To determine if the protein encoded by the as yet uncharacterized aflatoxin biosynthesis gene, hypB1, is an anthrone oxidase and to investigate whether this protein is required for formation of norsolorinic acid.
Approach: (1) Clone the codon-optimized coding sequence of hypB1 into pET28A vector and induce protein production in E. Coli. Purify the protein using nickel column purification procedures. (2) Prepare norsolorinic acid anthrone by reduction of norsolorinic acid using reagents used for preparation of other the anthrones from anthroquinones. Determine if the purified protein from step 1 catalyzes oxidation of the anthrone to norsolorinic acid. (3) Construct an expression vector containing the coding sequences for hypB1, the aflatoxin biosynthesis cluster polyketide synthase and the alpha and beta subunits of the hexanoyl synthase. Clone this vector into E. coli and induce protein production. Determine if norsolorinic acid is formed by the induced E. coli. Compare to polyketide production by controls containing various combinations of these initial aflatoxin biosynthetic genes.