Description:
<jats:title>Abstract</jats:title><jats:p>Norsolorinic acid synthase (NSAS) is a type I iterative polyketide synthase that occurs in the filamentous fungus <jats:italic>Aspergillus parasiticus</jats:italic>. PCR was used to clone fragments of NSAS corresponding to the acyl carrier protein (ACP), acyl transferase (AT) and β‐ketoacyl‐ACP synthase (KS) catalytic domains. Expression of these gene fragments in <jats:italic>Escherichia coli</jats:italic> led to the production of soluble ACP and AT proteins. Coexpression of ACP with <jats:italic>E. coli holo</jats:italic>‐ACP synthase (ACPS) let to production of NSAS <jats:italic>holo</jats:italic>‐ACP, which could also be formed in vitro by using <jats:italic>Streptomyces coelicolor</jats:italic> ACPS. Analysis by mass spectrometry showed that, as with other type I carrier proteins, self‐malonylation is not observed in the presence of malonyl CoA alone. However, the NSAS <jats:italic>holo</jats:italic>‐ACP serves as substrate for <jats:italic>S. coelicolor</jats:italic> MCAT, <jats:italic>S. coelicolor</jats:italic> actinorhodin <jats:italic>holo</jats:italic>‐ACP and NSAS AT domain‐catalysed malonate transfer from malonyl CoA. The AT domain could transfer malonate from malonyl CoA to NSAS <jats:italic>holo</jats:italic>‐ACP, but not hexanoate or acetate from either the cognate CoA or FAS ACP species to NSAS <jats:italic>holo</jats:italic>‐ACP. The NSAS <jats:italic>holo</jats:italic>‐ACP was also active in actinorhodin minimal PKS assays, but only in the presence of exogenous malonyl transferases.</jats:p>