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: zliaoyuan@126.com*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: yckang@korea.ac.kr*Corresponding author: Department of Chemical Engineering, Konkuk University, Seoul 05029, Republic of Korea Tel.: +82-2-4503505; Fax: +82-2-4583504;E-mail: jkrhee@konkuk.ac.kr

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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+,

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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.

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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.

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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