June 2011 135 23 3 4 1, 2 Aflatoxin Biosynthesis Kimiko Y67: 1, and Hiromitsu N6@6?>B6 2 1 National Food Research Institute, National Agricultural Research Organization (NARO), Tsukuba, Ibaraki 305 8642, Japan; 2 Faculty of Agriculture, Tottori University, Koyama, Tottori 680 8553, Japan; Corresponding author 1. 300 / / 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) 1 305 8642 2 1 12 7) 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
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 25 70 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) 2005 19 A. oryzae A. oryzae AF 22), 23) AF AF 24) AF 25) A. oryzae A. sojae A. flavus A. parasiticus
June 2011 137 norb cypa aflt pksa hypb1 nor-1 fas-2 fas-1 aflr aflj NI 26 NI 2 NI 5 1 1 R (R) adha esta nora ver-1 vera avna hypb2 verb avfa dmta 7 11 NI 16 16 6 NI 15 9 17 omta orda vbs cypx moxy ordb hypa nada vrda 18 19 25, (27) 8, 14 9 10 16 16 29 12 2. 3, 4 hypb1 hypb2 AF vrda R, NI, 3-1. A. flavus A. parasiticus 5. AF acetyl CoA 28 AF AF
138 Vol. 52, No. 3 3-2. 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
June 2011 139 AF NA reductase AF AF nor-1 10 31) 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-
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
June 2011 141 (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 1 11 -hydroxy-o-methylsterigmatocystin (HOMST) 61), 62) OMST HOMST, OMST dihydro DHOMST DHHOMST OrdA
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 1 3 2 1 62) 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
June 2011 143 AF 7. A. parasiticus AFB 1,AFB 2,AFG 1,AFG 2 A. flavus B-AF (AFB 1,AFB 2 ) AF A. flavus AF 1 1.5 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
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 11. 2011 1960 AF X 51 AF AF AF AF AF AF AF AflR AflR AF AflR AF AF 1) 1 2004, p. 69 11. 2) Bennett, J. W., Klich, M. Mycotoxins. Clin. Microbiol. Rev., 16, 497 516 (2003). 3) Cole, R. J., Cox, R. H. Handbook of Toxic Fungal Metabolites. Academic Press, New York, 1981. 4) Kumagai, S. The fate of aflatoxin B 1 in animals. Mycotoxins, 60, 7 16 (2010). 5) Bull. Natl. Inst. Health Sci., 124, 21 29 (2006). 6) B1, B2, G1 G2 http://www.mhlw.go.jp/shingi/2010/ 05/dl/s0518-10k 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, 1270 1277 (1994). 9) Ono, M., Sakuda, S., Suzuki, A., Isogai, A. Aflastatin A, a novel inhibitor of aflatoxin production by aflatoxigenic fungi. J. Antibiot., 50, 111 118 (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, 2738 2740 (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, 7466 7473 (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, 1111 1117 (2008). 13) Manabe, M., Tsuruta, O. Geographical distribution of aflatoxin-producing fungi in Southeast Asia. JARQ, 12, 224 227 (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, 77 81 (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, 2665 2673 (1995). 16) Yabe, K., Nakajima, H. Enzyme reactions and genes in aflatoxin biosynthesis. Appl. Microbiol. Biotechnol., 64, 745 755 (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, 1253 1262 (2004). 18) Aspergillus flavus and aflatoxin. http://www.aspergillusflavus.org/ 19) Ehrlich, K. C. Predicted roles of the uncharacterized clustered genes in aflatoxin biosynthesis. Toxins, 1, 37 58 (2009).
June 2011 145 20) Payne, G. A., Nystrom, G. J., Bhatnagar, D., Cleveland, T. E., Woloshuk, C. P. Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. Appl. Environ. Microbiol., 59, 156 162 (1993). 21) Smith, C. A., Woloshuk, C. P., Robertson, D., Payne, G. A. Silencing of the aflatoxin gene cluster in a diploid strain of Aspergillus flavus is suppressed by ectopic aflr expression. Genetics, 176, 2077 2086 (2007). 22) Dogan: http://www.bio.nite.go.jp/dogan/top 23) Machida, M. et al. Genome sequencing and analysis of Aspergillus oryzae. Nature, 438, 1157 1161 (2005). 24) Tominaga, M., Lee, Y.-H., Hayashi, R., Suzuki, Y., Yamada, O., Sakamoto, K., Gotoh, K., Akita, O. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Appl. Environ. Microbiol., 72, 484 490 (2006). 25) Matsushima, K., K. Yashiro, Matsushima, K. Yashiro, K. Hanya, Y. Abe, K. Yabe, K. Hamasaki, T. Absence of aflatoxin biosynthesis in koji mold (Aspergillus sojae). Appl. Microbiol., Biotech., 55, 771 776 (2001). 26) Mahanti, N., Bhatnagar D., Cary, J. W., Joubran, J., Linz, J. E. Structure and function of fas-1a, a gene encoding a putative fatty acid synthase directly involved in aflatoxin biosynthesis in Aspergillus parasiticus. Appl. Environ. Microbiol., 62, 191 195 (1996). 27) Townsend, C. A., Christensen, S. B., Trautwein, K. Hexanoate as a starter unit in polyketide synthesis. J. Am. Chem. Soc., 106, 3868 3869 (1984). 28) Feng, G. H., Leonard, T. J. Characterization of the polyketide synthase gene (pksl) required for aflatoxin biosynthesis in Aspergillus parasiticus. J. Bacteriol., 177, 6246 5624 (1995). 29) Chang, P. K., Cary, J. W., Yu, J., Bhatnagar, D., Cleveland, T. E. Aspergillus parasiticus polyketide synthetase gene, pksa, a homolog of Aspergillus nidulans wa, required for aflatoxin B 1. Mol. Gen. Genet., 248, 270 277 (1995). 30) Yabe, K., Nakamura, Y., Nakajima, H., Ando, Y., Hamasaki, T. Enzymatic conversion of norsolorinic acid to averufin in aflatoxin biosynthesis. Appl. Environ. Microbiol., 57, 1340 1345 (1991). 31) Trail, F., Chang, P. K., Cary, J, Linz, J. Structural and functional analysis of the nor-1 gene involved in the biosynthesis of aflatoxins by Aspergillus parasiticus. Appl. Environ. Microbiol., 60, 4078 4085 (1994). 32) Yabe, K., Matsuyama, Y., Ando, Y., Nakajima, H., Hamasaki, T. Stereochemistry during aflatoxin biosynthesis: Conversion of norsolorinic acid to averufin. Appl. Environ. Microbiol., 59, 2486 2492 (1993). 33) Yu, J., Chang, P. K., Cary, J. W., Bhatnagar, D., Cleveland, T. E. avna, a gene encoding a cytochrome P- 450 monooxygenase is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in Aspergillus parasiticus. Appl. Microbiol. Biotechnol., 63, 1349 1356 (1997). 34) Chang, P. K., Yu, J., Ehrlich, K. C., Boue, S. M., Montalbano, B. G., Bhatnagar, D., Cleveland, T. E. The aflatoxin biosynthesis gene adha in Aspergillus parasiticus is involved in conversion of 5 -hydroxyaverantin to averufin. Appl. Environ. Microbiol., 66, 4715 4719 (2000). 35) Sakuno, E., Yabe, K., Nakajima, H. Involvement of two cytosolic enzymes and a novel intermediate, 5 oxoaverantin, in the pathway from 5 -hydroxyaverantin to averufin in aflatoxin biosynthesis. Appl. Environ. Microbiol., 69, 6418 6426 (2003). 36) Sakuno, E., Wen, Y., Hatabayashi, H., Arai, H., Aoki, C., Yabe, K., Nakajima, H. Aspergillus parasiticus cyclase catalyzes two dehydration steps in aflatoxin biosynthesis. Appl. Environ. Microbiol., 71, 2999 3006 (2005). 37) Yabe, K., Chihaya, N., Hamamatsu, S., Sakuno, E., Hamasaki, T., Nakajima, H., Bennett, J. W. Enzymatic conversion of averufin to hydroxyversicolorone and elucidation of a novel metabolic grid involved in aflatoxin biosynthesis. Appl. Environ. Microbiol., 69, 66 73 (2003). 38) Yu, J., Woloshuk, C. P., Bhatnagar, D., Cleveland, T. E. Cloning and characterization of avfa and omtb genes involved in aflatoxin biosynthesis in three Aspergillus species. Gene, 248, 157 167 (2000). 39) Wen, Y., Hatabayashi, H., Arai, H., Kitamoto, H. K., Yabe, K. Function of the cypx and moxy genes in aflatoxin biosynthesis in Aspergillus parasiticus. Appl. Environ. Microbiol., 71, 3192 3198 (2005). 40) Miyamoto, M., Matsumoto, J., Iwaya, T., Itagaki, E. Bacterial steroid monooxygenase catalyzing the Baeyer Villiger oxidation of C21-ketosteroids from Rhodococcus rhodochrous: The isolation and characterization. Biochim. Biophys. Acta, 1251, 115 124 (1995). 41) Chang, P. K., Yabe, K., Yu, J. The Aspergillus parasiticus esta-encoded esterase converts versiconal hemiacetal acetate to versiconal and versiconol acetate to versiconol in aflatoxin biosynthesis. Appl. Environ. Microbiol., 70, 3593 3599 (2004). 42) Bennett, J. W., Lee, L. S., Cucullu, A. F. E#ect of dichlorvos on aflatoxin and versicolorin A production in Aspergillus parasiticus. Bot. Gaz., 137, 318 324 (1976). 43) Yabe, K., Ando, Y., Hamasaki, T. A metabolic grid among versiconal hemiacetal acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis. J. Gen. Microbiol., 137, 2469 2475 (1991). 44) Shima, Y., Shiina, M., Shinozawa, T., Ito, Y., Nakajima, H., Adachi, Y., Yabe, K. Participation in aflatoxin biosynthesis by a reductase enzyme encoded by vrda gene outside the aflatoxin gene cluster. Fungal. Genetics and Biology, 46, 221 231 (2009). 45) McGuire, S. M., Silva, J. C., Casillas, E. G., Townsend, C. A. Purification and characterization of versicolorin B synthase from Aspergillus parasiticus. Catalysis of the stereodi#erentiating cyclization in aflatoxin biosynthesis essential to DNA interaction. Biochemistry, 35, 11470 11486 (1996). 46) Silva, J. C., Minto, R. E., Barry, III C. E., Holland, K. A., Townsend, C. A. Isolation and characterization of the versicolorin B synthase gene from Aspergillus parasiticus: Expression of the aflatoxin B 1 biosynthetic clus-
146 Vol. 52, No. 3 ter. J. Biol. Chem., 271, 13600 13608 (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, 2493 2500 (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, 1907 1911 (1991). 49) Bhatnagar, D., Ehrlich, K. C., Cleveland, T. E. Molecular genetic analysis and regulation of aflatoxin biosynthesis. Appl. Microbiol. Biotechnol., 61, 83 93 (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, 3527 3537 (1992). 51) Ehrlich, K. C., Montalbano, B., Boue, S. M., Bhatnagar, D. An aflatoxin biosynthesis cluster gene encodes a novel oxidase required for conversion of versicolorin A to sterigmatocystin. Appl. Environ-Microbiol., 71, 8963 8965 (2005). 52) Cary, J. W., Ehrlich, K. C., Bland, J. M., Montalbano, B. G. The aflatoxin biosynthesis cluster gene, aflx, encodes an oxidoreductase involved in conversion of versicolorin A to demethylsterigmatocystin. Appl. Environ. Microbiol., 72, 1096 1101 (2006). 53) Yu, J., Bhatnagar, D., Cleveland, T. E. Completed sequence of aflatoxin pathway gene cluster in Aspergillus parasiticus. FEBS Lett., 564, 126 130 (2004). 54) Yabe, K., Ando, Y., Hashimoto, J., Hamasaki, T. Two distinct O-methyltransferases in aflatoxin biosynthesis. Appl. Environ. Microbiol., 55, 2172 2177 (1989). 55) Yabe, K., Matsushima, K. I., Koyama, T., Hamasaki, T. Purification and characterization of O-methyltransferase I involved in conversion of demethylsterigmatocystin to sterigmatocystin and of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. Appl. Environ. Microbiol., 64, 166 171 (1998). 56) Motomura, M., Chihaya, N., Shinozawa, T., Hamasaki, T., Yabe, K. Cloning and characterization of the O- methyltransferase I gene (dmta) from Aspergillus parasiticus associated with the conversion of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigatocystin in aflatoxin biosynthesis. Appl. Environ. Microbiol., 65, 4987 4994 (1999). 57) Yu, J., Cary, J. W., Bhatnagar, D., Cleveland, T. E., Keller, N. P., Chu, F. S. Cloning and characterization of a cdna from Aspergillus parasiticus encoding an O- methyltransferase involved in aflatoxin biosynthesis. Appl. Environ. Microbiol., 59, 3564 3571 (1993). 58) Prieto, R., Woloshuk, C. P. ord 1, an oxidoreductase gene responsible for conversion of O-methylsterigmatocystin to aflatoxin in Aspergillus flavus. Appl. Environ. Microbiol., 63, 1661 1666 (1997). 59) Yu, J., Chang, P. K., Ehrlich, K. C., Cary, J. W., Montalbano, B., Dyer, J. M., Bhatnagar, D., Cleveland, T. E. Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P-450 monooxygenase encoded by orda that is involved in the biosynthesis of aflatoxins B 1,G 1,B 2, and G 2. Appl. Environ. Microbiol., 64, 4834 4841 (1998). 60) Yabe, K., Nakamura, M., Hamasaki, T. Enzymatic formation of G-group aflatoxins and biosynthetic relationship between G- and B-group aflatoxins. Appl. Environ. Microbiol., 65, 3867 3872 (1999). 61) Udwary, D. W., Casillas, L. K., Townsend, C. A. Synthesis of 11 -hydroxyl O-methylsterigmatocystin and the role of a cytochrome P-450 in the final step of aflatoxin biosynthesis. J. Am. Chem. Soc., 124, 5294 5303 (2002). 62) Zeng, H., Hatabayashi, H., Nakagawa, H., Cai, J., Suzuki, R., Sakuno, E., Tanaka, T., Ito, Y., Ehrlich, K. C., Nakajima, H., Yabe, K. Conversion of 11-hydroxy- O-methylsterigmatocystin to aflatoxin G 1 in Aspergillus parasiticus. Appl. Microbiol. Biotechnol., 90, 635 650 (2011). 63) Ehrlich, K. C., Chang, P. K., Yu, J., Cotty, P. J. Aflatoxin biosynthesis cluster gene cypa is required for G aflatoxin formation. Appl. Environ. Microbiol., 70, 6518 6524 (2004). 64) Cai, J., Zeng, H., Shima, Y., Hatabayashi, H., Nakagawa, H., Ito, Y., Adachi, Y., Nakajima, H., Yabe, K. Involvement of the nada gene in formation of G-group aflatoxins in Aspergillus parasiticus. Fungal Genet. Biol., 45, 1081 1093 (2008). 65) Kusumoto, K., Nagata, Y., Ohta, H. Directed deletions in the aflatoxin biosynthesis gene homolog cluster of Aspergillus oryzae. Curr. Genet., 37, 104 111 (2000). 66) Schmidt-Heydt, M., Häckel, S., Rüfer, C. E., Geisen, R. A strain of Fusarium kyushuense is able to produce aflatoxin B 1 and G 1 Mycotox. Res., 25, 141 147 (2009). 67) Brown, D. W., Yu, J. H., Kelkar, H. S., Fernandes, M., Nesbitt, T. C., Keller, N. P., Adams, T. H., Leonard, T. J. Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans. Proc. Natl. Acad. Sci. U.S.A., 93, 1418 1422 (1996). 68) Keller, N. P., Hohn, T. M. Metabolic pathway gene clusters in filamentous fungi. Fungal Genet. Biol., 21, 17 29 (1997). 69) Hamasaki, T., Hatsuda, Y., Terashima, N., Renbutsu, M. Studies on the metabolites of Aspergillus versicolor (Vuillemin) Tiraboshi. Part V. Isolation and structure of three new metabolites, versicolorins A, B and C. Agric. Biol. Chem., 31, 11 17 (1967). 70) Aucamp, P. J., Holzapfel, C. W. Polyhydroxyanthraquinones from Aspergillus versicolor, Aspergillus nidulans and Bipolaris sp.: Their significance in relation to biogenetic theories on aflatoxin B 1. J. S. Afr. Chem. Inst., 23, 40 56 (1970). 71) Sakuno, E., Kameyama, M., Nakajima, H., Yabe, K. Purification and gene cloning of a dehydrogenase from Lactobacillus brevis that catalyzes a reaction involved
June 2011 147 in aflatoxin biosynthesis. Biosci. Biotechnol. Biochem. 72, 724 734 (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, 19533 19538 (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, 35 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, 911 920 (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, 3713 3717 (1998). 76) Shwab, E. K., Keller, N. P. Regulation of secondary metabolite production in filamentous ascomycetes. Micol. Res., 112, 225 230 (2008). 77) Amaike, S., Keller, N. P. Distinct roles for VeA and LaeA in development and pathogenesis of Aspergillus flavus. Eukaryotic Cell, 2009, 1051 1060 (2009).