Supplementalinformation
Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin biosynthesis and branched-chain α-keto acid dehydrogenase complex expression
Dong Wan Lee, Bee Gek Ng and Beom Seok Kim*
Plant Pharmacology Laboratory, Department of Biosystems and Biotechnology, College of Life Science & Biotechnology, KoreaUniversity, Seoul136-713, Republic of Korea
*Corresponding author: Beom Seok Kim. Division of Biotechnology, College of Life Science & Biotechnology, KoreaUniversity, Seoul136-713, Republic of Korea
Telephone: +82-2-3290-3047, Fax: +82-2-921-1715, E-mail:
Table S1 Primers used in this study for screening, disruption, complementation and RT-PCR of bkdABC and bkdFGH gene clustersPrimers / Nucleotide sequence / Predicted size
Screening
bkd-1F / 5'-GAATTCGGCGACGGCGCCACCTCCGAGGGCGAC-3' / 1.5 kb
bkd-1R / 5′-CGCCTCCAGATGGTGGAAGCAGCGCTCGGTGAT-3′
bkdF-F / 5′-AAGAATTCGAGCTCGGCGACGGCGCCACCTCCGAGGGCGAC-3′ / 0.3 kb
bkdF-R / 5′-AAGGATCCTCTAGAGGTSSWGTGGKGGCCGATSCGGWA-3′
Disruption
bafTE AprF / 5′- CCCGTACTCGTCTGCTTCGGCTCCGTCATG-ATTCCGGGGATCCGTCGACC-3′ / 1.5 kb
bafTE AprR / 5′-GAGCCAGTCGGCGACGGC-GTCGCCGGTTCATGTAGGCTGGAGCTGCTTC-3′
bafTE-F/R / 5′-ACGCCTCCGCCCTCCGTC-3′/ 5′-TCGTCGTACGGGGCCGCG-3′ / 0.9 kb
bkdA AprF / 5′- GAGCGGGCCAAGGAGGCCGCCGAGAGCATTCCGGGGATCCGTCGACC-3′ / 1.5 kb
bkdA AprR / 5′- AGACCGGCAGGGACGGGCCGTACGTGATGTAGGCTGGAGCTGCTTC-3′
bkdA-F/R / 5′- AGCGCGAGCTGACCGAGC -3′/ 5′- ACCAGCGAGCGCAGGTCC -3′ / 1.0 kb
bkdF AprF / 5′-TTATGCGATGCACTGGGTAACGTGCCTATGATTCCGGGGATCCGTCGACC-3′ / 1.5 kb
bkdF AprR / 5′-GGCGGGCGCGCTCCAGCGCGGAGCGGGTCATGTAGGCTGGAGCTGCTTC-3′
bkdF-F/R / 5′-ATCCGCGGGATAACGTGC-3′/ 5′-ACTTGGTCGGGTCGTCGG-3′ / 1.1 kb
Complementation
bkdRC-F/R / 5′-TATGGATCCAGAGACAGGATGGCAGCTGAACAAATGGCC-3′/5′-TTTCTAGATATAGAATTCTCAGGCCAGCTCCGTCGGGTG-3′ / 0.6 kb
RT-PCR
bkdF-RT-F/R / 5′-AGAAGCCGCAGAGTTCCGAG-3′/ 5′-ACATGCCCAGCAGGTTGGTG-3′ / 0.3 kb
16S rRNA-F/R / 5′-ACTGGGACTGAGACACGGCC-3′/ 5′-AGCTAGCCCGTATCGAATGC-3′ / 0.3 kb
Table S2 13C NMR and 1H NMR spectroscopic data for M10-1, 2, 3, 4, 5 and 6 (in CDCl3)
M10-1 & 2 / M10-3 & 4 / M10-5 & 6
Carbon / δ C / δ H (J in Hz) / δ C / δ H (J in Hz) / δ C / δ H (J in Hz)
1 / 167.5 / - / 167.4 / - / 167.5 / -
2 / 141.5 / - / 141.5 / - / 141.5 / -
2-OCH3 / 60.1 / 3.64 (3H, s) / 60.1 / 3.64 (3H, s) / 60.1 / 3.64 (3H, s)
3 / 133.6 / 6.67 (1H, s) / 133.6 / 6.66 (1H, s) / 133.6 / 6.65 (1H, br d, J=22.3)
4 / 133.2 / - / 133.1 / - / 133.2 / -
5 / 143.1 / 5.77 (1H, d, J=9.4) / 143.1 / 5.76 (1H, d, J=9.6) / 143.1 / 5.77 (1H, br d, J=9.2)
6 / 36.9 / 2.54 (1H, m) / 37 / 2.54 (1H, m) / 36.9 / 2.54 (1H, m)
7 / 81.4 / 3.30 (1H, d, J=7.5) / 81.3 / 3.29 (1H, d, J=6.4) / 81.4 / 3.29 (1H, d, J=6.3)
7-OH / - / 1.63 (1H, br s) / - / 1.61 (1H, br s) / - / 1.63 (1H, br s)
8 / 40.2 / 1.92 (1H, d, J=1.3) / 40.5 / 1.91 (1H, d, J=0.6) / 40.2 / 1.92 (1H, d, J=0.7)
9 / 41.4 / 2.15 (1H, m), 1.96 (1H, s) / 40.8 / 2.13 (1H, m), 1.96 (1H, d, J=0.6) / 41.4 / 2.13 (1H, m), 1.96 (1H, d, J=0.5)
10 / - / - / 133.1 / - / 133.1 / -
11 / 142.8 / 5.81 (1H, d, J=10.4) / 142.9 / 5.81 (1H, d, J=10.0) / 142.9 / 5.81 (1H, br d, J=9.7)
12 / 133.1 / 6.50 (1H, dd, J=14.8, 10.6) / 133.1 / 6.50 (1H, dd, J=14.1, 11.9) / 133.1 / 6.50 (1H, dd, J=14.3, 10.9)
13 / 127.5 / 5.17 (1H, dd, J=15.0, 9.5) / 127.4 / 5.17 (1H, dd, J=14.4, 9.4) / 127.4 / 5.16 (1H, br dd, J=15.2, 9.3)
14 / 82.4 / 3.89 (1H, t, J=8.9) / 82.3 / 3.85 (1H, ddd, J=21.5, 11.0, 5.0) / 82.4 / 3.88 (1H, t , J=8.8)
14-OCH3 / 55.7 / 3.25 (3H, s) / 55.7 / 3.24 (3H, s) / 55.7 / 3.24 (3H, s)
Table S2 (continued)
M10-1 & 2 / M10-3 & 4 / M10-5 & 6
Carbon / δ C / δ H (J in Hz) / δ C / δ H (J in Hz) / δ C / δ H (J in Hz)
15 / 77.2 / 4.96 (1H, d, J=7.7) / 77.2 / 4.94 (1H, d, J=8.4) / 77.2 / 4.95 (1H, d, J=8.4)
16 / 37.4 / 2.04 (1H, s) / 37.4 / 2.04 (1H, s) / 37.3 / 2.07 (1H, m)
17 / 70.8 / 4.14 (1H, d, J=10.3) / 70.9 / 4.13 (1H, d, J=10.8) / 70.8 / 4.13 (1H, br d, J=10.2)
17-OH / - / 4.62 (1H, s) / - / 4.56 (1H, s) / - / 4.64 (1H, s)
18 / 42.3 / 1.76 (1H, m) / 42.2 / 1.76 (1H, m) / 42.2 / 1.77 (1H, m)
19 / 99.1 / - / 99.0 / - / 99.0 / -
19-OH / - / 5.50 (1H, s) / - / 5.56 (1H, s) / - / 5.53 (1H, s)
19-OCH3a / - / 3.04 (1H, s) / - / 3.06 (1H, s) / - / 3.06 (1H, s)
20 / 41.2 / 2.30 (1H, dd, J=12.0, 4.6), 1.15 (1H, dd, J=14.2, 8.3) / 41.1 / 2.30 (1H, m), 1.14 (1H, m) / 41.4 / 2.33 (1H, m), 1.15 (1H, ddd, J=4.6, 1.0, 0.5)
21 / 77.1 / 3.70 (1H, s) / 77.1 / 3.70 (1H, s) / 77.0 / 3.70 (1H, s)
22 / 40.2 / 1.31 (1H, s) / 40.2 / 1.30 (1H, m) / 40.2 / 1.30 (1H, m)
23 / 76.0 / 3.50 (1H, d, J=6.4) / 75.6 / 3.49 (1H, s) / 75.8 / 3.47 (1H, d, J=10.6)
24 / 28.1 / 1.99 (1H, s) / 28.1 / 1.99 (1H, s) / 28.1 / 1.99 (1H, s)
25 / 12.3 / 0.86 (3H, d, J=6.6) / 12.5 / 0.86 (3H, d, J=6.4) / 12.5 / 0.86 (3H, d, J=6.4)
26 / 14.2 / 1.99 (3H, s) / 14.3 / 1.99 (3H, s) / 14.2 / 1.99 (3H, s)
27 / 17.4 / 1.07 (3H, d, J=7.0) / 17.4 / 1.07 (3H, d, J=6.7) / 17.4 / 1.07 (3H, d, J=6.5)
28 / 14.4 / 0.93 (3H, d, J=2.5) / 14.4 / 0.93 (3H, d, J=5.4) / 14.4 / 0.93 (3H, d, J=6.4)
Table S2 (continued)
M10-1 & 2 / M10-3 & 4 / M10-5 & 6
Carbon / δ C / δ H (J in Hz) / δ C / δ H (J in Hz) / δ C / δ H (J in Hz)
29 / 20.3 / 1.94 (3H, s) / 20.4 / 1.94 (3H, s) / 20.3 / 1.93 (3H, s)
30 / 10.0 / 0.83 (3H, d, J=6.8) / 10.0 / 0.83 (3H, d, J=8.2) / 10.0 / 0.83 (3H, d, J=6.4)
31 / 7.2 / 1.02 (3H, d, J=3.4) / 7.1 / 1.02 (3H, d, J=7.0) / 7.2 / 1.03 (3H, d, J=7.0)
32 / 21.8 / 0.99 (3H, d, J=6.6) / 21.7 / 0.99 (3H, d, J=3.0) / 21.8 / 1.00 (3H, d, J=7.4)
33 / 21.4 / 0.77 (3H, d, J=6.8) / 21.3 / 0.78 (3H, d, J=3.7) / 21.2 / 0.79 (3H, d, J=7.0)
1' / - / - / 164.5 / - / 164.3 / -
2' / - / - / 133.6 / 6.82 (1H, d, J=1.4) / 133.8 / 6.89 (1H, d, J=15.1)
3' / - / - / 133.2 / 7.05 (1H, d, J=1.6) / 133.6 / 7.12 (1H, d, J=15.3)
4' / - / - / 163.3 / - / 163.8 / -
4'-CONH / - / - / - / - / - / 8.30 (1H, s)
5' / - / - / - / - / 114.9 / -
6' / - / - / - / - / 174.9 / -
6'-OH / - / - / - / - / - / 13.34 (1H, m)
7' / - / - / - / - / 32.0 / 2.54 (m)
8' / - / - / - / - / 25.0 / 2.60 (2H, s)
9' / - / - / - / - / 197.5 / -
aNMR data of M10-2, 4 and 6 compounds; br, broad.
ORF / Geneproduct / No. of amino acid / Putative function / Nearest homologue
(enzyme, origin) / Identity (%) / Accession number
ORF1 / bafX / 530 / acyl-CoA ligase (partial) / Streptomyces griseus / 99 / AGK25217.1
acyl-CoA ligase / BafX (Streptomyces lohii) / 94 / ADC79613.1
ORF2 / bafY / 514 / amide synthetase / Streptomyces griseus / 99 / AGK25192.1
BafY (Streptomyces lohii) / 95 / ADC79614.1
ORF3 / bafZ / 414 / 5-aminolevulinate synthetase / Streptomyces griseus / 100 / AGK25193.1
BafZ (Streptomyces lohii) / 98 / ADC79615.1
ORF4 / bafI / 5007 / polyketide synthase I / Streptomyces griseus / 99 / AGK25194.1
PKS loading module + modules 1-3 / BafAI (Streptomyces lohii) / 92 / ADC79616.1
ORF5 / bafII / 5064 / polyketide synthase II / Streptomyces griseus / 99 / AGK25213.1
PKS modules 4-6 / BafAII (Streptomyces lohii) / 95 / ADC79617.1
ORF6 / bafIII / 3980 / polyketide synthase III / Streptomyces griseus / 99 / AGK25214.1
PKS modules 7,8 / BafAIII (Streptomyces lohii) / 94 / ADC79618.1
ORF7 / bafIV / 3614 / polyketide synthase IV / Streptomyces griseus / 98 / AGK25195.1
PKS modules 9,10 / BafAIV (Streptomyces lohii) / 90 / ADC79619.1
ORF8 / bafV / 2098 / polyketide synthase V / Streptomyces griseus / 99 / AGK25196.1
PKS module 11 + thioesterase / BafAV (Streptomyces lohii) / 92 / ADC79620.1
ORF9 / 296 / 3-hydroxyacyl-CoA dehydrogenase / Streptomyces griseus / 99 / AGK25191.1
glyceryl-ACP oxidoreductase / BafB (Streptomyces lohii) / 97 / ADC79621.1
ORF10 / 93 / acyl carrier protein (ACP) / Streptomyces griseus / 100 / AGK25197.1
BafC (Streptomyces lohii) / 97 / ADC79622.1
ORF11 / 363 / acyl-CoA dehydrogenase / Streptomyces griseus / 100 / AGK25198.1
acyl-ACP dehydrogenase / BafD (Streptomyces lohii) / 98 / ADC79623.1
ORF12 / 366 / FkbH-like protein / Streptomyces griseus / 99 / AGK25199.1
glycerate ACP ligase / BafE (Streptomyces lohii) / 95 / ADC79624.1
ORF13 / 220 / O-methyltransferase / Streptomyces griseus / 99 / AGK25200.1
BafF (Streptomyces lohii) / 96 / ADC79625.1
ORF14 / 609 / AfsR family transcriptional regulator / Streptomyces griseus / 99 / AGK25201.1
AfsR homologue / BafG (Streptomyces lohii) / 95 / ADC79626.1
ORF15 / 253 / thioesterase II / Streptomyces griseus / 100 / AGK25202.1
TEII / BafH (Streptomyces lohii) / 96 / ADC79627.1
ORF16 / 117 / LuxR-like protein / Streptomyces griseus / 99 / AGK25203.1
putative LuxR-like protein / Streptomyces lohii / 91 / ADC79628.1
ORF17 / 348 / malonyl transferase / Streptomyces griseus / 99 / AGK25204.1
putative malonyl transferase / Streptomyces lohii / 97 / ADC79629.1
ORF18 / 359 / putative CoA ligase / Streptomyces griseus / 99 / AGK25205.1
Streptomyces lohii / 97 / ADC79630.1
ORF19 / 486 / XRE family transcriptional regulator / Streptomyces griseus / 100 / AGK25206.1
Fig. S1UV absorption spectra of compound M10-1, 2, 3, 4, 5, and 6.
Fig. S2Organization of bafilomycin biosynthetic gene clusterinStreptomyces sp. M10 genome.
Fig. S3Architecture of modular polyketide synthases for bafilomycin biosynthesisinStreptomyces sp.M10.