Aflatoxin Biosynthesis

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1 June , 2 Aflatoxin Biosynthesis Kimiko Y67: 1, and Hiromitsu N6@6?>B6 2 1 National Food Research Institute, National Agricultural Research Organization (NARO), Tsukuba, Ibaraki , Japan; 2 Faculty of Agriculture, Tottori University, Koyama, Tottori , Japan; Corresponding author / / 1) 3) AF AF B 1 (AFB 1 ) AF AFB 1 P450 Q 1,P 1,M 1 AFB 1 P450 AFB 1-8,9- DNA 4) AFB 1 AFB 1-8,9- AF 250 AF AF 5), 6) ) AF AF AF AF AF AF 8) AF 9) 12) AF AF AF AF AF AF 2. AF A. flavus A. parasiticus Aspergillus flavus A. flavus A. parasiticus AF AF A. flavus AF 13) A. flavus

2 136 Vol. 52, No. 3 AF AF AF AF 14) AF AF AF AF AF AF AF AF 15 17) 3. AF AFB 1,AFB 2, AFG 1,AFG 2 AF AFM 1,AFM 2 1 AFB 1,AFB 2,AFM 1 difurocoumarocyclopentenone AFG 1,AFG 2, AFM 2 difurocoumaro-d-lactone 1 AF (AFB 1,AFG 1,AFM 1 ) 2 (AFB 2, AFG 2, AFM 2 ) AFM 1, AFM 2 14 AFB 1,AFB 2 AF 15) 17) AF AF 28 AF 37 AF AF AF AF kb A. flavus 8 AF III 16), 17) 2 AF ORF 19) AF aflr AF 1. AF AFM 1,AFM 2 AflR AF 20), 21) 4. A. oryzae A. sojae A. flavus AF FDA GRAS (Generally Recognized As Safe) A. oryzae A. oryzae AF 22), 23) AF AF 24) AF 25) A. oryzae A. sojae A. flavus A. parasiticus

3 June norb cypa aflt pksa hypb1 nor-1 fas-2 fas-1 aflr aflj NI 26 NI 2 NI R (R) adha esta nora ver-1 vera avna hypb2 verb avfa dmta 7 11 NI NI omta orda vbs cypx moxy ordb hypa nada vrda , (27) 8, , 4 hypb1 hypb2 AF vrda R, NI, 3-1. A. flavus A. parasiticus 5. AF acetyl CoA 28 AF AF

4 138 Vol. 52, No DMST HOMST VB (22) (24) G-AF 2 AF 3 17) (1) (4) Acetyl CoA norsolorinic acid (NA) AF 2 fas1 fas2 26) acetyl CoA malonyl CoA C6 hexanoyl CoA Hexanoyl CoA 27), pksa (polyketide synthase) CoA hexanoyltetrahydroxyanthrone (HexAT) 28), 29) HexAT C10 norsolorinic acid (NA) hypb1 19) NA NA NA AF 10) 12) (5) NA averantin (AVN) NA NA reductase NADPH NADH averantin (AVN) 30) AVN NAD(P) NA

5 June AF NA reductase AF AF nor ) Nor-1 AF (13R,14S)-( )-aflatoxin 1 AF AF AF AVN (1 S)-( ) NA 1 (HPLC) AVN NA (1 S)-( )-AVN (1 R)- ( )-AVN 32) NADP (1 S)-( )-AVN NA (1 R)-( )- AVN NA reductase NA AVN AVR (8) AF Emericella heterothallica AVN (1 S)-( )-AVN (1 R)-( )-AVN (6) AVN 5 -hydroxyaverantin (HAVN) AVN (AVN monooxygenase) 5 -hydroxyaverantin (HAVN) 30), 32) avna 33) (1 S)-( )-AVN (1 S, 5 S)-HAVN (1 S, 5 R)-HAVN 2:1 (1 R)-( )-AVN 32) AVN (monooxygenase) AVN 1 5 HAVN Emericella heterothallica 30) AF (7) HAVN 5 -oxoaverantin (OAVN) (1 S, 5 S)-HAVN (1 S, 5 R)-HAVN (HAVN dehydrogenase) (1 S)-5 -oxoaverantin (OAVN) 32) 5 adha 34) NAD NADP OAVN OAVN HAVN dehydrogenase 35) acetyl methionine sulfoxide 60 kda (8) OAVN averufin (AVR) (1 S)-OAVN OAVN (cyclase) (1 S, 5 S)-AVR 32) 158 kda 36) AF (1 S, 5 S)-AVR (1 S)-OAVN (1 S, 5 S)-AVR OAVN 1 S NA reductase (5) AVR 32), 36) OAVN (cyclase) VHOH cyclase (VBS) VHOH cyclase (14) 36) VHOH VB (9) AVR hydroxyversicolorone (HVN) avfa cypx AVR HVN 38), 39) AvfA CypX P450 2 cypx AF avfa AF Cai, (1 S, 5 S)-AVR NADPH hydroxyversicolorone (HVN) 37) AvfA (10) HVN versiconal hemiacetal acetate (VHA) HVN NADPH HVN (monooxygenase) versi-

6 140 conal hemiacetal acetate (VHA) 37) 40) moxy HVN versicolorone (VONE) moxy HVN monooxygenase 39) HVN VONE (12) Vol. 52, No. 3 AF VHA reductase vrda vrda AF 44) vrda aflr AF AF AF (11) VHA versiconal (VHOH) VHA esta VHA (esterase) versiconal (VHOH) 41) (DV) 42) DV AF AF VHA versiconol acetate (VOAc) 43) esta VHA VOAc 41) VOAc (12) Esterase VHA VHOH VOAc versiconol (VOH) esta 30 41) esterase VHOH VHA (12), (13) VHA reductase versicolorol (VONOL) HVN, VHA, VHOH NADPH VHA reductase VONE, VOAc, VOH 43), 44) VONE, VOAc, VOH feeding AF VONE, VOAc, VOH AF 43) VONE VOAc HVN VHA monooxygenase esterase HVN VHA VHOH VONE VOAc VOH VHA reductase 6 37), 43) VONE NADPH versicolorol (VONOL) (14) VHOH versicolorin B (VB) VHOH VHOH cyclase (1 R, 2 S)-versicolorin B (VB) 37), 45) AF vbs (versicolorin B synthetase) 46) VHOH cyclase AF OAVN cyclase VHOH cyclase 1 2 (8) (14) (8) OAVN AVR OAVN cyclase N VHOH VB VHOH cyclase (VBS) 36), 46) VBS vbs VHOH OAVN HAVN AF cyclase vbs 1 1 AF 2 36) AF AF AF HVN, VHA, VHOH (1 R, 2 S) (1 S, 2 R) AF HPLC VHOH cyclase VHOH (1 R, 2 S)-VB 47) VHOH VHOH (1 R, 2 S) (1 S, 2 R) versicolorin C (VC) VHOH (1 R, 2 S)- VB HPLC cyclase

7 June (1 R, 2 S)- (1 R, 2 S)-VB 47) (1 S, 2 R)-VHOH (1 R, 2 S)- VHOH (1 S, 2 R)- VHOH (1 R, 2 S)-VHOH VHOH cyclase VB (1 R, 2 S)- VB AF AF VHOH cyclase VHOH cyclase OAVN (15) VB versicolorin A (VA) (1 R, 2 S)-VB desaturase VA 48) (1 S, 2 R)- VB 47) Desaturase 48) VB AFB 2,AFG 2 tetrahhydrobisfuran versicolorin A (VA) AFB 1,AFG 1 dihydrobisfuran AF feeding VB 4 AF VA AFB 1 AFG 1 48) Desaturase verb 49) verb 1 AF (AFB 1,AFG 1 ) 2 AF (AFB 2,AFG 2 ) desaturase 2 AF 1 AF (16) VA demethylsterigmatocystin (DMST) VB dihydrodemethylsterigmatocystin (DHDMST) VA demethylsterigmatocystin (DMST) VA DMST VA DMST AF ver-1, ordb, hypx AF VA 50) 52) VA DMST 3 vera 53) VB DHDMST AF 2) (17) DMST sterigmatocystin (ST) DHDMST dihydrosterigmatocystin (DHST) DMST DHDMST 2 OH 6- OH 7-OH, S-adenosylmethionine O-methyltransferase I 6-OH ST DHST 54), 55) O- Methyltransferase I 90 kda K m 1 2 M 100 mm 55) AF O-Methyltransferase I dmta 56) (18) ST O-methylsterigmatocystin (OMST) DHST dihydro-o-methylsterigmatocystin (DHOMST) ST DHST 7-OH S-adenosylmethionine O-methyltransferase II OMST DHOMST O-Methyltransferase II DMST DHDMST 7-OH 6-OH 7-OH 55) O-Methyltransferase II omta 57) (19) OMST 11-hydroxy-O-methylsterigmatocystin (HOMST) DHOMST dihydro- 11-hydroxy-O-methylsterigmatocystin (DHHOMST) orda OrdA P450 OMST AFB 1 58) 60) orda OMST DHOMST 4 AF OrdA G-AF OMST AF OrdA OMST AFB hydroxy-o-methylsterigmatocystin (HOMST) 61), 62) OMST HOMST, OMST dihydro DHOMST DHHOMST OrdA

8 142 (20) (24), (25) HOMST AFB 1 DHHOMST AFB 2 HOMST AFB 1 DHHOMST AFB 2 OrdA 61), 62) aryl ring cleavage, demethylation, dehydration, decarboxylation OrdA (20) (24), (26) (29) HOMST AFG 1 DHHOMST AFG 2 HOMST DHHOMST AFB 1,AFB 2 AFG 1,AFG 2 HOMST feeding AFB 1 AFG 1 DHH- OMST AFB 2 AFG 2 62) HOMST AFG 1 AFB 1 cypa G-AF B-AF CypA 63) HOMST AFG 1 HOMST AFG ) OrdA CypA (29) NADA AFG 1 DHNADA AFG 2 nada G-AF NADA 64) NADA 360 AFG 1 NadA NAD(P)H NADA AFG 1 NADA dihydro DHNADA AFG 2 NadA G-AF HOMST AFG 1 NadA 64) HOMST AFG 1 6. AF NADPH acetyl CoA 1 AF 10 NADPH NAD 1 S-adenosylmethionine (SAM) 2 Vol. 52, No. 3 malonyl CoA SAM NADPH AF AF AF YES 20 5 AF NADPH NADPH AF AF AF AF AF AF AF nor-1 31) esta 41) AF vrda AF 44) vrda AflR AF AF vrda VONE AF VOROL 37) AF AF AF AF AF AF AF NA AVN OMT-I 1 cyclase 2 AF

9 June AF 7. A. parasiticus AFB 1,AFB 2,AFG 1,AFG 2 A. flavus B-AF (AFB 1,AFB 2 ) AF A. flavus AF kb G-AF cypa cypa Aspergillus flavus G-AF 63) B-AF G-AF HOMST AFB 1 OrdA HOMST AFG 1 OrdA 2 G-AF AF 4 AF AFM 1 AFM 2 AF M-AF AF M-AF 8. AF AF A. oryzae A. sojae AF 23), 24), 65) A. parasiticus A. flavus A. nomius, A. pseudotamarii, A. bombycis, A. ochraceoroseus, A. australis AF AF 66) A. nidulans A. versicolor 20 AF (ST) A. nidulans ST ST AF ST ST 67), 68) ST AF Mycosphaerella pini, A. fumigatus, Coccidioides immitis AF 19) A. versicolor E. hetherothallica AF AVR HAVN AF 3), 69), 70) AF 71) AF AF AF AF 9. AF AF AF AF ST OMST DHOMST AFG 1 NADA 64) AF AF 72), 73) AF feeding M AF aflt 74) 10. AF ph AF AF aflr AflR binuclear zinc cluster (Zn(II)2Cys6) DNA AF 53) aflr aflj 75) aflr camp Ca G 76) A. nidulans VeA LaeA A. flavus A. parasiticus AF

10 144 Vol. 52, No. 3 77) AF AF AF 9) Achromobacter xylosoxidans cyclo (A-leucyl-Aprolyl) AF cyclo (A-leucyl-A-prolyl) aflr 11) AF AF X 51 AF AF AF AF AF AF AF AflR AflR AF AflR AF AF 1) , p ) Bennett, J. W., Klich, M. Mycotoxins. Clin. Microbiol. Rev., 16, (2003). 3) Cole, R. J., Cox, R. H. Handbook of Toxic Fungal Metabolites. Academic Press, New York, ) Kumagai, S. The fate of aflatoxin B 1 in animals. Mycotoxins, 60, 7 16 (2010). 5) Bull. Natl. Inst. Health Sci., 124, (2006). 6) B1, B2, G1 G2 05/dl/s k 0001.pdf 7) Robens, J. The costs of mycotoxin management to the USA: Management of aflatoxins in the United States. http: //www. apsnet. org/ online/ feature/ mycotoxin/top.html (2001). 8) Cotty, P. J. Influence of field application of an atoxigenic strain of Aspergillus flavus on the population of A. flavus infecting cotton bolls and on the aflatoxin content of cottonseed. Phytopathology, 84, (1994). 9) Ono, M., Sakuda, S., Suzuki, A., Isogai, A. Aflastatin A, a novel inhibitor of aflatoxin production by aflatoxigenic fungi. J. Antibiot., 50, (1997). 10) Hua, S. S., Baker J. L., Flores-Espiritu, M. Interactions of saprophytic yeasts with a nor mutant of Aspergillus flavus. Appl. Environ. Microbiol., 65, (1999). 11) Yan, P.-S., Song Y., Sakuno, E., Nakajima H., Nakagawa H., Yabe K. Cyclo (A-leucyl-A-prolyl) produced by Achromobacter xylosoxidans inhibits aflatoxin production by Aspergillus parasiticus. Appl. Environ. Microbiol., 70, (2004). 12) Yabe, K., Yan, P.-S., Song, Y., Ichinomiya, M., Nakagawa, H., Shima, Y., Ando, Y., Sakuno, E. Nakajima, H. Isolation of microorganisms and substances inhibitory to aflatoxin production. Food Addit. Contam., Part A, 25, (2008). 13) Manabe, M., Tsuruta, O. Geographical distribution of aflatoxin-producing fungi in Southeast Asia. JARQ, 12, (1978). 14) Saito, M., Okazaki, H., Tanaka, K., Kushiro, M. Distribution of Aspergillus flavus and A. parasiticus in field soils from Ibaraki and Chiba Prefectures. Rep. Nat l. Food Res. Inst., 72, (2008). 15) Trail, F., Mahanti, N., Rarick, M., Mehigh, R., Liang, S. H., Zhou, R., Linz, J. E. Physical and transcriptional map of an aflatoxin gene cluster in Aspergillus parasiticus and functional disruption of a gene involved early in the aflatoxin pathway. Appl. Environ. Microbiol., 61, (1995). 16) Yabe, K., Nakajima, H. Enzyme reactions and genes in aflatoxin biosynthesis. Appl. Microbiol. Biotechnol., 64, (2004). 17) Yu, J., Chang, P. K., Ehrlich, K. C., Cary, J. W., Bhatnagar, D., Cleveland, T. E., Payne, G. A., Linz, J. E., Woloshuk, C. P., Bennett, J. W. Clustered pathway genes in aflatoxin biosynthesis. Appl. Environ. Microbiol., 70, (2004). 18) Aspergillus flavus and aflatoxin. 19) Ehrlich, K. C. Predicted roles of the uncharacterized clustered genes in aflatoxin biosynthesis. Toxins, 1, (2009).

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12 146 Vol. 52, No. 3 ter. J. Biol. Chem., 271, (1996). 47) Yabe, K., Hamasaki, T. Stereochemistry during aflatoxin biosynthesis: cyclase reaction in the conversion of versiconal to versicolorin B and racemization of versiconal hemiacetal acetate. Appl. Environ. Microbiol., 59, (1993). 48) Yabe, K., Ando, Y., Hamasaki, T. Desaturase activity in the branching step between aflatoxins B 1 and G 1 and aflatoxins B 2 and G 2. Agric. Biol. Chem., 55, (1991). 49) Bhatnagar, D., Ehrlich, K. C., Cleveland, T. E. Molecular genetic analysis and regulation of aflatoxin biosynthesis. Appl. Microbiol. Biotechnol., 61, (2003). 50) Skory, C. D., Chang, P. K., Cary, J., Linz, J. E. Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis. Appl. Environ. Microbiol., 58, (1992). 51) Ehrlich, K. C., Montalbano, B., Boue, S. M., Bhatnagar, D. 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13 June in aflatoxin biosynthesis. Biosci. Biotechnol. Biochem. 72, (2008). 72) Chanda, A., Roze, L. V., Kang, S., Artymovich, K. A., Hicks, G. R., Raikhel, N. V., Calvo, A. M., Linz, J. E. A key role for vesicles in fungal secondary metabolism. Proc. Natl. Acad. Sci. U.S.A., 106, (2009). 73) Roze, L. V., Chanda, A., Linz, J. E. Compartmentalization and molecular tra$c in secondary metabolism: A new understanding of established cellular processes. Fungal Genet. Biol., 48, (2011). 74) Chang, P. K., Yu, J., Yu, J.-H. aflt, a MFS transporterencoding gene located in the aflatoxin gene cluster, does not have a significant role in aflatoxin secretion. Fungal Genet. Biol., 41, (2004). 75) Meyers, D. M., O Brian, G., Du, W. L., Bhatnagar, E., Payne, G. A. Characterization of aflj, a gene required for conversion of pathway intermediates to aflatoxin. Appl. Environ. Microbiol., 64, (1998). 76) Shwab, E. K., Keller, N. P. Regulation of secondary metabolite production in filamentous ascomycetes. Micol. Res., 112, (2008). 77) Amaike, S., Keller, N. P. Distinct roles for VeA and LaeA in development and pathogenesis of Aspergillus flavus. Eukaryotic Cell, 2009, (2009).

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