Engineering Aspergillus oryzae for the Heterologous Expression of a Bacterial Modular Polyketide Synthase

Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may lead to molecules with enhanced biological activities. In this study, we modified the metabolism of the fungus Aspergillus oryzae to enable the synthesis of triketide lactone (TKL), the product of the modular polyketide synthase DEBS1-TE engineered from bacteria. We established (2S)-methylmalonyl-CoA biosynthesis via introducing a propionyl-CoA carboxylase complex (PCC); reassembled the 11.2 kb DEBS1-TE coding region from synthetic codon-optimized gene fragments using yeast recombination; introduced bacterial phosphopantetheinyltransferase SePptII; investigated propionyl-CoA synthesis and degradation pathways; and developed improved delivery of exogenous propionate. Depending on the conditions used titers of TKL ranged from <0.01–7.4 mg/L. In conclusion, we have demonstrated that A. oryzae can be used as an alternative host for the synthesis of polyketides from bacteria, even those that require toxic or non-native substrates. Our metabolically engineered A. oryzae may offer advantages over current heterologous platforms for producing valuable and complex natural products.

Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase

Polyketides, one of the largest classes of natural products, are often clinically relevant. The ability to engineer polyketide biosynthesis to produce analogs is critically important. Acyltransferases (ATs) of modular polyketide synthases (PKSs) catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. ATs have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, limiting the scope of AT engineering. Here, molecular dynamics simulations are used to prioritize ~50 mutations within the active site of EryAT6 from erythromycin biosynthesis, leading to identification of two previously unexplored structural motifs. Exchanging both motifs with those from ATs with alternative extender specificities provides chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy are expected to advance our ability to engineer PKSs towards designer polyketides.

Successes, surprises and pitfalls in modular polyketide synthase engineering: generation of ring-contracted stambomycins

The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combined multiple, state-of-the-art approaches including modification of docking domains, use of modules of varying domain composition, alternative interdomain fusion sites, and targeted adaptation of key domain-domain interfaces, to reprogram the stambomycin PKS by deleting seven internal modules – the most substantial modification to an intact system reported to date. One such system produced the target 37-membered mini-stambomycin metabolites, a reduction in chain length of 14 carbons relative to the 51-membered parental compounds, but also substantial quantities of shunt metabolites released from the multienzyme subunit upstream of the newly-installed junction. Our data also provide evidence for an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain acting on chains located four modules upstream. The yields of all metabolites were substantially reduced compared to the wild type compounds, likely reflecting the poor tolerance to the non-native substrates of the modules downstream of the introduced interfaces. Taken together, our data demonstrate that even ‘optimized’ PKS engineering strategies remain inadequate for efficient production of target polyketide derivatives, and highlight several areas for future investigation.

Structure and mechanism of a dehydratase/decarboxylase enzyme couple involved in polyketide β-methyl branch incorporation

Complex polyketides of bacterial origin are biosynthesised by giant assembly-line like megaenzymes of the type 1 modular polyketide synthase (PKS) class. The trans-AT family of modular PKSs, whose biosynthetic frameworks diverge significantly from those of the archetypal cis-AT type systems represent a new paradigm in natural product enzymology. One of the most distinctive enzymatic features common to trans-AT PKSs is their ability to introduce methyl groups at positions β to the thiol ester in the growing polyketide chain. This activity is achieved through the action of a five protein HCS cassette, comprising a ketosynthase, a 3-hydroxy-3-methylglutaryl-CoA synthase, a dehydratase, a decarboxylase and a dedicated acyl carrier protein. Here we report a molecular level description, achieved using a combination of X-ray crystallography, in vitro enzyme assays and site-directed mutagenesis, of the bacillaene synthase dehydratase/decarboxylase enzyme couple PksH/PksI, responsible for the final two steps in β-methyl branch installation in this trans-AT PKS. Our work provides detailed mechanistic insight into this biosynthetic peculiarity and establishes a molecular framework for HCS cassette enzyme exploitation and manipulation, which has future potential value in guiding efforts in the targeted synthesis of functionally optimised ‘non-natural’ natural products.