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Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds
Bioorganic & Medicinal Chemistry Letters 23 (2013) 5637–5640
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds Makoto Hashimoto a, Satomi Ishida a, Yasuyo Seshime a, Katsuhiko Kitamoto b, Isao Fujii a,⇑ a b
School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e
i n f o
Article history: Received 6 July 2013 Revised 5 August 2013 Accepted 6 August 2013 Available online 14 August 2013 Keywords: Aspergillus oryzae Type III polyketide synthase CsyB Csypyrone B
a b s t r a c t Csypyrones B1, B2 and B3 are a-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-13C] and [2-13C] acetate feeding experiments. 13C NMR analyses of the methyl esters of the csypyrone B compounds fed with the 13C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus. Ó 2013 Elsevier Ltd. All rights reserved.
Type III polyketide synthases (PKSs) are abundant in plants and microbes.1–3 In light of the significant progress of fungal genome analysis, a large number of type III PKS genes have been reported since our first report in 2005 concerning the discovery of csyAD from Aspergillus oryzae.4 We previously reported that CsyB expression in A. oryzae resulted in the production of csypyrones B1 (1), B2 (2) and B3 (3), which are acetyl a-pyrone compounds bearing carboxylic acid side-chains. Csypyrone B1 (1) is the major product and contains a propanoic acid side-chain, whereas csypyrones B2 (2) and B3 (3) are the minor compounds that contain butyric or pentanoic acid side-chains, respectively (Fig. 1).5,6 As type III PKS products, these csypyrone B compounds possess the rather unique feature of having two side-chains attached to their pyrone nucleus. To date, no other fungal type III PKSs have been reported that are capable of catalyzing the formation of pyrones or resorcylic acids bearing two side-chains.7–9 Based on the results of our previous feeding experiment with [1, 2-13C2] acetate,5 two possible mechanisms were proposed for the biosynthesis of B1 (1) (Fig. 2). In the first of these mechanisms, succinyl-CoA from the TCA cycle is used as the starter substrate for the left unit of 1. According to the second possible mechanism, butyryl-CoA could be used as a substrate to form the csypyrone B precursor with an alkyl side-chain, followed by oxidation of the side-chain terminus carbon to give 1. The production of the minor compounds B2 (2) and B3 (3) with side-chains of different carbon-lengths could support the latter of these two mechanisms. ⇑ Corresponding author. Tel.: +81 19 651 5111x5260; fax: +81 19 698 1923. E-mail address: [email protected] (I. Fujii). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.08.036
Csypyrone B precursors with alkyl side-chains, however, have not been detected in the A. oryzae transformant expressing CsyB. To differentiate between these possibilities, we have conducted a series of feeding experiments using [1-13C] or [2-13C] acetate. If fatty acyl-CoA is a precursor for the side chains of these molecules, the C-11 carbonyl carbon of 1 should be derived from the C-2 methyl carbon of the acetate. In contrast, the C-1 carbonyl carbon of the acetate should be incorporated into the C-11 carbon of 1 if succinyl-CoA is a precursor for the formation of the side chains (Fig. 2). The culture conditions for the A. oryzae transformant with pTA-csyB have been described previously.5 Following a preculture period in DPY medium, sodium [1-13C] or [2-13C] acetate (99 atom% 13C, ISOTEC Inc., Miamisburg, OH, USA) was added to induction culture to a final concentration of 1.5 g/L. Following a 3 day period of induction culture, the csypyrone B compounds were isolated as reported previously and then methylated with trimethylsilyldiazomethane10 to give the corresponding mono-methyl esters 1a, 2a, and 3a. The 13C enrichment levels of the individual carbons of the 13C-labeled csypyrone B compounds were calculated from the 13C signal intensity ratios with the natural abundance intensity signals using the O-methyl carbon intensity signals of the csypyrone B methyl esters as internal standards of natural abundance (Supplementary Figs. 1–9). When sodium [1-13C] acetate was used as the feed, significant levels of enrichment were observed at the C-2, C-4, C-6, C-7 and C-10 positions of 1a, whereas significant levels of enrichment were observed at the C-3, C-5, C-8, C-9 and C-11 positions of 1a when sodium [2-13C] acetate was used as the feed (Table 1). The
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M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5637–5640 11
9
ROOC 10
O
6
O
2
8 5
CH3
7
3 4
O
OH
R=H : csypyrone B1 (1) R=CH3 : csypyrone B1 methyl ester (1a) 9
11 12
10
O
6
ROOC
2
O 8
5
3
7
CH3
4
O
OH
R=H : csypyrone B2 (2) R=CH3 : csypyrone B2 methyl ester (2a) 13
ROOC 12
9
11 10
6
O
2
O 8
5
3
7
CH3
4
OH
O
R=H : csypyrone B3 (3) R=CH3 : csypyrone B3 methyl ester (3a) Figure 1. Structures of the csypyrone B compounds and the corresponding methyl esters.
incorporation of five acetate units was in agreement with the labeling pattern of [1, 2-13C2] acetate into 1 in the previous experiments.5 These results therefore revealed that the intact acetate units of the methyl carbon–carbonyl carbon had been incorporated into C-11–C-10 and C-9–C-6 positions of the side-
chain in 1, and indicated that butyryl or longer fatty acyl starter served as the starting materials for the biosynthesis of csypyrone B1. Similarly, the enrichment of the 13C levels at the C-13 position in 3a by [2-13C] acetate revealed that the C-13–C-12, C-11–C-10 and C-9–C-6 positions were the methyl carbon–carbonyl carbon acetate units of 3 (Table 1), and indicated that hexanoyl or a longer fatty acyl starter was involved in the biosynthesis of 3. In contrast to these results for 1a and 3a, the 13C enrichment pattern observed in 2a was quite unusual. When [2-13C] acetate was used as the feed, the relative intensity data for the 2a sidechain carbons at the C-12, C-11, C-10, C-9 and C-6 positions, were 3.8, 2.6, 5.3, 3.2, and 3.8, respectively, indicating that these five consecutive carbons had been enriched to a much greater extent with [2-13C] acetate. Furthermore, relatively lower enrichments of these carbons were observed when [1-13C] acetate was used as the feed (Table 1). With the exception of these side-chain carbons and the carbon at the C-6 position, the labeling patterns in the pyrone and acetyl carbons of 2a were comparable with those of 1a and 3a. These results indicated that the fatty acyl starter could be involved in the biosynthesis of 2 in an unusual way. Based on the carbon length of the side-chain, we have assumed that pentanoic acid could be the acyl starter in the biosynthesis of 2, with the pentanoic acid itself being formed via the a-oxidation of hexanoic acid. The pentanoic acid could also be formed from the propanoic acid derived from the a-oxidation of butanoic acid following its condensation with a malonate unit. In addition, it is possible that heptanoic acid could be involved in some way. A possible mechanism for the labeling randomization in the biosynthesis of 2 has been proposed based on the current results and is shown in Figure 3. Based on the results of our current feeding experiments with [1-13C] or [2-13C] acetate, it has been confirmed that fatty acyl starters are involved in the biosynthesis of the csypyrone B
Figure 2. Proposed biosynthesis of csypyrone B1 (1). Two possible biosynthetic schemes are shown: (1) succinyl-CoA serves as a CsyB substrate to form the csypyrone B carbon skeleton through a condensation with acetomalonyl-CoA. The bold lines indicate the acetate units and the filled circles represent the methyl carbon of the acetate unit; (2) the condensation of butyryl-CoA with malonyl-CoA followed by acetomalonyl-CoA would give the csypyrone B1 carbon skeleton, followed by the oxidation of the methyl carbon of the side chain to a carboxylic acid by host enzyme(s).
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M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5637–5640 Table 1 Incorporation of
13
C-labeled acetate into csypyrone Bs methyl ester (1a–3a)
Position
1a
2a a
Relative intensity 13
2 3 4 5 6 7 8 9 10 11 12 13 O-CH3 a
Intensity ratio of
3a a
Relative intensitya
Relative intensity
13
[1- C] acetate
[2- C] acetate
[1- C] acetate
[2- C] acetate
[1- C] acetate
[2-13C] acetate
2.0 0.7 3.3 0.8 4.4 2.6 1.0 0.8 4.1 0.8
1.3 3.1 1.1 4.1 1.1 1.2 3.0 5.0 1.8 6.6
2.0 1.0 2.5 1.0 1.3 2.0 1.0 1.9 1.5 2.7 1.6
1.6 3.6 2.1 10.3 3.8 1.1 5.7 3.2 5.3 2.6 3.8
1.0
1.0
1.0
1.0
2.2 1.0 3.3 0.7 4.8 2.4 0.9 0.9 5.6 0.8 6.8 1.1 1.0
1.3 3.6 1.1 3.9 1.0 1.1 3.0 6.1 1.2 7.7 1.2 7.4 1.0
13
C-enriched to natual abundance.
13
13
13
13
C NMR (125 MHz) data were measured in CDCl3 by JEOL ECA-500 spectrometer.
Figure 3. Proposed labeling randomization mechanism in the csypyrone B2 (2) biosynthesis. Filled circles represent the carbons derived from the methyl carbon of the acetate unit.
H 3C
SCoA m
O
H 3C
m
O
CoAS SCoA
malonyl-CoA
CH3
HOOC
fatty acyl-CoA
O
O
m = 1 ~ 3 or longer
acetomalonyl-CoA H3 C
O
O
m
CH3
CsyB reaction OH
O
O
oxidation by host fungus O
O
HO n n = 1 : B1 n = 2 : B2 n = 3 : B3
O
CH3 OH
O
csypyrone Bs Figure 4. Proposed CsyB reaction for the biosynthesis of csypyrone B.
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M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5637–5640
compounds. Csypyrone B precursors with alkyl side-chains formed by the CsyB reaction should be oxidized to the corresponding csypyrone B compounds by the host fungus oxidation system(s) (Fig. 4). Although a-pyrone compounds of this type bearing carboxylic acid side-chains have been isolated from fungi,11 the csypyrone B compounds have not been detected in the cultures of wild-type A. oryzae RIB40. Some fungi have been reported to metabolize 6pentyl a-pyrone to less toxic a-pyrones bearing carboxylic acid side-chains.12 Since csypyrone B precursors with alkyl side-chains could not be found in A. oryzae, it is possible that they could have some toxic effects on the host fungus. In conclusion, the current results indicate that a fatty acyl starter is involved in the CsyB reaction. The actual starter substrates of CsyB, however, have not yet been confirmed. Furthermore, the substrate for the diketide unit of the pyrone moiety remains unknown, although we believe that acetomalonyl-CoA could be acting as a diketide extender unit in the CsyB reaction. Further in vitro experiments are currently underway in our laboratory to fully characterize this unique type III PKS CsyB. Acknowledgment This work was supported in part by Grant from the Keiryokai Research Foundation.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 08.036. References and notes 1. Morita, H.; Abe, I.; Noguchi, H. In Comprehensive Natural Products II; Mander, L., Liu, H.-W., Eds.; Elsevier: Oxford, 2010; Vol. 1, p 171. 2. Austin, M. B.; Noel, J. P. Nat. Prod. Rep. 2003, 20, 79. 3. Katsuyama, Y.; Horinouchi, S. In Comprehensive Natural Products II; Mander, L., Liu, H.-W., Eds.; Elsevier: Oxford, 2010; Vol. 1, p 147. 4. Seshime, Y.; Juvvadi, P. R.; Fujii, I.; Kitamoto, K. Biochem. Biophys. Res. Commun. 2005, 331, 253. 5. Seshime, Y.; Juvvadi, P. R.; Kitamoto, K.; Ebizuka, Y.; Fujii, I. Bioorg. Med. Chem. 2010, 18, 4542. 6. Hashimoto, M.; Seshime, Y.; Kitamoto, K.; Uchiyama, N.; Goda, Y.; Fujii, I. Bioorg. Med. Chem. Lett. 2013, 23, 650. 7. Funa, N.; Awakawa, T.; Horinouchi, S. J. Biol. Chem. 2007, 282, 14476. 8. Seshime, Y.; Juvvadi, P. R.; Kitamoto, K.; Ebizuka, Y.; Fujii, I. Bioorg. Med. Chem. Lett. 2010, 20, 4785. 9. Li, J.; Luo, Y.; Lee, J.-K.; Zhao, H. Bioorg. Med. Chem. Lett. 2011, 21, 6085. 10. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981, 29, 1475. 11. Christensen, K. B.; Klink, J. W. V.; Weavers, R. T.; Larsen, T. O.; Andersen, B.; Phipps, R. J. Agric. Food Chem. 2005, 53, 9431. 12. Cooney, J. M.; Lauren, D. R. J. Nat. Prod. 1999, 62, 681.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds Makoto Hashimoto a, Satomi Ishida a, Yasuyo Seshime a, Katsuhiko Kitamoto b, Isao Fujii a,⇑ a b
School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e
i n f o
Article history: Received 6 July 2013 Revised 5 August 2013 Accepted 6 August 2013 Available online 14 August 2013 Keywords: Aspergillus oryzae Type III polyketide synthase CsyB Csypyrone B
a b s t r a c t Csypyrones B1, B2 and B3 are a-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-13C] and [2-13C] acetate feeding experiments. 13C NMR analyses of the methyl esters of the csypyrone B compounds fed with the 13C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus. Ó 2013 Elsevier Ltd. All rights reserved.
Type III polyketide synthases (PKSs) are abundant in plants and microbes.1–3 In light of the significant progress of fungal genome analysis, a large number of type III PKS genes have been reported since our first report in 2005 concerning the discovery of csyAD from Aspergillus oryzae.4 We previously reported that CsyB expression in A. oryzae resulted in the production of csypyrones B1 (1), B2 (2) and B3 (3), which are acetyl a-pyrone compounds bearing carboxylic acid side-chains. Csypyrone B1 (1) is the major product and contains a propanoic acid side-chain, whereas csypyrones B2 (2) and B3 (3) are the minor compounds that contain butyric or pentanoic acid side-chains, respectively (Fig. 1).5,6 As type III PKS products, these csypyrone B compounds possess the rather unique feature of having two side-chains attached to their pyrone nucleus. To date, no other fungal type III PKSs have been reported that are capable of catalyzing the formation of pyrones or resorcylic acids bearing two side-chains.7–9 Based on the results of our previous feeding experiment with [1, 2-13C2] acetate,5 two possible mechanisms were proposed for the biosynthesis of B1 (1) (Fig. 2). In the first of these mechanisms, succinyl-CoA from the TCA cycle is used as the starter substrate for the left unit of 1. According to the second possible mechanism, butyryl-CoA could be used as a substrate to form the csypyrone B precursor with an alkyl side-chain, followed by oxidation of the side-chain terminus carbon to give 1. The production of the minor compounds B2 (2) and B3 (3) with side-chains of different carbon-lengths could support the latter of these two mechanisms. ⇑ Corresponding author. Tel.: +81 19 651 5111x5260; fax: +81 19 698 1923. E-mail address: [email protected] (I. Fujii). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.08.036
Csypyrone B precursors with alkyl side-chains, however, have not been detected in the A. oryzae transformant expressing CsyB. To differentiate between these possibilities, we have conducted a series of feeding experiments using [1-13C] or [2-13C] acetate. If fatty acyl-CoA is a precursor for the side chains of these molecules, the C-11 carbonyl carbon of 1 should be derived from the C-2 methyl carbon of the acetate. In contrast, the C-1 carbonyl carbon of the acetate should be incorporated into the C-11 carbon of 1 if succinyl-CoA is a precursor for the formation of the side chains (Fig. 2). The culture conditions for the A. oryzae transformant with pTA-csyB have been described previously.5 Following a preculture period in DPY medium, sodium [1-13C] or [2-13C] acetate (99 atom% 13C, ISOTEC Inc., Miamisburg, OH, USA) was added to induction culture to a final concentration of 1.5 g/L. Following a 3 day period of induction culture, the csypyrone B compounds were isolated as reported previously and then methylated with trimethylsilyldiazomethane10 to give the corresponding mono-methyl esters 1a, 2a, and 3a. The 13C enrichment levels of the individual carbons of the 13C-labeled csypyrone B compounds were calculated from the 13C signal intensity ratios with the natural abundance intensity signals using the O-methyl carbon intensity signals of the csypyrone B methyl esters as internal standards of natural abundance (Supplementary Figs. 1–9). When sodium [1-13C] acetate was used as the feed, significant levels of enrichment were observed at the C-2, C-4, C-6, C-7 and C-10 positions of 1a, whereas significant levels of enrichment were observed at the C-3, C-5, C-8, C-9 and C-11 positions of 1a when sodium [2-13C] acetate was used as the feed (Table 1). The
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M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5637–5640 11
9
ROOC 10
O
6
O
2
8 5
CH3
7
3 4
O
OH
R=H : csypyrone B1 (1) R=CH3 : csypyrone B1 methyl ester (1a) 9
11 12
10
O
6
ROOC
2
O 8
5
3
7
CH3
4
O
OH
R=H : csypyrone B2 (2) R=CH3 : csypyrone B2 methyl ester (2a) 13
ROOC 12
9
11 10
6
O
2
O 8
5
3
7
CH3
4
OH
O
R=H : csypyrone B3 (3) R=CH3 : csypyrone B3 methyl ester (3a) Figure 1. Structures of the csypyrone B compounds and the corresponding methyl esters.
incorporation of five acetate units was in agreement with the labeling pattern of [1, 2-13C2] acetate into 1 in the previous experiments.5 These results therefore revealed that the intact acetate units of the methyl carbon–carbonyl carbon had been incorporated into C-11–C-10 and C-9–C-6 positions of the side-
chain in 1, and indicated that butyryl or longer fatty acyl starter served as the starting materials for the biosynthesis of csypyrone B1. Similarly, the enrichment of the 13C levels at the C-13 position in 3a by [2-13C] acetate revealed that the C-13–C-12, C-11–C-10 and C-9–C-6 positions were the methyl carbon–carbonyl carbon acetate units of 3 (Table 1), and indicated that hexanoyl or a longer fatty acyl starter was involved in the biosynthesis of 3. In contrast to these results for 1a and 3a, the 13C enrichment pattern observed in 2a was quite unusual. When [2-13C] acetate was used as the feed, the relative intensity data for the 2a sidechain carbons at the C-12, C-11, C-10, C-9 and C-6 positions, were 3.8, 2.6, 5.3, 3.2, and 3.8, respectively, indicating that these five consecutive carbons had been enriched to a much greater extent with [2-13C] acetate. Furthermore, relatively lower enrichments of these carbons were observed when [1-13C] acetate was used as the feed (Table 1). With the exception of these side-chain carbons and the carbon at the C-6 position, the labeling patterns in the pyrone and acetyl carbons of 2a were comparable with those of 1a and 3a. These results indicated that the fatty acyl starter could be involved in the biosynthesis of 2 in an unusual way. Based on the carbon length of the side-chain, we have assumed that pentanoic acid could be the acyl starter in the biosynthesis of 2, with the pentanoic acid itself being formed via the a-oxidation of hexanoic acid. The pentanoic acid could also be formed from the propanoic acid derived from the a-oxidation of butanoic acid following its condensation with a malonate unit. In addition, it is possible that heptanoic acid could be involved in some way. A possible mechanism for the labeling randomization in the biosynthesis of 2 has been proposed based on the current results and is shown in Figure 3. Based on the results of our current feeding experiments with [1-13C] or [2-13C] acetate, it has been confirmed that fatty acyl starters are involved in the biosynthesis of the csypyrone B
Figure 2. Proposed biosynthesis of csypyrone B1 (1). Two possible biosynthetic schemes are shown: (1) succinyl-CoA serves as a CsyB substrate to form the csypyrone B carbon skeleton through a condensation with acetomalonyl-CoA. The bold lines indicate the acetate units and the filled circles represent the methyl carbon of the acetate unit; (2) the condensation of butyryl-CoA with malonyl-CoA followed by acetomalonyl-CoA would give the csypyrone B1 carbon skeleton, followed by the oxidation of the methyl carbon of the side chain to a carboxylic acid by host enzyme(s).
5639
M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5637–5640 Table 1 Incorporation of
13
C-labeled acetate into csypyrone Bs methyl ester (1a–3a)
Position
1a
2a a
Relative intensity 13
2 3 4 5 6 7 8 9 10 11 12 13 O-CH3 a
Intensity ratio of
3a a
Relative intensitya
Relative intensity
13
[1- C] acetate
[2- C] acetate
[1- C] acetate
[2- C] acetate
[1- C] acetate
[2-13C] acetate
2.0 0.7 3.3 0.8 4.4 2.6 1.0 0.8 4.1 0.8
1.3 3.1 1.1 4.1 1.1 1.2 3.0 5.0 1.8 6.6
2.0 1.0 2.5 1.0 1.3 2.0 1.0 1.9 1.5 2.7 1.6
1.6 3.6 2.1 10.3 3.8 1.1 5.7 3.2 5.3 2.6 3.8
1.0
1.0
1.0
1.0
2.2 1.0 3.3 0.7 4.8 2.4 0.9 0.9 5.6 0.8 6.8 1.1 1.0
1.3 3.6 1.1 3.9 1.0 1.1 3.0 6.1 1.2 7.7 1.2 7.4 1.0
13
C-enriched to natual abundance.
13
13
13
13
C NMR (125 MHz) data were measured in CDCl3 by JEOL ECA-500 spectrometer.
Figure 3. Proposed labeling randomization mechanism in the csypyrone B2 (2) biosynthesis. Filled circles represent the carbons derived from the methyl carbon of the acetate unit.
H 3C
SCoA m
O
H 3C
m
O
CoAS SCoA
malonyl-CoA
CH3
HOOC
fatty acyl-CoA
O
O
m = 1 ~ 3 or longer
acetomalonyl-CoA H3 C
O
O
m
CH3
CsyB reaction OH
O
O
oxidation by host fungus O
O
HO n n = 1 : B1 n = 2 : B2 n = 3 : B3
O
CH3 OH
O
csypyrone Bs Figure 4. Proposed CsyB reaction for the biosynthesis of csypyrone B.
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M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5637–5640
compounds. Csypyrone B precursors with alkyl side-chains formed by the CsyB reaction should be oxidized to the corresponding csypyrone B compounds by the host fungus oxidation system(s) (Fig. 4). Although a-pyrone compounds of this type bearing carboxylic acid side-chains have been isolated from fungi,11 the csypyrone B compounds have not been detected in the cultures of wild-type A. oryzae RIB40. Some fungi have been reported to metabolize 6pentyl a-pyrone to less toxic a-pyrones bearing carboxylic acid side-chains.12 Since csypyrone B precursors with alkyl side-chains could not be found in A. oryzae, it is possible that they could have some toxic effects on the host fungus. In conclusion, the current results indicate that a fatty acyl starter is involved in the CsyB reaction. The actual starter substrates of CsyB, however, have not yet been confirmed. Furthermore, the substrate for the diketide unit of the pyrone moiety remains unknown, although we believe that acetomalonyl-CoA could be acting as a diketide extender unit in the CsyB reaction. Further in vitro experiments are currently underway in our laboratory to fully characterize this unique type III PKS CsyB. Acknowledgment This work was supported in part by Grant from the Keiryokai Research Foundation.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013. 08.036. References and notes 1. Morita, H.; Abe, I.; Noguchi, H. In Comprehensive Natural Products II; Mander, L., Liu, H.-W., Eds.; Elsevier: Oxford, 2010; Vol. 1, p 171. 2. Austin, M. B.; Noel, J. P. Nat. Prod. Rep. 2003, 20, 79. 3. Katsuyama, Y.; Horinouchi, S. In Comprehensive Natural Products II; Mander, L., Liu, H.-W., Eds.; Elsevier: Oxford, 2010; Vol. 1, p 147. 4. Seshime, Y.; Juvvadi, P. R.; Fujii, I.; Kitamoto, K. Biochem. Biophys. Res. Commun. 2005, 331, 253. 5. Seshime, Y.; Juvvadi, P. R.; Kitamoto, K.; Ebizuka, Y.; Fujii, I. Bioorg. Med. Chem. 2010, 18, 4542. 6. Hashimoto, M.; Seshime, Y.; Kitamoto, K.; Uchiyama, N.; Goda, Y.; Fujii, I. Bioorg. Med. Chem. Lett. 2013, 23, 650. 7. Funa, N.; Awakawa, T.; Horinouchi, S. J. Biol. Chem. 2007, 282, 14476. 8. Seshime, Y.; Juvvadi, P. R.; Kitamoto, K.; Ebizuka, Y.; Fujii, I. Bioorg. Med. Chem. Lett. 2010, 20, 4785. 9. Li, J.; Luo, Y.; Lee, J.-K.; Zhao, H. Bioorg. Med. Chem. Lett. 2011, 21, 6085. 10. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981, 29, 1475. 11. Christensen, K. B.; Klink, J. W. V.; Weavers, R. T.; Larsen, T. O.; Andersen, B.; Phipps, R. J. Agric. Food Chem. 2005, 53, 9431. 12. Cooney, J. M.; Lauren, D. R. J. Nat. Prod. 1999, 62, 681.