Characterization of the function of the ver-1A and ver-1B genes, involved in aflatoxin biosynthesis in Aspergillus parasiticus

Abstract

The ver-1A gene was cloned and its nucleotide sequence was determined as part of a previous study on aflatoxin B 1 (AFB 1) biosynthesis in the filamentous fungus Aspergillus parasiticus SU-1. A second copy of this gene, ver-1B, was tentatively identified in this fungal strain. In this study, ver-1B was cloned by screening an A. parasiticus cosmid library with a ver-1A probe. The nucleotide sequence of ver-1B was determined. The predicted amino acid sequence of ver-1B had 95% identity with ver-1A. A translational stop codon, found in the ver-1B gene coding region, indicated that it encodes a truncated polypeptide. To confirm the function of the ver-1 genes in AFB 1 synthesis, a plasmid (pDV-VA) was designed to disrupt ver-1A and/or ver-1B by transformation of the AFB 1 producer A. parasiticus NR-1. One disruptant, VAD-102, which accumulated the pathway intermediate versicolorin A was obtained. Southern hybridization analysis of VAD-102 revealed that ver-1A but not ver-1B was disrupted. A functional ver-1A gene was transformed back into strain VAD-102. Transformants which received ver-1A produced AFB 1 , confirming that ver-1A is the only functional ver-1 gene in A. parasiticus SU-1 and that its gene product is involved in the conversion of versicolorin A to sterigmatocystin in AFB 1 biosynthesis. A duplicated chromosomal region (approximately 12 kb) was identified upstream from ver-1A and ver-1B by Southern hybridization analysis. This duplicated region contained the aflR gene, which is proposed to be one regulator of AFB 1 synthesis. A similar gene duplication was also identified in several other strains of A. parasiticus.

Genetic organization and function of the aflatoxin B1 biosynthetic genes

“…A second gene, ver1B, was identi¢ed in A. parasiticus that has 95 percent identity to ver1A. ver1B is truncated rendering it non-functional and the gene is not located in the gene cluster [18]. Gene-disruption of stcU, a homolog of ver1A, in a sterigmatocystin-producing strain of A. nidulans resulted in VER A accumulation [20].…”

“…£avus and A. parasiticus, and they estimated the cluster size to be 75 kb. The relative position of genes within the clusters and the nucleotide sequence of the genes are similar between the two species, although A. parasiticus appears to have an additional copy of several genes indicating that at least part of the cluster is duplicated [17,18].…”

Analysis of mechanisms regulating expression of the ver-1 gene, involved in aflatoxin biosynthesis

“…It is a complex pathway which requires 17 or more different enzymatic activities (5,16). Several genes directly involved in AFB 1 syn-thesis have been cloned, and the functions of their gene products have been characterized; these genes include nor-1, which is involved in conversion of norsolorinic acid to averantin (11,47); ver-1A, involved in the conversion of versicolorin A to demethylsterigmatocystin (26,42); omtA, involved in the conversion of sterigmatocystin to O-methyl-sterigmatocystin (49); and the fas-1A (28) and pksA genes (10,48), which are involved in the synthesis of the AFB 1 polyketide backbone. The aflR gene, encoding a protein which is proposed to be a positive regulator of aflatoxin pathway function, was identified in A. flavus (33) and A. parasiticus (9).…”

Tetrahydroxynaphthalene Reductase: Catalytic Properties of an Enzyme Involved in Reductive Asymmetric Naphthol Dearomatization

“…While mono‐ or dioxygenases are involved in the aerobic pathways, oxidoreductases are required for the anaerobic metabolism, thus leading to the formation of acetyl coenzyme A. Additionally, polyphenolic polyketide synthase (PKS) products degrade by a reduction–dehydration sequence during secondary metabolite synthesis 1. This deoxygenation strategy was found as a key step in 1,8‐dihydroxynaphthalene (DHN, 1 )‐melanin biosynthesis of various fungi2–6 and is also proposed for the formation of aflatoxin,7, 8 actinorhodin,9 and chrysophanol 10. 11 Furthermore, polyhydroxynaphthalenes represent branching points in several secondary metabolite syntheses 12–14.…”