Biosynthesis of Resorcylic Acid Lactone Lasiodiplodin in Lasiodiplodia Theobromae
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Biosci. Biotechnol. Biochem., 73 (5), 1118–1122, 2009 Biosynthesis of Resorcylic Acid Lactone Lasiodiplodin in Lasiodiplodia theobromae y Takasumi KASHIMA, Kosaku TAKAHASHI, Hideyuki MATSUURA, and Kensuke NABETA Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Received December 10, 2008; Accepted January 26, 2009; Online Publication, May 7, 2009 [doi:10.1271/bbb.80878] The biosynthesis of lasiodiplodin (1) and its (5S)-5- as an example, the initial PKS was of the reducing hydroxylated derivative (2) were investigated by the type (R-PKS) named PKS4, containing minimal PKS administration of 13C-labeled acetates to Lasiodiplodia condensation domains of ketosynthase (KS), malonyl- theobromae. The labeling patterns of biosynthetically CoA:ACP acyltransferase (MAT), and acyl carrier 13C-labeled 1 and 2 were determined by 13C-NMR and protein (ACP); additional domains include those of INADEQUATE spectra, demonstrating the octaketide dehydratase (DH), enoylreductase (ER), and ketoreduc- origins of 1 and 2. Taking into account the biosynthetic tase (KR). The subsequent PKS was nonreducing PKS study of resorcylic acid lactones, the involvement of (NR-PKS) named PKS13, containing a putative starter highly reduced acyl intermediates in the biosynthesis of unit-ACP transacylase (SAT), a putative product tem- lasiodiplodins was presumed; thus, we synthesized 2H- plate (PT), and C-terminal thioesterase (TE) domains in labeled hypothetical acyl intermediates of 1, 9-hydrox- addition to the minimal domains. The biosynthetic ydecanoic acid (4) and its N-acetylcysteamine thioester system for zearalenone starts with the reduced hexake- (SNAC, 5). When L. theobromae was incubated with tide acyl chain being synthesized by PKS4 and trans- 5mM of a 2H-labeled intermediate, the 2H-label from ferred to PKS13. PKS13 then extends the acyl chain the intermediate was incorporated at the expected with three acetyl-CoA units, and then facilitates an aldol position of 1. These incorporation studies revealed that condensation to yield the resorcylic acyl intermediate. 1 was produced via a pathway which closely resembles Finally, it undergoes macrocyclization, resulting in the that of resorcylic acid lactone biosynthesis. production of zearalenone. In the case of hypothemycin, the polyketide skeleton was presumably synthesized Key words: lasiodiplodin; biosynthesis; resorcylic acid along almost the same pathway as that for zearalenone. lactone; Lasiodiplodia theobromae The structures of lasiodiplodins are classified as those of a resorcylic acid lactone; however the 12-membered Resorcylic acid lactones (RALs) are members of a macrolactone ring is two carbons shorter than with the unique family of macrolides that possess such potent other RALs just described. Taking into consideration this biological activities as radicicol (Hsp90 inhibitor),1) structural similarity, lasiodiplodins are probably synthe- LL-Z1640-2 (TAK1 inhibitor),2) hypothemycin (MAP sized via the same biosynthetic system as other RALs. kinase inhibitor),3) and zearalenone (estrogen receptor We demonstrate in this report that lasiodiplodin (1) agonist).4) Despite their structural similarity, a highly and (5S)-5-hydroxylasiodiplodin (2) were synthesized reduced 14-membered macrolactone with resorcylate via a polyketide biosynthetic pathway in L. theobromae (2,4-dihydroxybenzoate) in its lactone ring, these RALs by the incorporation of 13C-labeled acetate. The incor- are mycotoxins produced by a variety of different fungal poration of the chemically synthesized 2H-labeled species via polyketide biosynthesis.5) precursors into 1 clearly indicates that lasiodiplodin Lasiodiplodin 1 (Fig. 1) was first reported as a was biosynthesized by a similar biosynthetic system to metabolite produced by the fungus, Lasiodiplodia such other RALs as zearalenone and hypothemycin. theobromae, functioning as a potent plant growth inhibitor,6) and potent antileukemic activity was later Results and Discussion characterized.7) Lasiodiplodia theobromae is a well- known fungus that produces the plant hormone, jas- Lasiodiplodia theobromae was statically incubated in monic acid,6) as well as other physiologically active a 1% potato-glucose medium for 7 d. Three kinds of 13 13 13 13 compounds such as theobroxide and its derivatives C-labeled sodium acetate ([1- C], [2- C], and [ C2]) which are active in flower bud formation and/or potato were separately supplemented to the culture at a micro-tuber formation,8–11) and a variety of lasiodiplodin concentration of 10 mM. After an additional 10-d derivatives active in potato micro-tuber formation.12–15) incubation, the culture was filtered to separate the The biosynthesis of zearalenone and hypothemycin mycelia and supernatant. The mycelia and supernatant has recently been genetically characterized, indicating were extracted with EtOAc. After those extracts had that these RALs were mainly synthesized by only two been combined and evaporated, the resulting residue polyketide synthases (PKSs).16–19) Taking zearalenone was separated twice by PTLC, affording pure biosyn- y To whom correspondence should be addressed. Tel/Fax: +81-11-706-2505; E-mail: [email protected] Abbreviations: SNAC, N-acetylcysteamine thioester; RALs, resorcylic acid lactones; PKS, polyketide synthase; R-PKS, reducing PKS; KS, ketosynthase; MAT, malonyl-CoA:ACP acyltransferase; ACP, acyl carrier protein; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; NR-PKS, nonreducing PKS Lasiodiplodin Biosynthesis in Lasiodiplodia theobromae 1119 13 13 13 Table 2. Incorporation of Sodium [1- C], [2- C], and [ C2] Acetate into 1 13C-atom% C Position C 13 13 13 (acetylated) [1- C] [2- C] [ C2] JC{C acetate acetate acetate (Hz) 1 169.5 167.8 3.70 0.97 3.77 77.1 3 72.6 72.3 3.43 1.12 3.37 39.1 4 32.3 32.3 0.90 4.78 3.63 35.2 5 21.3 21.2 3.81 1.12 3.59 35.2 6 26.3 26.4 0.88 5.58 3.53 35.2 7 24.1 24.2 3.84 1.17 3.80 34.6 Fig. 1. Structures of Lasiodiplodin 1 and (5S)-5-Hydroxylasiodiplo- 8 25.4 25.4 0.88 5.61 3.45 34.6 din 2. 9 30.0 29.9 3.53 1.21 3.61 34.6 10 30.4 30.4 0.87 4.95 3.48 42.5 10a 142.9 142.5 3.51 1.00 2.93 42.5 Table 1. Administration Experiments Using the 13C-Labeled Acetate 11 108.4 114.5 0.87 5.41 3.32 67.0 and 2H-Labeled Acyl Intermediate of Compounds 1 and 2 12 157.9 151.9 3.69 1.18 3.51 67.0 Yield 13 97.0 102.7 0.93 5.05 3.61 70.4 Amount Concentration 14 157.9 157.2 3.41 1.05 3.40 70.4 Precursor (mg/150 ml) (mg/150 ml) (mM) 14a 117.0 122.6 0.94 4.59 3.73 77.1 12 15 19.5 19.4 0.88 6.02 3.82 39.1 Sodium [1-13C] acetate 125 10 14.4 4.7 PhOMe 55.7 56.0 1.01 3.41 2.67 Sodium [2-13C] acetate 125 10 6.3 2.2 CH3CO–Ph 21.3 1.11 1.11 1.11 13 CH3C(=O)–Ph 169.0 Sodium [ C2] acetate 126 10 6.6 4.4 Compound 4 144 5 14.4 Compound 5 219 5 1.6 13 13 13 Table 3. Incorporation of Sodium [1- C], [2- C], and [ C2] Acetate into 2 thetically 13C-labeled 1 and 2. The yields of 1 and 2 were in the range of 6.3–14.4 mg and 2.2–4.7 mg, 13C-atom% C respectively, per 150 ml of culture (Table 1). Position C 13 13 13 (acetylated) [1- C] [2- C] [ C2] JC{C The 13C{1H}-NMR spectra of [1-13C] acetate-derived acetate acetate acetate (Hz) 1 and 2 showed several enhanced signals at C-1, C-3, 1 168.3 167.5 3.53 1.23 4.76 77.1 C-5, C-7, C-9, C-10a, C-12, and C-14. In contrast, those 3 69.6 69.4 3.78 1.23 5.18 39.1 13 of [2- C] acetate-derived 1 and 2 revealed enhanced 4 40.4 36.9 0.85 5.79 4.93 38.5 signals at C-4, C-6, C-8, C-10, C-11, C-13, C-14a, and 5 66.7 69.8 3.99 1.22 5.19 38.5 C-15. Labeled acetate-derived 1 and 2 were then 6 35.7 31.9 0.90 5.96 4.69 34.1 acetylated, and the 13C abundance ratio was calculated, 7 22.1 21.5 3.78 1.38 5.47 34.6 referencing the relative intensity of the acetyl methyl 8 24.9 24.8 0.86 5.99 4.64 34.6 9 30.1 29.8 3.77 1.59 4.73 34.1 signal of the C-12 hydroxyl group as 1.11% of natural 10 30.1 30.0 1.02 5.40 4.88 43.6 13 abundance. The specific incorporation of Cto1 and 2 10a 143.0 142.3 3.47 1.07 3.89 42.5 was in the range of 3.4–6.0% and 3.5–6.0%, respectively 11 108.4 114.6 0.84 5.65 4.65 67.0 (Tables 2 and 3). Moreover, the 13C{1H}-NMR spec- 12 157.5 152.1 3.83 1.25 4.75 67.0 13 13 97.0 102.8 0.83 5.38 4.66 70.4 trum of [ C2] acetate-derived 1 and 2 showed intense 13C–13C coupling signals besides a methoxyl carbon 14 158.2 157.3 3.82 1.24 4.47 70.4 14a 117.4 122.2 0.89 5.96 5.00 77.1 signal at C-14.