SUPPLEMENTARY INFORMATION
Alkyldihydropyrone s , new polyketide s synthesized by a type III polyketide synthase from Streptomyces reveromyceticus
Teruki Aizawa 1 , Seung-Young Kim 1 , Shunji Takahashi 2, 3 , Masahiko Koshita 1 , Mioka
Tani 1 , Yushi Futamura 3 , Hiroyuki Osada 2, 3 , and Nobutaka Funa 1, *
1 Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka,
Shizuoka, Japan
2 Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science,
Saitama, Japan
3 Antibiotics Laboratory, RIKEN, Saitama, Japan
*Corresponding author
Mailing address: 52-1, Yada, Suruga, Shizuoka 422-8526, Japan
Phone: (+81) 54-264-5552
Fax: (+81) 54-264-5555 e-mail: funa@u-shizuoka-ken.ac.jp

FIGURE LEGENDS OF SUPPLEMENTAL FIGURES
Supplemental figure S1. SDS-PAGE analysis of recombinant DpyA. The g el was stained with coomassie brilliant blue R-250. The soluble fraction and insoluble fraction prepared from S. lividans TK21 harboring pIJ4123-SRE2_11 are shown, together with the
DpyA purified by affinity chromatography with His-binding resin.
Supplemental figure S2. Chiral HPLC analysis of 3-hydroxyoctanoic acid
S -(2'-acetamidoethyl) ester (5). CHIRALCEL OD-3 (4.6  150 mm; Daicel Co.,
Osaka, Japan) was eluted using hexane/isopropylalcohol (9:1, v/v) at a flow rate of 0.6 ml/min.
Supplemental figure S3. Kinetic analysis of DpyA. Hanes-Woolf plots of three independent experiments for ( R )5 ( a , b , c ) and 6 ( d , e , f ) are shown.
Supplemental figure S 4 . Phylogenetic tree of type III polyketide synthases.
Using DpyA sequence as a query, 50 hits with an E-value less than 1  e -39 were selected. Phylogenetic tree of the selected proteins with PhlD 1 , SrsA 2 , and RppA 3 was constructed by CLUSTALW (http://clustalw.ddbj.nig.ac.jp) and was drawn by Njplot.
Functionally characterized proteins, DpyA, GCS 4 , Cpz6 5 , PhlD, RppA, Fur1 6 , SaRppA 7 ,
SrsA are highlighted. LpmD 8 and LipD 9 are identified in the biosynthetic gene cluster of caprazamycin-related compounds. DNA sequence of DpyA has been deposited at
DDBJ/EMBL/GenBank under the project accession AB898686.
Supplemental figure S 5 . 1 H-NMR spectrum of
4-hydroxy-6-isopentyl-3-methyl-5,6-dihydro-2 H -pyran-2-one (1) in CD
3
OD.
Supplemental figure S 6 . 13 C-NMR spectrum of
4-hydroxy-6-isopentyl-3-methyl-5,6-dihydro-2 H -pyran-2-one (1) in CD
3
OD.
Supplemental figure S 7 . 1 H13 C-HSQC-NMR spectrum of
4-hydroxy-6-isopentyl-3-methyl-5,6-dihydro-2 H -pyran-2-one (1) in CD
3
OD.

Supplemental figure S 8 . 1 H13 C-HMBC-NMR spectrum of
4-hydroxy-6-isopentyl-3-methyl-5,6-dihydro-2 H -pyran-2-one (1) in CD
3
OD.
Supplemental figure S 9 . 1 H-NMR spectrum of
4-hydroxy-3-methyl-6-pentyl-5,6-dihydro-2 H -pyran-2-one (2) in CD
3
OD.
Supplemental figure S 10 . 13 C-NMR spectrum of
4-hydroxy-3-methyl-6-pentyl-5,6-dihydro-2 H -pyran-2-one (2) in CD
3
OD.
Supplemental figure S 11 . 1 H13 C-HSQC-NMR spectrum of
4-hydroxy-3-methyl-6-pentyl-5,6-dihydro-2 H -pyran-2-one (2) in CD
3
OD.
Supplemental figure S 12 . 1 H13 C-HMBC-NMR spectrum of
4-hydroxy-3-methyl-6-pentyl-5,6-dihydro-2 H -pyran-2-one (2) in CD
3
OD.
Supplemental figure S 13 . 1 H-NMR spectrum of
4-hydroxy-3-methyl-6-(3'-methylpentyl)-5,6-dihydro-2 H -pyran-2-one (3) in
CD
3
OD.
Supplemental figure S 14 . 13 C-NMR spectrum of
4-hydroxy-3-methyl-6-(3'-methylpentyl)-5,6-dihydro-2 H -pyran-2-one (3) in
CD
3
OD.
Supplemental figure S 15 . 1 H13 C-HSQC-NMR spectrum of
4-hydroxy-3-methyl-6-(3'-methylpentyl)-5,6-dihydro-2 H -pyran-2-one (3) in
CD
3
OD.
Supplemental figure S 16 . 1 H13 C-HMBC-NMR spectrum of
4-hydroxy-3-methyl-6-(3'-methylpentyl)-5,6-dihydro-2 H -pyran-2-one (3) in
CD
3
OD.
Supplemental figure S 17 . 1 H-NMR spectrum of
4-hydroxy-3-methyl-6-(4'-methylpentyl)-5,6-dihydro-2 H -pyran-2-one (4) in

CD
3
OD.
Supplemental figure S 18 . 13 C-NMR spectrum of
4-hydroxy-3-methyl-6-(4'-methylpentyl)-5,6-dihydro-2 H -pyran-2-one (4) in
CD
3
OD.
Supplemental figure S 19 . 1 H13 C-HSQC-NMR spectrum of
4-hydroxy-3-methyl-6-(4'-methylpentyl)-5,6-dihydro-2 H -pyran-2-one (4) in
CD
3
OD.
Supplemental figure S 20 . 1 H13 C-HMBC-NMR spectrum of
4-hydroxy-3-methyl-6-(4'-methylpentyl)-5,6-dihydro-2 H -pyran-2-one (4) in
CD
3
OD.
Supplemental figure S 21 . Selected HMBC collerations of alkyldihydropyrones (1
– 4).
Supplemental figure S 22 . Chiral HPLC analysis of in vitro reaction of DpyA.
CHIRALCEL OD-3 (4.6  150 mm; Daicel Co., Osaka, Japan) was eluted using hexane/isopropylalcohol (9:1, vol/vol) at a flow rate of 0.6 ml/min. The reactions are performed according to the condition described in “ In vitro reaction of DpyA” of
MATERIALS AND METHODS section. The enzymes and substrates used were;
DpyA, (±)5 , and methylmalonyl-CoA ( a ); DpyA, ( S )5 , and methylmalonyl-CoA ( c );
DpyA, ( R )5 , and methylmalonyl-CoA ( e ). Chromatograms of authentic (±)5 ( b ),
( S )5 ( d ), and ( R )5 ( f ) are also shown. ( S )5 probably decreased due to the recovery rate of the extraction from the reaction mixture.
Supplemental figure S 23 . HPLC chromatograms of in vitro reactions of DpyA.
Compound 6 ( 100 µM) and methylmalonyl-CoA (100 µM) were used as substrates.
Compound 7 was produced in the reaction using recombinant DpyA ( a ), whereas the reaction did not proceed when boiled enzyme was used ( b ). Compound 7 was determined by comparison of retention time and UV spectrum with authentic sample that was prepared from a recombinant Streptomyces expressing a type III polyketide

synthase from S. reveromyceticus (unpublished result).
4-hydroxy-3-methyl-6-pentyl-2 H -pyran-2-one ( 7 ). 1 H NMR (400 MHz, CDCl
3
): 
0.86 (t, 3H, J = 6.9 Hz, C5'H
3
), 1.26 (m, 2H, C3'H
2
), 1.28 (m, 2H, C4'H
2
), 1.61 (m, 2H,
C2'H
2
), 1.94 (s, 3H, C3-CH
3
), 2.42 (t, 2H, J = 7.7, C1'H
2
), 6.01 (s, 1H, C5H), 8.41 (br s,
1H, C4-OH); 13 C NMR (100 MHz, CDCl
3
):  8.1 (C3-CH
3
), 13.9 (C5'), 22.3 (C4'), 26.5
(C2'), 31.1 (C3'), 33.5 (C1'), 98.5 (C3), 100.0 (C5), 163.6 (C6), 165.5 (C2), 167.5 (C4);
Positive mode DART/TOF-MS, m/z 197.11503 (calcd for C
11
H
17
O
3
, 197.11777).
Supplemental figure S24. HPLC chromatograms of in vitro reactions of DpyA from acyl-CoA substrates. 100 µM of hexanoyl-CoA and 100 µM methylmalonyl-CoA ( a ); 100 µM of hexanoyl-CoA and 100 µM malonyl-CoA ( b ); 100
µM of hexanoyl-CoA, 100 µM malonyl-CoA, and 100 µM methylmalonyl-CoA ( e ); 10
µM of hexanoyl-CoA, 10 µM malonyl-CoA, and 10 µM methylmalonyl-CoA ( f ); were used as substrates. Cromatograms of authentic 7 ( d ) and 7 spiked with the reaction from 100 µM of hexanoyl-CoA, 100 µM malonyl-CoA, and 100 µM methylmalonyl-CoA ( c ) are also shown. The structures of the unknown products are postulated.
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