On the production of α,β-heterodimeric acyl-coenzyme A: isopenicillinN-acyltransferase ofPenicillium chrysogenum

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Abstract
A high level E. coli expression system has been constructed for the Penicillium chrysogenum penDE gene, which encodes the acyl‐coenzyme A: isopenicillin N‐acyltransferase (AT) enzyme. Induction of overexpression of recombinant AT (recAT) by increasing the growth temperature of the host adversely affected solubility and activity of the AT enzyme. Addition of isopropylthio‐β‐d‐galactopyranoside (IPTG) at decreased growth temperatures (less than 32°C) resulted in the overproduction of soluble, active recAT. When purified to homogeneity, recAT was an α,β‐heterodimer, comprised of 11 kDa (α) and 29 kDa (β) subunits, derived from a 40 kDa precursor polypeptide by a posttranslational cleavage. The recAT enzyme contained both the acyl‐coenzyme A: isopenicillin N‐acyltransferase and the acyl‐coenzyme A: 6‐aminopenicillanic acid acyltransferase activities. The processing event that generated the two subunits of recAT from the 40 kDa precursor polypeptide occurred between Gly102/Cys103. This expression system produced a large amount of soluble, active recAT that is identical to native AT, making it a suitable source of AT enzyme for further characterization.