Hybrid Transcription Factor Engineering Activates the Silent Secondary Metabolite Gene Cluster for (+)-Asperlin in Aspergillus nidulans

1

Hybrid Transcription Factor Engineering Activates the Silent Secondary Metabolite Gene Cluster for (+)-Asperlin

in Aspergillus nidulans

Michelle F. Grau

, Ruth Entwistle

, Yi-Ming Chiang

, Manmeet Ahuja

, C.

Elizabeth Oakley

, Tomohiro Akashi

, Clay C. C. Wang*

, Richard B. Todd*

, Berl R. Oakley*

†

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90089, United States

‡

Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, United States

§

Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan City 71710, Taiwan

∥

Division of OMICS analysis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan

⊥

Department of Chemistry, University of Southern California, Dornsife College of Letters, Arts, and Sciences, Los Angeles, California 90089, United States

#

Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center, Manhattan, KS 66506, United States

∇

Industrial Biotechnology Division, Reliance Technology Group, Reliance Industries Limited, Reliance Corporate Park, Thane Belapur Road, Ghansoli, Navi, Mumbai 400701, India.

*Corresponding authors: [email protected]; [email protected]; [email protected]

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Table of Contents

1. SUPPLEMENTAL METHODS S3 1.1 Isolation of Secondary Metabolites S3 1.2 Detailed Structural Characterization S4

2. FIGURES S6

Figure S1. Correct coding sequence for afoA and corresponding amino acid sequence of its protein product (AfoA). S6 Figure S2. Corrected annotation of AN11200. Intron is shown in red. S10 Figure S3. CAGE RNA-seq data for misannotated AN9221. S11 Figure S4. Coding sequence of alnR and predicted amino acid sequence. S12 Figure S5. Coding sequence of alnG and amino acid sequence of its predicted product. S14 Figure S6. HRESIMS spectra of (2Z, 4Z, 6E)-octa-2,4,6-trienoic acid (1) and (+)-Asperlin (2). S16 Figure S7. (2Z, 4Z, 6E)-octa-2,4,6-trienoic acid (1) and (+)-Asperlin (2)

1

H and

C assignments. S17 Figure S8.

H NMR of (2Z,4Z,6E)-octa-2,4,6-trienoic acid (1) in CDCl

(400MHz). S18

Figure S9.

C NMR of (2Z, 4Z, 6E)-octa-2,4,6-trienoic acid (1) in CDCl

(100MHz). S19

Figure S10.

H NMR of (+)-Asperlin (2) in CDCl

(400MHz). S20

Figure S11.

C NMR of (+)-Asperlin (2) in CDCl

(100MHz). S21

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1. SUPPLEMENTAL METHODS 1.1 Isolation of Secondary Metabolites

For scaling up to isolate compound 1, 1 L of LMM (20 125-mL flasks were used containing 50 mL of medium each) inoculated with 1.0 x 10

spores L

of A. nidulans strain LO4909 was incubated at 37°C with shaking at 180 rpm. For alcA(p) induction, 50mM of MEK was added to the culture(s) 42 h after inoculation. Culture medium were collected 72 h after induction by vacuum filtration. The culture medium partitioned with ethyl acetate (EtOAc; 1 L) after acidification by 1N HCl to pH = 3. The EtOAc layer was collected and evaporated in vacuo to yield compound 1 without further purification.

For scaling up to isolate compound 2, 2 L of LMM (2 2-Liter flasks were used containing 1 L of

medium each) inoculated with 1.0 x 10

spores L

of A. nidulans strain LO9721 was incubated

at 37°C with shaking at 180 rpm. For alcA(p) induction, 50 mM of MEK was added to the

culture(s) 42 h after inoculation. Culture medium and hyphae were collected 72 h after induction

by vacuum filtration. The culture medium partitioned with ethyl acetate (EtOAc; 2 L), and the

EtOAc layer was evaporated in vacuo (crude extract 184.7 mg). Thin Layer Chromatography

was carried out (Merck TLC Silica Gel 60 RP-C

F

glass plates 20 x 20 cm) on the crude

extract, with the correct compound identified by UV visualization. A razor blade was used to

scrape the silica containing the product off the plate. The silica was placed in a fritted funnel and

flushed with EtOAc. The filtrate was collected and the solvent was removed in vacuo resulting in

the isolation of (2) (114.4 mg).

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1.2 Detailed Structural Characterization

Compound 1 was isolated as a white amorphous powder. The molecular formula was found to be C

H

O

by its

H NMR,

C NMR (Figures S7-S9) and HRESIMS spectral data (Figure S6), representing four indices of hydrogen deficiency (IHD). The

H and

C NMR in CDCl

exhibited signals for three disubstituted olefins [δ

5.67, 5.97, 6.37, 6.62, 7.15, and 7.18 (each 1H); δ

116.0, 122.0, 126.0, 136.1, 138.7, 141.0], one carboxylic acid [δ

10.87 (1H, br s); δ

172.3], and one methyl group [1.85 (3H, br d, J = 6.8 Hz)]. This together with the molecular formula of 1 indicated that 1 is a linear trienoic acid. 2D NMR correlations (

H-

H COSY, gHMQC and gHMBC) also support the structure (data not shown). The double bond

configurations were determined to be 2Z, 4Z, and 6E based on the coupling constants of H-2 and H-3 (J = 11.3 Hz), H-3 and H-4 (J = 11.1 Hz), and H-4 and H-5 (J = 14.6 Hz). Therefore,

compound 1 was assigned as (2Z,4Z,6E)-octa-2,4,6-trienoic acid.

Compound 2 was isolated as a colorless oil. The molecular formula was found to be C

H

O

from HRESIMS (Figure S6) and both

H and

C NMR data (Figures S7, and S10-S11),

indicating 2 has four IHD.

C NMR spectrum of 2 exhibited one olefin (δ

124.8 and 140.5) and two ester or carboxylic acid carbonyl carbons (δ

161.46 and 169.70) in the down-field region.

1

H,

C, gHSQC NMR spectra indicated compound 2 contains two methyl [δ

1.36 (3H, d, J = 5.2 Hz), δ

17.0 (q) and δ

2.11 (3H, s), δ

20.5 (q)] and four oxymethine groups [δ

3.03 (1H, dd, J = 7.0, 2.1 Hz), δ

54.9 (d); δ

3.06 (1H, dq, J = 5.2, 2.1 Hz), δ

54.5 (d); δ

4.09 (1H, dd, J

=7.2, 2.8 Hz), δ

78.8 (d); and δ

5.29 (1H, dd, J = 5.7, 2.8 Hz), δ

62.1 (d)]. Because compound

2 has four IHD but only contains one olefin and two ester or carboxylic acid carbonyl carbons, 2

must contain a cyclic ether or an epoxide moiety. This, together with the fact that two

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oxymethine groups (δ

3.03, δ

54.8 and δ

3.06, δ

54.5) coupling to each other has a relatively high field chemical shift, indicated that 2 has an epoxide functional group. HSQC and long-range HMBC correlations allowed full assignment of the structure (Figure S7). Comparison of

H and

13

C NMR data of 2 with (+)-Asperlin in the literature

confirmed the identity of compound 2.

References:

1. Argoudelis, A. D.; Zieserl, J. F. The Structure of U-13,933, a New Antibiotic.

Tetrahedron Lett. 1966, 7 (18), 1969–1973.

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2. FIGURES

Figure S1. Correct coding sequence for afoA and corresponding amino acid sequence of its protein product (AfoA). Intron sequences are shown in red.

M A C P T R R G R Q Q P G F A C E ATGGCGTGTC CCACCAGACG AGGACGACAG CAGCCCGGCT TTGCATGCGA

1 ---+ ---+ ---+ ---+ ---+ 50 E C R R R K A R C D R V R P K C G

GGAGTGTCGC CGCCGCAAAG CGCGCTGTGA TCGCGTGCGT CCGAAATGCG

51 ---+ ---+ ---+ ---+ ---+ 100 F C T E N E L Q C V F V D K R Q

GGTTCTGCAC TGAGAATGAG CTGCAGTGTG TGTTCGTTGA CAAGAGGCAG

101 ---+ ---+ ---+ ---+ ---+ 150 Q R G P I K G Q I T S M Q S Q L A

CAGAGGGGTC CGATCAAAGG GCAGATCACC TCGATGCAGT CGCAGCTGGG

151 ---+ ---+ ---+ ---+ ---+ 200 TAGGTGTTTG TCTTGTCTCA TTGTATCTCG TCTCGTCTGC GCTTTTGTGA

201 ---+ ---+ ---+ ---+ ---+ 250 TTATGGGGCT GCCATGTTTC CGGTCCGGAC ACAGGCATCT GCAAGGCCCG

251 ---+ ---+ ---+ ---+ ---+ 300 CCGCTGTGCT CCCCCGATCT GCAGGGACCA ATGCAGCTGG TTCTGGAGCT

301 ---+ ---+ ---+ ---+ ---+ 350 TGTGCTGTGC TGCTTCCCTG TCTTTCCACA TGGTCGAGTC GAGCGAGCTA

351 ---+ ---+ ---+ ---+ ---+ 400

T L R W Q L D GCTAACATGG GATGCCTCAT GCTTTCAGCA ACGCTTCGAT GGCAGCTTGA

401 ---+ ---+ ---+ ---+ ---+ 450

R Y L R H R P P P S I T M A G E L TCGATACCTG CGACATCGAC CTCCCCCGTC CATAACCATG GCCGGCGAGC

451 ---+ ---+ ---+ ---+ ---+ 500 D E P P A D I Q T M L D D F D V

TCGATGAGCC ACCAGCGGAT ATCCAGACGA TGCTGGATGA CTTTGATGTA

501 ---+ ---+ ---+ ---+ ---+ 550 Q V A A L K Q D A T A T T T M S T

CAGGTCGCCG CGCTGAAGCA GGATGCCACG GCAACCACCA CAATGTCGAC

551 ---+ ---+ ---+ ---+ ---+ 600

S T A L M P A P A I S S K D A A P GTCGACAGCT CTCATGCCTG CCCCAGCCAT CTCATCTAAA GATGCTGCTC

601 ---+ ---+ ---+ ---+ ---+ 650

A G A G L S W P D P T W L D R Q CTGCTGGTGC TGGTTTATCG TGGCCTGACC CAACCTGGCT GGATCGCCAG

651 ---+ ---+ ---+ ---+ ---+ 700

7

W Q D V S S T S L V P P S D L T V TGGCAGGATG TCAGCAGTAC CAGCCTCGTC CCTCCATCAG ACCTGACAGT 701 ---+ ---+ ---+ ---+ ---+ 750

S S A T T L T D P L S F D L L N E CTCGTCGGCC ACTACCCTAA CCGACCCTCT CAGCTTCGAC CTTTTGAACG 751 ---+ ---+ ---+ ---+ ---+ 800

T P P P P S T T T T T S T T R R AGACTCCTCC TCCTCCTTCT ACGACGACAA CAACGTCGAC GACGAGGCGA

801 ---+ ---+ ---+ ---+ ---+ 850 D S C T K V M L T D L I R A E L

GACTCATGTA CTAAGGTCAT GTTAACTGAC CTCATCCGGG CTGAATTGTA

851 ---+ ---+ ---+ ---+ ---+ 900

CACTACCTAA CTGATTTGTC TACCATGACA CCTGACTGAC AATGTGCAGA

901 ---+ ---+ ---+ ---+ ---+ 950

D Q L Y F D R V H A F C P I I H R GACCAACTCT ACTTCGACCG GGTCCACGCC TTCTGCCCCA TCATCCACCG

951 ---+ ---+ ---+ ---+ ---+ 1000

R R Y F A R V A R D S H T P A Q A GCGACGGTAC TTTGCGCGGG TCGCCCGAGA TAGCCATACC CCAGCACAGG

1001 ---+ ---+ ---+ ---+ ---+ 1050

C L Q F A M R T L A A A M S A H CATGTCTGCA GTTCGCCATG CGAACGCTCG CAGCGGCAAT GTCTGCTCAC

1051 ---+ ---+ ---+ ---+ ---+ 1100 C H L S E H L Y A E T K A L L E T

TGCCATCTTA GCGAGCATCT CTATGCCGAG ACCAAGGCCC TCTTGGAGAC

1101 ---+ ---+ ---+ ---+ ---+ 1150

H S Q T P A T P R D K V P L E H I GCACAGCCAG ACGCCCGCCA CACCGCGAGA CAAGGTCCCG CTCGAGCACA

1151 ---+ ---+ ---+ ---+ ---+ 1200

Q A W L L L S H Y E L L R I G V TCCAGGCCTG GCTGTTGTTA AGCCACTACG AGCTGCTGCG GATCGGCGTG

1201 ---+ ---+ ---+ ---+ ---+ 1250

H Q A M L T A G R A F R L V Q M A CACCAGGCTA TGCTCACGGC TGGCCGGGCC TTTCGTCTCG TGCAGATGGC

1251 ---+ ---+ ---+ ---+ ---+ 1300

R L S E L D A G S D R Q L S P P S ACGACTGTCA GAGCTGGATG CCGGGTCAGA TCGACAGCTC TCGCCGCCGT

1301 ---+ ---+ ---+ ---+ ---+ 1350 S S P P S S L T L S P S G E N A

CTTCGTCGCC GCCGTCTTCG CTAACCCTAT CTCCTTCGGG GGAGAATGCT

1351 ---+ ---+ ---+ ---+ ---+ 1400

8

E N F V D A E E G R R T F W L A Y GAGAACTTCG TCGACGCCGA AGAAGGCCGG CGGACGTTCT GGCTTGCTTA

1401 ---+ ---+ ---+ ---+ ---+ 1450

C F D R L L C L Q N E W P L T L Q TTGCTTTGAT CGTTTGCTTT GCTTGCAGAA TGAGTGGCCG TTAACGTTAC

1451 ---+ ---+ ---+ ---+ ---+ 1500

E E M

AAGAAGAGAT GGTACGTCGC GCTTCTTTTA TTCTATTTAC CTCAGAATTT 1501 ---+ ---+ ---+ ---+ ---+ 1550

I L T R L P ATATTCAGTT ATTTTTTATT CTAACCCTGC TAGATATTAA CCCGCCTCCC

1551 ---+ ---+ ---+ ---+ ---+ 1600

S L E H N Y Q N N L P A R T P F L CTCCCTCGAA CACAACTACC AGAACAATCT CCCCGCACGC ACGCCCTTTC

1601 ---+ ---+ ---+ ---+ ---+ 1650

T E A M A Q T G Q S T M S P F A TCACTGAAGC CATGGCCCAG ACCGGGCAGA GCACAATGTC CCCGTTTGCC

1651 ---+ ---+ ---+ ---+ ---+ 1700 E C I I M A T L H G R C M T H R R

GAATGCATTA TCATGGCCAC CCTTCACGGC CGATGTATGA CGCACCGCCG

1701 ---+ ---+ ---+ ---+ ---+ 1750

F Y A N S N S T A S G S E F E S G CTTCTACGCA AACAGCAACT CGACTGCGTC CGGCTCCGAG TTCGAGTCTG

1751 ---+ ---+ ---+ ---+ ---+ 1800

A A T R D F C I R Q N W L S N A GCGCCGCGAC GCGAGACTTC TGTATCCGCC AGAATTGGCT GTCGAATGCA

1801 ---+ ---+ ---+ ---+ ---+ 1850

V D R R V Q M L Q Q V S S P A V D GTGGACCGGC GAGTCCAGAT GCTACAGCAG GTCTCCTCGC CCGCTGTTGA

1851 ---+ ---+ ---+ ---+ ---+ 1900

S D P M L L F T Q T L G Y R A T M CAGCGACCCG ATGCTGCTCT TCACGCAGAC GCTCGGCTAC CGCGCGACCA

1901 ---+ ---+ ---+ ---+ ---+ 1950

H L S D T V Q Q V S W R A L A S TGCACCTGAG CGATACCGTC CAGCAAGTCT CCTGGCGGGC TCTCGCCAGC

1951 ---+ ---+ ---+ ---+ ---+ 2000

S P V D Q Q L L S P G A T M S L S TCGCCCGTTG ACCAGCAGCT ACTGAGCCCG GGCGCGACGA TGTCGCTGTC

2001 ---+ ---+ ---+ ---+ ---+ 2050

A A A Y H Q M A S H A A G E I V R GGCCGCCGCG TACCACCAGA TGGCCAGCCA CGCAGCCGGC GAGATCGTCC

2051 ---+ ---+ ---+ ---+ ---+ 2100

9

L A K A V P S L S P F K A H P F GCCTGGCGAA GGCCGTCCCC TCGCTGAGTC CGTTCAAGGC GCACCCGTTC

2101 ---+ ---+ ---+ ---+ ---+ 2150

L P D T L A C A A T F L S T G S P CTACCCGATA CGTTGGCGTG CGCCGCCACG TTCCTCTCGA CGGGCAGTCC

2151 ---+ ---+ ---+ ---+ ---+ 2200

D P T G G E G V Q H L L R V L S E CGATCCCACG GGCGGCGAGG GGGTGCAGCA TCTGCTACGA GTGTTAAGCG

2201 ---+ ---+ ---+ ---+ ---+ 2250 L R D T H S L A R D Y L Q G L S

AGCTGCGCGA TACACACAGC CTGGCGCGGG ATTATTTGCA GGGGTTGTCG

2251 ---+ ---+ ---+ ---+ ---+ 2300

V Q T Q D E D H R Q D T R W Y C T GTGCAGACGC AGGACGAAGA TCATAGACAG GATACGAGGT GGTATTGTAC

2301 ---+ ---+ ---+ ---+ ---+ 2350

ATAG

2351 ---- 2354 TATC

10

Figure S2. Corrected annotation of AN11200. Intron is shown in red.

ATGTTCTCAA GTACCCGGCG GGTAAGTAAC TCTTTCCATC ATCTGGCCCA TCTTCTTTTC TTTTTTGTTT TCAATTGTAA GCTCTCGACT AACGACGCCC GGCACCTAGG CAGAAGGCCC CTGTGCAACC GAACTGACGC AGGTATCATC GCTGCTACCT CCGCGCGGGC CATACGAGTT CAGCCTCCTG CCAACACTCA CTCGACCGTT AGAGGACCTC TCGAAATGCA TCGAAGGTGC GAGACAGACC TCTGCGACTG CAAATGGTTA CAGCCCCACA GGGCTCGTCC CGCTAGCGGA TTCGATTCTG GAAATCTGTC AGGCTGCTTG TACAGCTTAT GGTCTTGTTG ACGGTGCTAT TGCTGCAGGT GTGGGTACAG GAAGCAGTGA TAATAGCCCT ACTGCCACAG GAATAGGAGC AGCAGGACTT ACAGGAGACC GCCCCTCCTC TTCCGGCGCA TCGACCTGGC GCTGTGTAAA AACCCCCATG ACGCTGGGAT CGCTTACGCT ACAGAATGAA GAAGAGTCGC TGCTCGCAAG GCAGATCGTG TACGCCGTGT TGACAAGCTT GAGCGCATTA CTGCGAGAAG TTTATGTTCG AGAGAAGGAC GTTGTTTCAG AGACTGATGT GGTGGGGGAA GGAGGGGTAG GAGCTGGAGC GGCACTGTAT GGGCGTGAAG GGGCTGGAGC CGTTAGTCAG TGTCTCTCGA GGGTTTTAGC GCTCTTGGGA AAGATAGTAC CTGAGTGA

11

Figure S3. CAGE RNA-seq data showing the transcription start site for alnR. The AspGD gene models for AN11192 and AN9221 are shown. Blue genes are transcribed right to left and orange genes are transcribed left to right. In CAGE RNA-seq the 5’ cap structures of mRNAs are captured and used for library construction. Sequencing of the library reveals transcription start sites. The CAGE RNA-seq library used in the current study was made from a mixture of mRNAs from two sources, wild-type hyphae cultured for one day at 37ºC in liquid glucose minimal medium and hyphae carrying a deletion of the mcrA gene grown for four days at 37ºC in liquid glucose minimal medium. Deletion of mcrA upregulates many genes including secondary metabolism genes.

Deletion of mcrA did not upregulate the (+)-asperlin cluster but there were enough reads in the region of the gene annotated as AN9221 to allow us to determine the transcription start site. The CAGE RNA-seq reads are shown as blue or orange lines below the gene models. The great majority of the reads in AN9221 map to the beginning of what was annotated as the third exon. This result indicates that the third exon is a separate transcription unit (i.e. gene). These and additional RNA-seq data reveal that AN9221 is actually two genes that we are now designating alnR and alnG.

Reference:

2. Oakley, C. E., M. Ahuja, W. W. Sun, R. Entwistle, T. Akashi, J. Yaegashi, C. J. Guo, G. C.

Cerqueira, J. R. Wortman, C. C. Wang, Y. M. Chiang, and B. R. Oakley. 2017. Discovery of

McrA, a master regulator of Aspergillus secondary metabolism. Mol Microbiol 103:347-365.

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Figure S4. Coding sequence of alnR along with predicted amino acid sequence. The position of the Zn(II)2Cys6 zinc binuclear cluster DNA-binding domain is underlined with the cysteine residues in bold.

M S T V N Q S S T R S E L A G N W

1 ATGAGCACGG TGAACCAATC TTCCACGCGT TCAGAGCTAG CCGGTAACTG 50 E R L R K S C D T C Q E A K V K C

51 GGAACGCCTG CGCAAGTCCT GCGATACCTG TCAGGAGGCC AAGGTCAAAT 100 S Q H K P S C H R C L R H R Q P

101 GCAGTCAACA CAAGCCGTCC TGCCACCGAT GCCTTCGACA TCGTCAGCCC 150 C V Y S P Q R R S G R P P K R P S

151 TGCGTCTACA GCCCGCAACG TCGGTCGGGA CGTCCTCCCA AGAGGCCCAG 200 P S S R L G P E S N N S G D D I H

201 TCCCTCCAGT CGCTTAGGAC CTGAATCAAA CAATTCCGGA GATGACATTC 250 N E N T I Q R T N L N A N D S A

251 ACAATGAAAA CACCATACAG CGAACGAATC TAAATGCCAA TGACTCTGCC 300 M T D A G A V D P R V L T G D F A

301 ATGACTGACG CCGGGGCAGT CGATCCCCGG GTGCTAACCG GCGACTTCGC 350 A S T G I D P V D D I F Q T S F E

351 CGCAAGTACT GGCATAGATC CTGTCGACGA TATCTTCCAA ACATCCTTTG 400 S F L A A S L S P K G G L L P G

401 AATCCTTCCT CGCAGCCTCA TTGTCTCCTA AAGGTGGACT CCTGCCAGGA 450 S H S N P T T P N G F S M N S P S

451 TCTCATAGCA ATCCAACCAC ACCCAACGGC TTCTCGATGA ATTCGCCCTC 500 I T D P F G A F P F L I T D H N L

501 CATCACTGAT CCATTCGGCG CCTTTCCGTT TCTCATAACG GACCACAACT 550 P I A A L S S H V P P I D Q L P

551 TGCCTATCGC CGCGCTCTCA TCGCATGTTC CTCCAATTGA TCAGCTACCC 600 V L S T G A S N T S S E C G D C G

601 GTACTAAGCA CCGGAGCCTC AAATACAAGC AGCGAGTGCG GCGACTGCGG 650 A K C Y S S L L Q H L L F L R Q T

651 TGCGAAGTGC TACAGCTCAC TATTACAGCA CCTTTTGTTC CTCCGCCAGA 700 L P E S T R P S I D V I M Q A E

701 CGCTCCCCGA GTCCACCAGG CCATCAATAG ACGTGATAAT GCAGGCTGAG 750 G H V R A L L D R V L G C N A C L

751 GGCCATGTGC GTGCTTTACT TGATCGGGTA TTAGGCTGCA ACGCATGCCT 800 G N R S S I L L I S A I T E R I V

801 TGGCAATCGG TCGTCTATCC TGCTCATATC AGCGATAACA GAGCGCATAG 850

13

Q M L D W I I E E K T L L D T E

851 TCCAGATGTT AGACTGGATC ATCGAGGAGA AGACTCTTTT GGATACCGAG 900 N M R Y N R R T F S S W G R P P R

901 AATATGCGTT ACAACCGACG AACGTTTAGT TCATGGGGTC GCCCTCCCCG 950 L P P H G L N G M R R N V C H V S

951 GTTACCACCT CATGGCCTTA ATGGTATGCG GAGGAACGTC TGCCACGTTT 1000 L R V G N T E L D E D A K Q Y F

1001 CACTTCGCGT GGGTAATACT GAATTGGATG AGGACGCCAA ACAGTATTTT 1050 L K N F I L L R L K K L A V K V Q

1051 CTTAAGAATT TCATTTTGCT TCGACTAAAG AAACTCGCAG TTAAGGTGCA 1100 E V R R T A T T R P G D C I Y R A

1101 GGAAGTGCGA CGGACAGCTA CCACCCGTCC TGGCGATTGC ATATACCGCG 1150 A E L V L A D S I Q R L D Y L R

1151 CTGCGGAATT GGTGCTGGCG GATTCGATTC AACGACTGGA TTATCTTCGT 1200 G Q C Q L W E *

1201 GGCCAGTGTC AGTTATGGGA GTGA 1224

14

Figure S5. Coding sequence of alnG and amino acid sequence of its predicted product. The intron is shown in red.

M T R Q I P L L A L S W L E L I F 1 ATGACGCGGC AAATCCCGCT CCTAGCGCTA TCGTGGCTTG AATTGATTTT 50 F S C Y Y G G L A G L G Y H S L W

51 CTTCAGCTGC TACTACGGCG GACTAGCGGG ACTGGGATAC CATTCCCTCT 100 R I A L R R R N V A P A I K S V

101 GGAGGATTGC ACTTCGCCGA AGGAATGTGG CACCCGCTAT CAAGTCTGTT 150 L Q T G R F A D G T P L T R R Y T

151 CTGCAGACTG GGCGCTTTGC GGATGGAACG CCCCTAACGC GCCGGTATAC 200 N L E F L D K K L V P A V I F Y D

201 TAACTTGGAA TTTTTGGATA AGAAATTGGT TCCTGCGGTA ATCTTCTACG 250 G L L T G A C P L Y R L L L V D

251 ACGGATTGTT GACTGGAGCA TGCCCACTTT ATCGCTTGTT ACTGGTGGAC 300 I H S T M Q A M A L C M L V S T R

301 ATCCATTCGA CCATGCAAGC GATGGCACTC TGCATGCTTG TCAGCACCAG 350 S K S L S T I S L L

351 ATCCAAGTCG TTATCGACTA TATCTTTGCT GTGAGTCGGG TCCTTCTGCC 400 L P T

401 TTTGAGTATA ACGAAGCTCT AATAATCTAC CGAGGGACAG CTTGCCAACT 450 F W N V F N Q F Y G A A F V Y P L

451 TTTTGGAATG TCTTCAACCA GTTTTACGGT GCTGCCTTCG TCTACCCCCT 500 Y L L L E A V T T G F N P L Y P V

501 CTACCTCTTA TTAGAGGCAG TAACGACTGG CTTTAACCCT CTGTATCCGG 550 E T E T S R S A L L V S A M I G

551 TCGAGACCGA GACATCTCGT TCTGCGTTAC TGGTGAGCGC TATGATCGGC 600 S F L P F T F L W P A F L R S G T

601 TCTTTTTTAC CGTTCACCTT TCTCTGGCCA GCTTTTCTTC GGTCTGGCAC 650 E S R Q R A I A L Y R F A P V V F

651 GGAGAGCCGA CAACGTGCTA TTGCATTATA CCGATTTGCT CCGGTAGTGT 700 S L L Q I V G E K V L G A Q M I

701 TCTCACTTCT GCAGATTGTT GGAGAGAAGG TGCTGGGCGC GCAGATGATC 750 P Q P T S Q A S P Y L V A G C A A

751 CCTCAGCCAA CTTCTCAGGC TAGCCCTTAT TTGGTTGCCG GCTGCGCTGC 800 T V G H W Y A L G G A L G L A M R

801 CACAGTGGGG CATTGGTACG CTCTTGGGGG AGCTTTAGGT CTCGCCATGC 850 L S H R K G R L G A L T L V L K

851 GGCTGTCTCA CAGAAAGGGC CGCTTGGGGG CTCTCACCTT AGTCCTCAAA 900

15

R L Y L P R S A E E T T R L D A S

901 CGGCTTTATC TGCCTCGCTC GGCTGAAGAA ACTACTCGCT TGGACGCCTC 950 V L A R A A H E F L Q Y D V L V L

951 TGTACTCGCT CGCGCAGCGC ACGAATTTCT GCAATACGAT GTCCTCGTGC 1000 I A A Y I P Y A Y Y L L A P L N

1001 TCATTGCAGC TTATATTCCG TACGCATACT ATCTGCTCGC GCCCCTCAAT 1050 L A S P F A M V V S L V L G T I F

1051 CTGGCATCGC CCTTTGCGAT GGTTGTGTCC CTTGTACTTG GCACCATTTT 1100 L G P G A V L A F A Y R V R W H L

1101 TTTAGGGCCG GGGGCGGTTC TGGCTTTCGC GTACCGGGTT CGCTGGCATC 1150 A I S D *

1151 TAGCTATCTC AGATTAG 1167

16

Figure S6. HRESIMS spectra of (2Z, 4Z, 6E)-octa-2,4,6-trienoic acid (1) (negative mode) and

(+)-asperlin (2) (positive mode).

17

Figure S7. (2Z,4Z,6E)-octa-2,4,6-trienoic acid (1) and (+)-asperlin (2)

H and

C assignments.

18

Figure S8.

H NMR of (2Z,4Z,6E)-octa-2,4,6-trienoic acid (1) in CDCl

(400 MHz).

19

Figure S9.

C NMR of (2Z,4Z,6E)-octa-2,4,6-trienoic acid (1) in CDCl

(100 MHz).

20

Figure S10.

H NMR of (+)-asperlin (2) in CDCl

(400 MHz).

21

Figure S11.

C NMR of (+)-asperlin (2) in CDCl

(100 MHz).