Combinatorial Biosynthesis of (+)-Daurichromenic ... - ACS Publications

May 25, 2017 - (20) Nirma, C.; Eparvier, V.; Stien, D. J. Nat. Prod. 2015, 78, 159−. 162. (21) Wipf, P.; Weiner, W. S. J. Org. Chem. 1999, 64, 5321â...
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Combinatorial Biosynthesis of (+)-Daurichromenic Acid and Its Halogenated Analogue Masahiro Okada,*,† Kai Saito,† Chin Piow Wong,† Chang Li,† Dongmei Wang,† Miu Iijima,‡ Futoshi Taura,‡ Fumiya Kurosaki,‡ Takayoshi Awakawa,† and Ikuro Abe*,† †

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Sugitani, Toyama 930-0194, Japan



S Supporting Information *

ABSTRACT: Daurichromenic acid is a meroterpenoid with various pharmacological activities that is biosynthesized from grifolic acid in Rhododendron dauricum. Heterologous expression of grifolic acid synthases from Stachybotrys bisbyi and a daurichromenic acid synthase from R. dauricum in Aspergillus oryzae mediated three-step combinatorial biosynthesis of (+)-daurichromenic acid through enantioselective 6-endo-trig cyclization. Additional introduction of a halogenase from Fusarium sp. into the strain resulted in the biosynthesis of (+)-5-chlorodaurichromenic acid, which exceeds the antibacterial activity of the original compounds.

T

in biosynthetic studies of fungal natural products.13 Notably, LL-Z1272β is biosynthesized from grifolic acid, similar to 1, through StbB-catalyzed reduction of the carboxylic acid of grifolic acid to form aldehyde. The stbA is the PKS gene responsible for the synthesis of orsellic acid from acetyl-CoA and three molecules of malonyl-CoA and the stbC is UbiA-like prenyltransferase gene14 responsible for the synthesis of grifolic acid from orsellic acid and farnesyl diphosphate. A strain coexpressing stbA and stbC was already prepared in that study, and it was confirmed that the transformant efficiently produced grifolic acid (2) in its mycelia. Then, in order to construct the platform to produce 1, we planned to express the gene encoding DCA synthase (DCAS, accession number: LC184180) derived from R. dauricum in the A. oryzae expressing stbA and stbC, which have some homology to cannabinoid synthase, a flavin adenine dinucleotide oxidase.15 Another benefit of heterologous expression is the possible production of unnatural compounds by constructing artificial biosynthetic pathway with introducing additional biosynthetic genes into the host. Herein, we report the heterologous production of DCA (1) in A. oryzae via combination of fungal grifolic acid synthases and plant DCAS (Scheme 1). Further production of unnatural analogues, chlorinated DCA (3) and chlorinated grifolic acid, named ilicicolinic acid (4),16 was also accomplished by introducing a halogenase17 derived from fungus Fusarium sp., a strain found in our laboratory that produces chlorinated meroterpenoids, into the DCA producer strain.18 These chlorides 3 and 4 exceeded original compounds 1 and 2 on antibacterial activity.

raditional medicinal plants are one of the most attractive sources of naturally derived drugs, and a number of revolutionary drugs and promising drug leads originated from plants. Classes of compounds such as alkaloids, nonribosormal peptides (NRPs), polyketides (PKs), terpenoids, and meroterpenoids are particularly appealing.1 In many cases, supply of the plant sources plagued many researchers, hindering further scientific developments. Scarcity and designation as protected plant species often causes inaccessibility or reduction in supply for researchers. Daurichromenic acid (DCA, 1) is one of the meroterpenoids2 and was isolated from the leaves of Rhododendron dauricum collected in Hokkaido, Japan.3 The Rhododendron species including R. dauricum has been used in traditional Chinese medicinal herb for treatment of various diseases.4 DCA (1) was originally identified as an antibacterial compound for Gram-positive bacteria,3 and 1 has attracted remarkable attention because it exhibits various pharmacological activities,5 in particular, highly potent anti-HIV activity.6 However, R. dauricum is designated as a protected species in Hokkaido; therefore, unrestricted supply and economical preparation of 1 is desirable for further pharmacological studies and clinical applications. Previously reported enantioselective synthesis of 1 involves complex synthetic scheme with over 10 steps. To develop an improved method to synthesize 1, we focused on the combinatorial biosynthesis in microorganisms.8 DCA (1) is biosynthesized from grifolic acid (2) through cyclization of the farnesyl moiety to form a chromene skeleton.9 Recently, we identified the biosynthetic gene cluster of LLZ1272β10 (also known as ilicicolin B) from the fungus Stachybotrys bisbyi PYH05-711 by using a heterologous expression of the stbA-C genes (accession no. LC125467) in Aspergillus oryzae NSAR1,12 which has been successfully applied © 2017 American Chemical Society

Received: May 2, 2017 Published: May 25, 2017 3183

DOI: 10.1021/acs.orglett.7b01288 Org. Lett. 2017, 19, 3183−3186

Letter

Organic Letters Scheme 1. Schematic Representation of Engineered Biosynthesis of DCA and Its Analogues

Successively, absolute stereochemistry of the purified 1 was determined by a chiral LC−HRMS analysis. As a result, it was confirmed that the biosynthesized DCA was the (3′S)(+)-form, which is same as naturally occurring DCA (Figure 2). In addition, optical rotation of biosynthesized DCA was

In order to obtain 1, we prepared an expression vector harboring a DNA fragment, with optimized codon for Aspergillus species, encoding DCAS, and the vector was transformed into the A. oryzae harboring stbA and stbC. After the resulting transformant was cultivated in induction medium culture, mycelial extract was analyzed by HPLC. As a result, a new metabolite was detected in addition to grifolic acid (2) (Figure 1A,C). Comparison of the retention time with

Figure 2. Chiral LC−HRMS profiles of authentic chiral DCA enantiomers [(A) (+)-DCA, (B) (−)-DCA], and (C) purified DCA from bioengineered strain. These spectra are negative-extracted ion chromatography (EIC) of m/z 369.21 ± 0.1 (calcd for C23H29O4, 369.2071).

measured, and the value [α]D +30.6 (c 0.40, CHCl3) was almost same as that of naturally occurring DCA [lit.6 [α]D +30.4 (c 0.46, CHCl3)]. The reaction catalyzed by DCAS corresponds to 6-endo-trig Wacker-type cyclization initiated by deprotonation of the 1′proton.8 Notably, such cyclization is difficult to accomplish with a chemical synthetic method because this cyclization usually proceeds in a 5-exo-trig fashion to give 2,3-dihydrobenzofuran skeleton instead of 2H-chromene (2H-1-benzopyran) skeleton. Furthermore, enantioselective 6-endo-trig Wacker-type cyclization is extremely difficult. Actually, only one report including enantioselective 6-endo-trig Wacker-type cyclization to produce the 2H-chromene (2H-1-benzopyran) skeleton, moreover, with moderate enantioselectivity, has been published.19 Therefore,

Figure 1. HPLC (UV 254 nm) of authentic samples [(A) grifolic acid (2) and (B) DCA (1)] and mycelial extract of A. oryzae transformants harboring genes ((C) stbA, stbC, and DCAS and (D) stbA, stbC, DCAS, and AscD).

authentic DCA (Figure 1B,C) and the HRMS and NMR analyses confirmed that the new metabolite 1 was DCA. Notably, any other metabolites derived from 2, besides 1 (and 2 itself), were not obtained from the extract. The optimization of cultivation condition resulted in the titers of 1.23 mg/L for 1, and purification from the mycelial extract afforded analytically pure 1. 3184

DOI: 10.1021/acs.orglett.7b01288 Org. Lett. 2017, 19, 3183−3186

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Organic Letters

MIC value of 3.13 μg/mL and S. aureus with an MIC value of 6.25 μg/mL. In addition, 3 was also found to possess a weak antimicrobial activity against Candida albicans at 100 μg/mL, where 1, 2, and 4 are inactive at similar concentrations. Our results indicated that antibacterial activity against B. subtilis and S. aureus of chlorinated compounds 3 and 4 was enhanced by 2fold, compared to their nonchlorinated compounds 1 and 2, respectively. The weak activity of 3 against C. albicans could also be attributed to chlorination. Taken together, our efforts in generating chlorinated derivatives of 3 and 4 by introducing a halogenase derived from Fusarium sp. into the DCA producer strain successfully improved the antibacterial activity. In conclusion, simple preparation of (+)-1, which could be hardly realized by chemical synthesis, was accomplished by redesign of biosynthetic pathway. In addition, a novel analogue (+)-3 with more active antibacterial activity than original compound was created through a halogenation. Although the enzyme effectively worked with structurally altered substrates, to obtain more the compound, optimization of expression level of each enzyme is required. This is the first study to construct an artificial biosynthetic pathway in A. oryzae using biosynthetic genes derived from a plant. Our method opens a new path for synthetic biology to produce bioactive compounds.

we have accomplished the total synthesis of (+)-DCA through enantioselective 6-endo-trig Wacker-type cyclization by redesign of biosynthetic pathway. Next, in order to apply our approach to produce new molecules, we conceived that a novel DCA analogue was biosynthesized by expressing a gene encoding additional tailoring enzyme into the DCA producer strain. Therefore, we chose a halogenase as a tailoring enzyme because it was previously reported that a number of 5-chlorinated meroterpenoids derived from orsellic acid or its analogues possessed a wide range of biological activities20 and 4 exhibited slightly higher inhibitory activity against glucose-6-phosphatase than 1.16 After a halogenase (AscD) (accession no. LC228576) derived from Fusarium species was introduced and heterologously expressed in the DCA producer strain,18 the resulting transformant was cultivated in the supplemented culture medium with 5% NaCl. As a result of LC−HRMS analyses for the mycelial extract, two chlorinated metabolites were detected in addition to a small amount of DCA (1) and grifolic acid (2) (Figure 1A,D and Figure S1). The molecular formula of the major product 3 was confirmed to be C23H29ClO4 according to the HRMS, and the UV spectrum of 3 was almost the same as that of DCA (1). 1H and 13C NMR spectra of 3 were indeed very similar to those of 1 except for the disappearance of the 1H signal corresponding to the 5 position. Further the two-dimensional NMR analyses successfully established the chemical structure of 3 to be 5-chloro DCA. Similarly, another minor product 4 was confirmed to be 5chlorogrifolic acid, based on the HRMS, UV, and NMR analyses. Successively, absolute stereochemistry of 3 was analyzed by a chiral HPLC−HRMS analysis. As a result, it was revealed that the biosynthesized 3 is the (+)-form which is same as naturally occurring DCA with the 97:3 ratio of (+)/(−)-3 on the basis of the peak area in the EIC spectrum (Figure S4). The incomplete enantioselectivity is probably attributed to racemization through retro-Claisen rearrangement7,21 rather than a different substrate 4 from the original grifolic acid (2). Notably, 3 is not so stable in some cases; for example, 3 was decomposed during the purification by silica gel column chromatography with a CHCl3−MeOH solvent system. However, a simple two-step purification from the mycelial extract using only CH3CN as a solvent afforded analytically pure 3 with titers of 2.06 mg/L broth. The antimicrobial activity of the compounds 1−4 was investigated by measuring the minimum inhibitory concentration (MIC) according to the Clinical & Laboratory Standards Institute protocol.22 All compounds 1−4 exhibited antimicrobial activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus but were inactive against Gram-negative Escherichia coli (Table 1). Notably, 3 exhibited the most potent antibacterial activity against B. subtilis with an



S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b01288. Experimental procedures and supplementary data (PDF)



*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Ikuro Abe: 0000-0002-3640-888X Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Professor K. Gomi (Tohoku University) and Professor K. Kitamoto (The University of Tokyo) for the expression vectors and the fungal strain. This work was supported in part by a Grant-in-Aid for Scientific Research from MEXT, Japan (JSPS KAKENHI Grant Nos. JP15H01836, JP16H06443, JP26303005, JP24688011, and JP17H05429), Takeda Science Foundation, Kobayashi International Scholarship Foundation, SUNBOR Grant, Noda Institute for Scientific Research, Asahi Glass Foundation, Institute for Fermentation, and Suzuken Memorial Foundation.



MIC (μg/mL) compd

B. subtilis

S. aureus

C. albicans

E. coli

6.25 25.00 3.13 12.50 6.25

12.50 50.00 6.25 25.00 12.50

>100.00 >100.00 100.00 >100.00 >100.00

>100.00 >100.00 >100.00 >100.00 >100.00

AUTHOR INFORMATION

Corresponding Authors

Table 1. Antimicrobial Activities of DCA and Its Analogues

1 2 3 4 ampicillin

ASSOCIATED CONTENT

REFERENCES

(1) (a) Helfrich, E. J.; Piel, J. Nat. Prod. Rep. 2016, 33, 231−316. (b) Abe, I.; Morita, H. Nat. Prod. Rep. 2010, 27, 809−838. (2) (a) Geris, R.; Simpson, T. J. Nat. Prod. Rep. 2009, 26, 1063− 1094. (b) Matsuda, Y.; Abe, I. Nat. Prod. Rep. 2016, 33, 26−53. (3) Japan Kokai Tokkyo Koho JP 82-28080, 1982. (4) Chinese Materia Medica; Jiangsu New Medical College, Ed.; Shanghai People’s Publishing House: Shanghai, 1977; p 2506. 3185

DOI: 10.1021/acs.orglett.7b01288 Org. Lett. 2017, 19, 3183−3186

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Organic Letters (5) Asakawa, Y.; Hashimoto, T.; Quang, D. N.; Nukada, M. Heterocycles 2005, 65, 2431−2439. (6) Kashiwada, Y.; Yamazaki, K.; Ikeshiro, Y.; Yamagishi, T.; Fujioka, T.; Mihashi, K.; Mizuki, K.; Cosentino, L. M.; Fowke, K.; MorrisNatschke, S. L.; Lee, K. H. Tetrahedron 2001, 57, 1559−1563. (7) Liu, K.; Woggon, W. D. Eur. J. Org. Chem. 2010, 2010, 1033− 1036. (8) (a) Paddon, C. J.; Keasling, J. D. Nat. Rev. Microbiol. 2014, 12, 355−367. (b) Fischbach, M. A.; Walsh, C. T. Chem. Rev. 2006, 106, 3468−3496. (9) (a) Taura, F.; Iijima, M.; Lee, J. B.; Hashimoto, T.; Asakawa, Y.; Kurosaki, F. Nat. Prod. Commun. 2014, 9, 1329−1332. (b) Taura, F.; Iijima, M.; Yamanaka, E.; Takahashi, H.; Kenmoku, H.; Saeki, H.; Morimoto, S.; Asakawa, Y.; Kurosaki, F.; Morita, H. Front. Plant Sci. 2016, 7, 1452. (10) Ellestad, G. A.; Evans, R. H., Jr.; Kunstmann, M. P. Tetrahedron 1969, 25, 1323−1334. (11) Li, C.; Matsuda, Y.; Gao, H.; Hu, D.; Yao, X. S.; Abe, I. ChemBioChem 2016, 17, 904−907. (12) (a) Jin, F. H.; Maruyama, J.; Juvvadi, P. R.; Arioka, M.; Kitamoto, K. FEMS Microbiol. Lett. 2004, 239, 79−85. (b) Jin, F.; Maruyama, J.; Juvvadi, P.; Arioka, M.; Kitamoto, K. Biosci., Biotechnol., Biochem. 2004, 68, 656−662. (c) Fujii, T.; Yamaoka, H.; Gomi, K.; Kitamoto, K.; Kumaga, C. Biosci., Biotechnol., Biochem. 1995, 59, 1869− 1874. (13) (a) Itoh, T.; Tokunaga, K.; Matsuda, Y.; Fujii, I.; Abe, I.; Ebizuka, Y.; Kushiro, T. Nat. Chem. 2010, 2, 858−864. (b) Matsuda, Y.; Awakawa, T.; Wakimoto, T.; Abe, I. J. Am. Chem. Soc. 2013, 135, 10962−10965. (c) Matsuda, Y.; Wakimoto, T.; Mori, T.; Awakawa, T.; Abe, I. J. Am. Chem. Soc. 2014, 136, 15326−15336. (d) Matsuda, Y.; Iwabuchi, T.; Wakimoto, T.; Awakawa, T.; Abe, I. J. Am. Chem. Soc. 2015, 137, 3393−3401. (e) Matsuda, Y.; Iwabuchi, T.; Fujimoto, T.; Awakawa, T.; Nakashima, Y.; Mori, T.; Zhang, H.; Hayashi, F.; Abe, I. J. Am. Chem. Soc. 2016, 138, 12671−12677. (f) Okada, M.; Matsuda, Y.; Mitsuhashi, T.; Hoshino, S.; Mori, T.; Nakagawa, K.; Quan, Z.; Qin, B.; Zhang, H.; Hayashi, F.; Kawaide, H.; Abe, I. J. Am. Chem. Soc. 2016, 138, 10011−10018. (g) Matsuda, Y.; Mitsuhashi, T.; Lee, S.; Hoshino, M.; Mori, T.; Okada, M.; Zhang, H.; Hayashi, F.; Fujita, M.; Abe, I. Angew. Chem., Int. Ed. 2016, 55, 5785−5788. (h) Qin, B.; Matsuda, Y.; Mori, T.; Okada, M.; Quan, Z.; Mitsuhashi, T.; Wakimoto, T.; Abe, I. Angew. Chem., Int. Ed. 2016, 55, 1658−1661. (i) Ye, Y.; Minami, A.; Mandi, A.; Liu, C.; Taniguchi, T.; Kuzuyama, T.; Monde, K.; Gomi, K.; Oikawa, H. J. Am. Chem. Soc. 2015, 137, 11846−11853. (14) Tanner, M. E. Nat. Prod. Rep. 2015, 32, 88−101. (15) (a) Taura, F.; Hashimoto, T.; Asakawa, Y. Planta Med. 2011, 77, PI1. (b) Taura, F.; Sirikantaramas, F.; Shoyama, Y.; Shoyama, Y.; Morimoto, S. Chem. Biodiversity 2007, 4, 1649−1663. (16) Kuroda, M.; Takatsu, T.; Takahashi, H.; Hosoya, T.; Furuya, K. Jpn. Kokai Tokkyo Koho JP 05-255184, 1993. (17) Brown, S.; O’Connor, S. E. ChemBioChem 2015, 16, 2129− 2135. (18) (a) Hijikawa, Y.; Matsuzaki, M.; Suzuki, S.; Inaoka, D. K.; Tatsumi, R.; Kido, Y.; Kita, K. J. Antibiot. 2017, 70, 304−307. (b) Tamura, G.; Suzuki, S.; Takatsuki, A.; Ando, K.; Arima, K. J. Antibiot. 1968, 21, 539−544. (19) Takenaka, K.; Tanigaki, Y.; Patil, M. L.; Rao, C. V. L; Takizawa, S.; Suzuki, T.; Sasai, H. Tetrahedron: Asymmetry 2010, 21, 767−770. (20) Nirma, C.; Eparvier, V.; Stien, D. J. Nat. Prod. 2015, 78, 159− 162. (21) Wipf, P.; Weiner, W. S. J. Org. Chem. 1999, 64, 5321−5324. (22) Balouiri, M.; Sadiki, M.; Ibnsouda, S. K. J. Pharm. Anal. 2016, 6, 71−79.

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DOI: 10.1021/acs.orglett.7b01288 Org. Lett. 2017, 19, 3183−3186