Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli

Abstract

Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKSWJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKSWJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.cyclase ͉ ketosynthase ͉ megasynthase

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

“…Indeed, while compound 4 can be recovered from the cell pellets of E. coli expressing PKSH1, no product can be detected from E. coli expressing different domains as stand-alone proteins. This was similarly demonstrated using PKS4 minimal PKS in E. coli, where artificially linking the KS-MAT with ACP led to significant improvement in product turnover in vivo (19).…”

“…rantly cyclized products 2 and 3, confirming the role of the PT domain in catalyzing the C2-C7 aldol condensation (19).…”

“…The G. fujikuroi PKS4 is well-suited to serve as the template for PT domain swapping because (i) the megasynthases can be expressed at high levels and the minimal PKS (KS-MAT, ACP) can produce ample amounts of the nonaketide products in vitro, which facilitates product structure elucidation; (ii) the minimal PKS domain does not significantly influence the cyclization regioselectivity of the nascent polyketide (19). We therefore anticipated that a heterologous PT could dictate the cyclization outcome; and (iii) other built-in cyclization domain, such as the TE/CLC domain can be removed from the assembly line without affecting the product elongation and turnover.…”

“…Ma et al (23) have reconstituted the intact bikaverin synthase PKS4 (from Gibberella fujikuroi) in E. coli to synthesize the C2-C7 cyclized nonaketide SMA76a. Systematic removal of individual domains from the intact PKS4 was then conducted to verify the functional roles of the TE and PT domains (18,19). Removal of the TE/CLC domain (named PKS4 -99) produced the C2-C7 cyclized SMA93 1 and confirmed the role of the TE/CLC in catalyzing the C1-C10 Claisen reaction (18) (Fig.…”

A comprehensive overview on genomically directed assembly of aromatic polyketides and macrolide lactones using fungal megasynthases

“…Furthermore, mixing single-domain enzymes from PKS4 and type II PKS, revealed both distinct enzyme cross talk allowing biosynthesis of aromatic compounds along with regioselectively cyclization reaction. 28 The results reported by Tang and co-workers showcased a b-ketoreductase (KR) domain of a bacterial PKS, actinorhodin biosynthetic enzyme, and its capacity to catalyze a reduction at C9 of a poly-b-keton core structure that is produced by a fungal PKS. Additionally, the stand-alone KR domain was able to produce 5 in the absence of a starter unit that is pre-cyclized at the C7-C12 position ( Figure 7).…”