Roles of type II thioesterases and their application for secondary metabolite yield improvement

Abstract

A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an "assembly line" and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such "unnatural" natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)--discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.

Fig. 1

Roles of type II thioesterases (TEIIs) associated with polyketide synthases and nonribosomal peptide synthetases. Wavy line represents 4′-phosphopantetheine (4′PP). KS ketosynthase, AT acyltransferase, ACP acyl carrier protein, PCP peptidyl carrier protein. a Normal polyketide chain elongation starts with a decarboxylative condensation catalyzed by the KS domain. Incorrect decarboxylation of an extender unit without condensation gives rise to an acyl residue attached to the ACP. Editing TEII hydrolyzes the acyl residue and leaves the ACP free to accept a new, dicarboxylated extender unit transferred by the AT domain. b ACP and PCP domains are primed with a cofactor—4′PP—which is transferred by a 4′PP transferase from coenzyme A (CoA). 4′PP transferases can also accept acyl-CoA as a substrate and misprime carrier domains with acyl-4′PP, thus making the attachment site inaccessible for an extender unit. Editing TEII hydrolyzes the acyl residue and leaves the carrier domain free to accept a correct substrate. c TEII influences the choice of the starter unit by specific hydrolysis of some residues attached to the ACP of the loading module. The residue which is not cleaved becomes a starting unit for the polyketide chain. d Promiscuous TEIIs can remove amino acids and polyketide synthesis intermediates, including correct dicarboxylated extender units, if they are not readily processed by downstream domains. e TEIIs specific for certain moieties which are required for the construction of the full structure of the metabolite release them from specific carriers to make them available for the next biosynthetic step. Some TEIIs release complete PKS/NRPS products from the multienzyme