGGCGGCGTAATTTCTAACATCAACTACCATGGAAGCGGTGATACTTTACCAATA
CCTCGTGTTCAATGGGGCTGCGGCATGGCTCACAAAACTATAAGAGGAGGATTATGCCCCGGCGGACGT
CTTAGAATGGAAATGCTAAGAGATCTTGTTCTATATAAACGTGTTGATTTGAGTAAACTTGTTACTCAT
GTATTTGATGGTGCAGAAAATATTGAAAAGGCCCTTTTGCTTATGAAAAATAAGCCAAAAGATTTAATT
AAATCAGTAGTTACATTCTAA
Full-length DDH and its variantDDHATGAAACGTCAAAAACGATTTGAAGAATTAGAGAAACGCCCGATCCATTTAGACGGTTTCGTTAAGGAA
25
TGGCCTGAAGAAGGCTTCGTTGCCATGATGGGGCCTAATGACCCAAAGCCAAGCATCAAGATTGAAAAC
GGCAAGGTTACTGAAATGGATAGTAAACCAGCTGCTGACTTCGATCTGATTGATCTCTACATCGCAAAA
TATGGCATTAAGCTTGAAAATGCTGAGAAAGTAATGGCGATGGATTCCACTAAGATCGCCAATATGCTC
TGTGACCCCAATGTGCCACGTAAAGACATCATTGAGATTACAACGGCGATGACGCCGGCCAAAGCTGAA
GAAGTTATCAGCAAATTGAACTTTGCCGAAATGATTATGGCAACGCAAAAAATGCGGCCACGGCGGACA
CCAGCAACGCAATGTCACGTTACCAATATTCGGGATAATCCCGTTCAAATTGCTGCTGACGCTGCCGAT
GCTGCCTTACGGGGTTTCCCGGAACAAGAAACCACGACGGCCGTTGCCCGGTATGCCCCATTGAATGCC
ATCTCGTTGATGGTGGGGGCGCAAACCGGTCGTCCTGGTGTTATCACCCAATGCTCGGTTGAAGAAGCA
GAAGAATTGAGTTTAGGGATGCGGGGCTTCACTGGCTACGCCGAAACCATTTCTGTTTACGGTACCGAT
AAGGTCTTCACTGATGGTGATGATACACCATGGTCCAAAGGCTTCTTAGCTTCCTGCTATGCTTCGCGG
GGGTTGAAGATGCGGTTTACGTCTGGTTCTGGTTCCGAAGTTATGATGGGTTATACCGAAGGTAAGTCC
ATGTTATACCTCGAATCACGTTGTATCTTCATTACCAAAGCGTCCGGTGTTCAAGGCCTCCAAAACGGT
GGGGTTAGTTGTATTGGGATTCCAGGGTCTGTTCCTTCTGGGATTCGCTCCGTCTTGGGTGAAAACCTA
TTGTGCATGATGCTTGACCTTGAATGTGCGTCTGCTAATGACCAAGCGTTCTCCCATTCTGATATGCGG
CGGACAGAACGGTTATTAGGCCAATTCATTGCCGGAACCGATTACATTTCTTCTGGTTACTCCTCAACA
CCTAACTATGACAACACGTTTGCGGGGTCAAACACCGATGGCTTGGACTACGATGATTACTACGTTATG
GAACGCGACTTGGCCATCAACGGTGGGATTCACCCAGTTGATGAACAAACAATCATCAAAGCCCGCAAC
AAGGCTGCACGGGCCCTTCAAGGTGTCTTTGAAGATCTAGGTTTGCCTAAGATTACCGATGAAGAAGTG
GAAGCGGCAACTTACGCCAACACCTCTAAGGATATGCCAGAACGGAACATGGTTGAAGATATGAAGGCC
GCCCAAGATCTGATGGATCGCGGCATTACCGGGGTCGATATTGTTAAAGCCTTGTTCAACCACGGATTT
AAGGATGTTGCCCAAGCCGTTTTAGATTTGCAAAAGCAAAAGGTTTGTGGGGACTTCTTACAGACATCC
GCTATTTTCGACAGCAAGTGGCATGTCATTTCCGCCGTCAACGATGCCAATGACTATCAAGGTCCTGGT
ACGGGTTACCGGTTGGAAGAAGATACGGAAGAATGGGAACGCATCAAGAACTTACCGTTTGCCATTGAT
CCACAAAACATGCAGCTTTAGTCGAAAAGGGGGTTAACACTATGGCTCAAGAAATTGATGAAAACTTAT
TGCGGAATATTATCCGTGATGTGATTGCGGAAACCCAAACGGGGGACACGCCAATCTCATTTAAAGCTG
ATGCACCAGCAGCGTCATCAGCTACGACGGCAACGGCTGCACCAGTTAATGGTGACGGCCCAGAACCGG
AAAAACCAGTTGACTGGTTCAAACACGTTGGGGTTGCCAAGCCCGGCTATTCACGTGATGAAGTCGTGA
TTGCTGTGGCACCAGCCTTTGCAGAAGTGATGGACCATAACTTGACCGGAATCAGTCATAAAGAAATTT
TACGACAGATGGTTGCTGGTATTGAAGAAGAAGGACTGAAGGCCCGAATTGTGAAAGTCTACCGGACTT
CTGACGTTTCCTTCTGTGGTGCCGAAGGGGATCATTTATCAGGTTCTGGCATCGCCATTGCCATTCAAT
CCAAGGGGACGACGATCATTCACCAAAAGGACCAAGAACCATTGTCCAACTTGGAATTATTCCCACAAG
CACCTGTCTTGGATGGTGATACCTACCGGGCTATCGGCAAGAATGCAGCCGAATACGCTAAAGGAATGT
CACCAAGCCCCGTTCCAACGGTTAATGACCAAATGGCTCGGGTTCAATACCAGGCCTTGTCTGCCTTGA
TGCATATCAAGGAAACGAAGCAGGTCGTTATGGGGAAACCCGCTGAACAAATCGAAGTCAACTTTAACT
AGGAGGAATGGGTCATGAGTGAAATTGATGACTTAGTAGCAAAAATCGTCCAACAAATTGGTGGCACTG
AGGCCGCTGATCAGACGACTGCCACGCCTACGTCAACGGCGACGCAGACGCAGCATGCAGCATTATCGA
AACAAGATTATCCACTGTACTCTAAGCACCCAGAGCTCGTACATTCACCGTCTGGGAAAGCTTTGAACG
ATATCACTTTGGATAATGTTCTCAACGATGATATTAAGGCCAATGATTTACGAATTACGCCGGATACCT
TACGGATGCAAGGTGAAGTGGCCAACGATGCTGGTCGGGATGCGGTTCAACGTAACTTCCAGCGGGCGT
CAGAATTGACCTCTATTCCGGATGATCGGTTACTGGAAATGTACAACGCCTTACGACCATACCGGTCTA
CTAAAGCGGAATTATTAGCGATTTCAGCCGAGTTAAAGGATAAATATCATGCCCCAGTGAACGCCGGAT
GGTTTGCGGAAGCGGCCGACTACTACGAATCCCGTAAGAAGCTGAAGGGTGATAACTAG
DDH:Q337A/F375I
26
ATGAAACGTCAAAAACGATTTGAAGAATTAGAGAAACGCCCGATCCATTTAGACGGTTTCGTTAAGGAA
TGGCCTGAAGAAGGCTTCGTTGCCATGATGGGGCCTAATGACCCAAAGCCAAGCATCAAGATTGAAAAC
GGCAAGGTTACTGAAATGGATAGTAAACCAGCTGCTGACTTCGATCTGATTGATCTCTACATCGCAAAA
TATGGCATTAAGCTTGAAAATGCTGAGAAAGTAATGGCGATGGATTCCACTAAGATCGCCAATATGCTC
TGTGACCCCAATGTGCCACGTAAAGACATCATTGAGATTACAACGGCGATGACGCCGGCCAAAGCTGAA
GAAGTTATCAGCAAATTGAACTTTGCCGAAATGATTATGGCAACGCAAAAAATGCGGCCACGGCGGACA
CCAGCAACGCAATGTCACGTTACCAATATTCGGGATAATCCCGTTCAAATTGCTGCTGACGCTGCCGAT
GCTGCCTTACGGGGTTTCCCGGAACAAGAAACCACGACGGCCGTTGCCCGGTATGCCCCATTGAATGCC
ATCTCGTTGATGGTGGGGGCGCAAACCGGTCGTCCTGGTGTTATCACCCAATGCTCGGTTGAAGAAGCA
GAAGAATTGAGTTTAGGGATGCGGGGCTTCACTGGCTACGCCGAAACCATTTCTGTTTACGGTACCGAT
AAGGTCTTCACTGATGGTGATGATACACCATGGTCCAAAGGCTTCTTAGCTTCCTGCTATGCTTCGCGG
GGGTTGAAGATGCGGTTTACGTCTGGTTCTGGTTCCGAAGTTATGATGGGTTATACCGAAGGTAAGTCC
ATGTTATACCTCGAATCACGTTGTATCTTCATTACCAAAGCGTCCGGTGTTCAAGGCCTCCAAAACGGT
GGGGTTAGTTGTATTGGGATTCCAGGGTCTGTTCCTTCTGGGATTCGCTCCGTCTTGGGTGAAAACCTA
TTGTGCATGATGCTTGACCTTGAATGTGCGTCTGCTAATGACGCAGCGTTCTCCCATTCTGATATGCGG
CGGACAGAACGGTTATTAGGCCAATTCATTGCCGGAACCGATTACATTTCTTCTGGTTACTCCTCAACA
CCTAACTATGACAACACGATTGCGGGGTCAAACACCGATGGCTTGGACTACGATGATTACTACGTTATG
GAACGCGACTTGGCCATCAACGGTGGGATTCACCCAGTTGATGAACAAACAATCATCAAAGCCCGCAAC
AAGGCTGCACGGGCCCTTCAAGGTGTCTTTGAAGATCTAGGTTTGCCTAAGATTACCGATGAAGAAGTG
GAAGCGGCAACTTACGCCAACACCTCTAAGGATATGCCAGAACGGAACATGGTTGAAGATATGAAGGCC
GCCCAAGATCTGATGGATCGCGGCATTACCGGGGTCGATATTGTTAAAGCCTTGTTCAACCACGGATTT
AAGGATGTTGCCCAAGCCGTTTTAGATTTGCAAAAGCAAAAGGTTTGTGGGGACTTCTTACAGACATCC
GCTATTTTCGACAGCAAGTGGCATGTCATTTCCGCCGTCAACGATGCCAATGACTATCAAGGTCCTGGT
ACGGGTTACCGGTTGGAAGAAGATACGGAAGAATGGGAACGCATCAAGAACTTACCGTTTGCCATTGAT
CCACAAAACATGCAGCTTTAGTCGAAAAGGGGGTTAACACTATGGCTCAAGAAATTGATGAAAACTTAT
TGCGGAATATTATCCGTGATGTGATTGCGGAAACCCAAACGGGGGACACGCCAATCTCATTTAAAGCTG
ATGCACCAGCAGCGTCATCAGCTACGACGGCAACGGCTGCACCAGTTAATGGTGACGGCCCAGAACCGG
AAAAACCAGTTGACTGGTTCAAACACGTTGGGGTTGCCAAGCCCGGCTATTCACGTGATGAAGTCGTGA
TTGCTGTGGCACCAGCCTTTGCAGAAGTGATGGACCATAACTTGACCGGAATCAGTCATAAAGAAATTT
TACGACAGATGGTTGCTGGTATTGAAGAAGAAGGACTGAAGGCCCGAATTGTGAAAGTCTACCGGACTT
CTGACGTTTCCTTCTGTGGTGCCGAAGGGGATCATTTATCAGGTTCTGGCATCGCCATTGCCATTCAAT
CCAAGGGGACGACGATCATTCACCAAAAGGACCAAGAACCATTGTCCAACTTGGAATTATTCCCACAAG
CACCTGTCTTGGATGGTGATACCTACCGGGCTATCGGCAAGAATGCAGCCGAATACGCTAAAGGAATGT
CACCAAGCCCCGTTCCAACGGTTAATGACCAAATGGCTCGGGTTCAATACCAGGCCTTGTCTGCCTTGA
TGCATATCAAGGAAACGAAGCAGGTCGTTATGGGGAAACCCGCTGAACAAATCGAAGTCAACTTTAACT
AGGAGGAATGGGTCATGAGTGAAATTGATGACTTAGTAGCAAAAATCGTCCAACAAATTGGTGGCACTG
AGGCCGCTGATCAGACGACTGCCACGCCTACGTCAACGGCGACGCAGACGCAGCATGCAGCATTATCGA
AACAAGATTATCCACTGTACTCTAAGCACCCAGAGCTCGTACATTCACCGTCTGGGAAAGCTTTGAACG
ATATCACTTTGGATAATGTTCTCAACGATGATATTAAGGCCAATGATTTACGAATTACGCCGGATACCT
TACGGATGCAAGGTGAAGTGGCCAACGATGCTGGTCGGGATGCGGTTCAACGTAACTTCCAGCGGGCGT
CAGAATTGACCTCTATTCCGGATGATCGGTTACTGGAAATGTACAACGCCTTACGACCATACCGGTCTA
CTAAAGCGGAATTATTAGCGATTTCAGCCGAGTTAAAGGATAAATATCATGCCCCAGTGAACGCCGGAT
GGTTTGCGGAAGCGGCCGACTACTACGAATCCCGTAAGAAGCTGAAGGGTGATAACTAG
27
Full-length dhaRATGCAAAAGGTGATAGGTGTAGATATTGGTAATTCCTCAACGGAAGTTGCTTTGGCTGATATTTCAGAT
CAAGGTGCGGTTGATTTTATCAATTCTGACATTGCAGAGACCACGGGGATCAAAGGAACTAAGCAAAAC
CTCATTGGGATCAAAAAAGCCATTACGCAGGTGTTAAATAAAAGTCATTTAGCCTTGAGTGATATTGAC
CTGATTCGGATTAACGAAGCAACACCGGTTATTGGGGATGTAGCGATGGAAACCATCACGGAAACAGTG
ATTACTGAATCCACGATGATTGGGCATAACCCGAACACACCAGGTGGCGTCGGCATTGGTTCTGGCTAT
ACGGTGAATTTGCTACAACTGCTTCAAGAAACCGATAAGACTCGTCCGTACATTGTTCTGGTACCGGCT
GAAGTTGATTTCGAAGATGCGGCTAAGCTGATCAATCTATACCAGCAAAGTGGTTATCAAATAACTGCG
GCCATCCTGCAAAATGACGATGGGGTGTTAATTGATAACCGATTGGAACATAAGATACCAATTGTAGAT
GAAGTGGCGCGGATTGATAAGGTTCCCATGGGGATGATGGCTGGCGTTGAGGTTGCTGGTAAAGGGCAA
GTTATTTCGCAGTTATCTAATCCGTATGGGATCGCCACGCTCTTTGATTTGACGGCCGATGAAACCAAA
AACATCGTGCCGGTTTCTCGGGCGTTAATTGGCAACCGGTCTGCGGTGGTCATCAAGACGCCTAAGGGG
GATGTGAAGGCCCGGGTTATTCCGGCCGGGAGCATCCAAATCGAAGGTGATCGAGATTCTGACAAGGTT
AACGTGGCCGCTGGTGCTGAAGCAATTATGAAGAAGGTCAATCAGTTTGACCGGATTCAAGATATTACA
GGTGAAGCGGGAACCAATGTTGGTGGGATGTTGGAAAAGGTTCGGCAGACGATGGCGGACCTCACCAAC
AAACAGAACAGAGATATCGCTATTCAAGATCTGTTAGCCGTCAACACGGCGGTTCCAGTCAAGGTACAA
GGTGGACTGGCTGGCGAGTTCTCAACCGAACAAGCCGTTGGGATTGCCGCAATGGTAAAATCTGACCAC
CTCCAGATGCAACAGATTGCTGATTTGATTCAAGACGAGCTTCACATTTCCGTTGAGATTGGCGGAGCT
GAAGCTGAGGCCGCAATCTTGGGAGCTTTGACGACACCAGGAACAACCAAGCCCATTGCGATTCTTGAC
TTGGGTGCCGGTTCAACAGATGCCTCGATTATCAATCAGCAAGATGATATTGTGGCGATTCACTTGGCT
GGTGCCGGGGACATGGTCACCATGATTATCAATTCTGAGTTAGGCCTAGACGATGTGTACTTGGCCGAG
GATATTAAGAAATATCCGCTGGCCCGAGTTGAAAATCTATTCCAAATTCGGCATGAAGATGGCACGGTT
CAGTTTTTTGAAGACCCACTGCCAGCAGACATTTTTGCCCGCACAGTGGTCATTAAGCCGGACGGTTAC
GTCCCATTACCAGGGAATATGAACATTGAAAAGGTTAAGCAGATTCGCCAGACCGCTAAGAAGCGGGTG
TTTGTGGAAAATGCACGGCGGGCCTTACAACACGTGAGTCCCACTGGTAATATCCGTGACATCCCGTTT
GTCGTGATCGTTGGGGGATCGGCGCTGGACTTTGAAATCCCACAATTGGTCACGGATGAGTTGTCTCAC
TATAACCTTGTTGCCGGACGGGGAAATATTCGGGCCGTGGAAGGACCGCGAAATGCGGTTGCTACGGGA
TTGATCCTGTCTTATGCCCGGGAGAGAAGGGACGCCTATGACCAACACAATGGATAAACCGGCAATTTT
TATTGCCGTGCCGACAGCGGCGTCCGATTTACCGGTGACTTTGAAACCGTTACTTAACGGGATTGAAGA
AGAAGCCATTCCGGTACAGACCAAGGTGATTGCAGAAGACGATGTCACCATGCGCACTTATCAGGCGGC
TTTGGCTTCACGGTTGTCCGTGGGCATTGGTTTTGACGATCAGCATGTGGTTGTTCACTACAAGAACCT
GCATGCCGAGCAGCCGTTATTCACGGTGACCCGTGATTCGGCGGACCGCCTGCGCCGGTTAGGTGCTAA
TGCAGCTCGCTTGGTGAAGGGCGTGCCCTTTAAGACATTAGATTAG
Full-length NOXATGAAGATTCTTGTCATTGGTGCTACCCATGCCGGTACATTTGCAACCCAGCAGATCCTAACCGACCAT
CCAGATGCAGAGGTTACTGTCTACGAACGCAATAACAACCTGTCCTTCCTCTCGTGCGGCATTGCCTTG
TGGGTTGGTGATCATGTCAGTGACCCGGATAAAATGTTCTATTCCAGTCCCGAAGCACTCGCTAAACTC
GGTGCTAATATGCAAATGGAACATGATGTGCTCAATATTGATCCAGCAACTAAAACAGTTGAAGTCAAG
GATCTAAAAACCGGAACCGTTACTACCGATACTTATGACAAATTAGTCTACACAACCGGATCGACGCCA
ATCATTCCAAATATTCCCGGTATCCACGATTCAAACGTCTACTTATGCAAAAATTGGTCCGACGCCAAG
ACGCTAAAAGATCTGGCCCCGTCCATTAAAAGCGCCATTGTCATCGGTGCAGGCTACATCGGTGCAGAA
TTAGCCGAACAATTTGCGTTAACCGACAAAGAAGTCACGTTAATCGATGGACTTCCACGGGTTTTGGCG
AAAAACTTTGACGCCACTATCACGGATCGCGTTGAAAAGCTTTACACCGATCACGGGGTTCACTTGGCA
CTCAATGAGATGGTTACCGAGTTCGCACAAGCTGATCAGGGTATCAAGGTTACAACCAATAAAGGCGAC
28
TATACCGCGGATATTGCAATTTTATGTACCGGCTTCCGTCCGAACACGGATCTGCTAAAGGACCATCTG
GACACCCTGCCTAATGGCGCTGTCATAACAAATGCATATATGCAGACCAGTGACCCCGACATTTTCGCT
GCTGGTGATACCGCTACCGTCCACTATAATCCGACTGGCAAAAATGACTACATCCCGCTTGCGACCAAC
GCAGTCCGTCAAGGCATTCTTGTTGGTAAAAATATCATGACCCCCACGGAAAAATACCTGGGAACACAA
TCTAGCTCGGCCGTTGAACTTTTTGATCACGCCATTGCGGCAAGCGGCCTAACGGTGGAAGGCGCTCAC
ACACGTGGACTTGAGCTTGATAGTGTCACGATCGAACAGGATTATCGCCCCGATTTCATGTTAACCACA
ACGCCGGTGCTCTGCAGCCTGACATGGGATCCCAAGACGCATGAAGTTAAAGGAGGTGCCTTTTTCTCC
AAGCACGATATCAGCCAAAGCGCTAATGTCATTTCGCTTGCGATCCAGACCCACATGACGATCGAAACA
CTTGCGATGGTTGACATGCTCTTCCAACCTAACTTCGATCAGCCGATTAACTGGGTAAATGCCGTGGCT
ATGGCGGCAGTTGACAAGGCTAAAAAGAAGCCGACAACACCGGTAGCCTAA
29