The Biosynthetic Gene Cluster for Sestermobaraenes—Discovery of a Geranylfarnesyl Diphosphate Synthase and a Multiproduct Sesterterpene Synthase from Streptomyces mobaraensis

Abstract A biosynthetic gene cluster from Streptomyces mobaraensis encoding the first cases of a bacterial geranylfarnesyl diphosphate synthase and a type I sesterterpene synthase was identified. The structures of seven sesterterpenes produced by these enzymes were elucidated, including their absolute configurations. The enzyme mechanism of the sesterterpene synthase was investigated by extensive isotope labeling experiments.

The mass spectra on the left are from compounds in CLSA headspace extracts and the mass spectra on the right are from enzyme products. Figure S2 (continued). EI mass spectra of A) sestermobaraene A (6), B) sestermobaraene B (7), C) sestermobaraene C (8), D) sestermobaraene D (9), sestermobaraene E (10), F) sestermobaraene F (11), G) sestermobaraol (12), and further unknown compounds (a, b, c). The mass spectra on the left are from compounds in CLSA headspace extracts and the mass spectra on the right are from enzyme products.  Figure S3. A) Phylogenetic tree constructed from 3267 amino acid sequences of bacterial terpene synthase homologs using the tree builder function of Geneious (alignment type: global alignment with free end gaps, cost matrix: Blosum45, genetic distance model: Jukes-Cantor, tree build method: neighbor-joining, gap open panelty: 8, gap extension penalty: 2). The largest branches representing functionally characterized enzymes [2][3][4][5][6][7][8][9][10][11][12][13][14] and their closest relatives with likely the same function are shown in green and blue. B) Expansion for the branch containing SmTS1 and its closest relatives. The red arrows indicate the terpene synthases from S. mobaraensis. The scale bar indicates the number of substitutions per site.

Compound purification
The crude products obtained from the preparative scale enzyme incubation were purified via silica gel chromatography to afford pure 10 (0.4 mg) and 12 (0.7 mg). Compound 6 (0.9 mg) was obtained via HPLC purification. HPLC purifications were performed on a Smartline series HPLC system (Knauer, Berlin, Germany), equipped with a UV/Vis-Detector S-2550 (190-1000 nm) and a Knauer Eurospher II 100-5 C18P column (5 m; 8 × 250 mm). Elution was performed with acetonitrile at 6 mL/min (86 bar). The UV/Vis absorption was monitored at 205 nm. The other compounds were purified via repeated preparative TLC using AgNO3 coated TLC plates. The AgNO3 coated TLC plates were obtained by treatment of commercial TLC plates (TLC Silica gel 60 F254, 20 x 20 cm, Merck, Darmstadt, Germany) with a solution of AgNO3 in methanol (5 g/100 mL) for 10 min, followed by drying at 65 °C for 40 min. After TLC separation with a mixture solvent of cyclohexane and ethyl acetate, a small stripe of the TLC plate was cut off and stained with molybdophosphoric acid in EtOH (10 g/100 mL). The silica of regions containing the target compounds was scratched off and extracted with diethyl ether. After evaporation of the solvent, the pure compounds 7 (1. IR spectroscopy and optical rotations IR spectra were recorded on an ALPHA II FTIR Spectrometer (Bruker Optics, MA, USA), and the scan range was set to 500 to 4000 cm -1 . Optical rotations were recorded on a MCP 150 Modular Circular Polarimeter (Anton Paar GmbH, Graz, Austria).
[c] For assignment of H and H cf. Figure S101. [a] Carbon numbering as shown in Figure S15. [b] Chemical shifts  in ppm, multiplicity: s = [a] Carbon numbering as shown in Figure S23. [b] Chemical shifts  in ppm, multiplicity: s = [a] Carbon numbering as shown in Figure S31. [b] Chemical shifts  in ppm, multiplicity: s = singlet, d = doublet, q = quartet, br =broad, m = multiplet, coupling constants J are given in [a] Carbon numbering as shown in Figure S39. [b] Chemical shifts  in ppm, multiplicity: s = [a] Carbon numbering as shown in Figure S47. [b] Chemical shifts  in ppm, multiplicity: s = [a] Carbon numbering as shown in Figure S55. [b] Chemical shifts  in ppm, multiplicity: s = singlet, d = doublet, q = quartet, m = multiplet, coupling constants J are given in Hertz.
[c] For assignment of H and H cf. Figure S107.

S81
S82 Figure S65. Enzymatic conversion of (3-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (3-13 C)GFPP.
S83 Figure S66. Enzymatic conversion of (4-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (4-13 C)GFPP.
S84 Figure S67. Enzymatic conversion of (5-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (5-13 C)GFPP.
S85 Figure S68. Enzymatic conversion of (6-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (6-13 C)GFPP.
S86 Figure S69. Enzymatic conversion of (7-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (7-13 C)GFPP.
S87 Figure S70. Enzymatic conversion of (8-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (8-13 C)GFPP.
S88 Figure S71. Enzymatic conversion of (9-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (9-13 C)GFPP.
S89 Figure S72. Enzymatic conversion of (10-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (10-13 C)GFPP.
S90 Figure S73. Enzymatic conversion of (11-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (11-13 C)GFPP. Figure S74. Enzymatic conversion of (12-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (12-13 C)GFPP.

S91
S92 Figure S75. Enzymatic conversion of (13-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (13-13 C)GFPP.
S93 Figure S76. Enzymatic conversion of (14-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (14-13 C)GFPP.
S94 Figure S77. Enzymatic conversion of (15-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (15-13 C)GFPP.
S95 Figure S78. Enzymatic conversion of (16-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (16-13 C)GFPP.
S96 Figure S79. Enzymatic conversion of (17-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (17-13 C)GFPP.
S97 Figure S80. Enzymatic conversion of (18-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (18-13 C)GFPP.
S98 Figure S81. Enzymatic conversion of (19-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (19-13 C)GFPP.
S99 Figure S82. Enzymatic conversion of (20-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (20-13 C)GFPP. S100 Figure S83. Enzymatic conversion of (21-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (21-13 C)GFPP. S101 Figure S84. Enzymatic conversion of (22-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (22-13 C)GFPP. S102 Figure S85. Enzymatic conversion of (23-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (23-13 C)GFPP. S103 Figure S86. Enzymatic conversion of (24-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (24-13 C)GFPP. S104 Figure S87. Enzymatic conversion of (25-13 C)GFPP with SmTS1. Coloured dots indicate labeled carbons and the corresponding peaks in the 13 C-NMR spectra. Figures A) -G) show the 13 C-NMR spectra of unlabeled 6 -12, H) shows the 13 C-NMR spectrum of the enzyme products from (25-13 C)GFPP.