Combinatorialization
of Fungal Polyketide Synthase–Peptide
Synthetase Hybrid Proteins
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Abstract
The
programming of the fungal polyketide synthase (PKS) is quite
complex, with a simple domain architecture leading to elaborate products.
An additional level of complexity has been found within PKS-based
pathways where the PKS is fused to a single module nonribosomal peptide
synthetase (NRPS) to synthesize polyketides conjugated to amino acids.
Here, we sought to understand the communication between these modules
that enable correct formation of polyketide-peptide hybrid products.
To do so, we fused together the genes that are responsible for forming
five highly chemically diverse fungal natural products in a total
of 57 different combinations, comprising 34 distinct module swaps.
Gene fusions were formed with the idea of testing the connection and
compatibility of the PKS and NRPS modules mediated by the acyl carrier
protein (ACP), condensation (C) and ketoreductase (KR) domains. The
resulting recombinant gene fusions were analyzed in a high-yielding
expression platform to avail six new compounds, including the first
successful fusion between a PKS and NRPS that make highly divergent
products, and four previously reported molecules. Our results show
that C domains are highly selective for a subset of substrates. We
discovered that within the highly reducing (hr) PKS class, noncognate
ACPs of closely related members complement PKS function. We intercepted
a pre-Diels–Alder intermediate in lovastatin synthesis for
the first time, shedding light on this canonical fungal biochemical
reaction. The results of these experiments provide a set of ground
rules for the successful engineering of hr-PKS and PKS-NRPS products
in fungi