Evolutionary Imprint of Catalytic Domains in Fungal PKS–NRPS Hybrids

Abstract

Fungal hybrid enzymes consisting of a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) module are involved in the biosynthesis of a vast array of ecologically and medicinally relevant natural products. Whereas a dozen gene clusters could be assigned to the requisite PKS-NRPS pathways, the programming of the multifunctional enzymes is still enigmatic. Through engineering and heterologously expressing a chimera of PKS (lovastatin synthase, LovB) and NRPS (cytochalasin synthase, CheA) in Aspergillus terreus, we noted the potential incompatibility of a fungal highly reducing PKS (hrPKS) with the NRPS component of fungal PKS-NRPS hybrids. To rationalize the unexpected outcome of the gene fusion experiments, we conducted extensive bioinformatic analyses of fungal PKS-NRPS hybrids and LovB-type PKS. From motif studies and the function of the engineered chimeras, a noncanonical function of C-terminal condensation (C) domains in truncated PKS-NRPS homologues was inferred. More importantly, sequence alignments and phylogenetic trees revealed an evolutionary imprint of the PKS-NRPS domains, which reflect the evolutionary history of the entire megasynthase. Furthermore, a detailed investigation of C and adenylation (A) domains provides support for a scenario in which not only the A domain but also the C domain participates in amino acid selection. These findings shed new light on the complex code of this emerging class of multifunctional enzymes and will greatly facilitate future combinatorial biosynthesis and pathway engineering approaches towards natural product analogues.

Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering

“…Exchange of amino acids in fungal polyketide-nonribosomal peptide products have been attempted a number of times by swapping entire NRPS modules [9,16,37]. An alternative and perhaps easier approach when working with these large genes, could be to simply change the A domain specificity by introduction of point mutations.…”

“…An alternative and perhaps easier approach when working with these large genes, could be to simply change the A domain specificity by introduction of point mutations. Several models for prediction of the specificity-conferring amino acids of bacterial and fungal NRPS A domains have been published [37–41]. The specificity of bacterial NRPS A domains is well-established; however, the identity of the specificity-conferring amino acids of fungal A domains is much less characterized, which is perhaps due to a more complex mechanism of amino acid selectivity compared to bacterial A domains.…”

Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi

“…Occasionally, epimerase (E) and N -methyltransferase (M) domains that convert L- to D-amino acids and N -methylate peptide bonds, respectively, are present within the NRPS [14, 15]. Deviations of the classical NRPS composition can be found in hybrid PKS/NRPS enzymes [16] (e.g., Fus1 responsible for fusarin C biosynthesis) and stand-alone monomodular NRPS-like enzymes [17] (e.g., TdiA responsible for terrequinone A biosynthesis) where not all canonical domains are present (Fig. 1).…”

Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea

“…PKS modules are programmed and produce polyketide chains with different reduction and methylation patterns (Fig. 3) and NRPS modules are responsible for fusing the polyketide chain to an amino acid (tyrosine) (Boettger et al, 2012) and an offloading domain for release (Du and Lou, 2010). In this case, acetate is extended seven times with seven unites of malonate by PKS modules, programmed C-methylations occur after the first three and the sixth extensions, and cycles of full reduction occur after the first two extensions (Fisch et al, 2011).…”

“…A typical PKS-NRPS hybrid consists of an iterative PKS module followed by a single NRPS module. The hybrid prudently combines the versatile nature of acetate-derived polyketide chains with the more than 300 proteinogenic and nonproteinogenic amino acids that could be incorporated by NRPS modules (Boettger et al, 2012;Fisch, 2013). Among this class of natural products, an array of pyridones is well known to be produced by various fungi and more importantly even by entomogenous fungi (Haga et al, 2013;Hosoya et al, 2013;Ma et al, 2011;Peng et al, 2011).…”