Genetic engineering of non-reducing polyketide synthases by domain swap strategy in Aspergillus nidulans

2013-07-30Filamentous fungi are potent producers of secondary metabolites. Polyketides have been recognized as an important class of fungal secondary metabolites, which comprise polyphenols, polyenes and macrolides with diverse structures and bioactivies, such as the anti-cholesterol compound, lovastatin and the potent mycotoxin, aflatoxin B1. Fungal polyketides are synthesized by iterative multi-domain type I polyketide synthases (PKSs), which minimally consist of the KS, AT and ACP domain. Based the reductive degree during the biosynthesis, fungal PKSs can be grouped into three major subclasses: non-reducing PKS (NR-PKS), partially-reducing PKS (PR-PKS) and highly-reducing PKS (HR-PKS). NR-PKSs exclusively contain the starter unit ACP transacylase domain (SAT), the product template domain (PT) and the releasing domains. Although the catalytic activity of each domain is known, it remains mysterious how fungal PKSs diversify the chemical features of each extension cycle by iteratively using a single multi-domain module. In the case of NR-PKSs, the major question is how NR-PKSs control the product chain length and methylation. In light of the technological developments, rational domain swap strategy grows into a potential approach to expand the diversity of non-reduced polyketides and decipher the programming rules of NR-PKSs. ❧ Asperfuranone belongs to the azaphilone class of fungal natural product that has displayed a broad range of bioactivities such as inhibition of monamine oxidase, acyl-CoA:cholesterol acyltransferase, and cholesteryl ester transfer protein. Through a genome mining effort in Aspergillus nidulans, our group was able to uncover the biosynthetic pathway of asperfuranone. We reasoned that if we could engineer AfoE, the NR-PKS mediating asperfuranone biosynthesis, to accept different starter units, we would have the opportunity to create "unnatural" polyketide products in vivo. Therefore, we replaced the SAT domain in AfoE with the SAT domain from StcA, the NR-PKS in the sterigmatocystin biosynthesis pathway. SAT domain swap between AfoE and StcA resulted in the production of a naphthaquinone compound with a hexyl side chain. Interestingly, this hybrid metabolite presented the same chain length as asperfuranone. Further feeding studies supported our hypothesis that the chain length of non-reduced polyketides may be under stringent control, but not controlled by the SAT domain. ❧ To explore the potential of SAT swap, we conducted extensive SAT domain swaps between AfoE and nine other NR-PKSs in Aspergillus nidulans. Results revealed that SAT domain swap provided an efficient tool to alter the polyketide side chain, but might be restricted by the substrate specificity of the KS domain. Moreover, regardless of incorporating a longer or shorter starter unit, the chain length of non-reducing polyketides remained stringently controlled. ❧ Further extended domain swap between AfoE and AN3386 revealed that the KS domain is the principle determinant of not only the chain length but also the methylation pattern of non-reduced polyketides. Thus far the programming of NR-PKS has been fully deciphered, providing valuable guidance on fungal NR-PKS combinatorial biosynthesis in future