Engineered polyketides: Synergy between protein and host level engineering

Abstract

Metabolic engineering efforts toward rewiring metabolism of cells to produce new compounds often require the utilization of non-native enzymatic machinery that is capable of producing a broad range of chemical functionalities. Polyketides encompass one of the largest classes of chemically diverse natural products. With thousands of known polyketides, modular polyketide synthases (PKSs) share a particularly attractive biosynthetic logic for generating chemical diversity. The engineering of modular PKSs could open access to the deliberate production of both existing and novel compounds. In this review, we discuss PKS engineering efforts applied at both the protein and cellular level for the generation of a diverse range of chemical structures, and we examine future applications of PKSs in the production of medicines, fuels and other industrially relevant chemicals.

Keywords: ACP, Acyl carrier protein; AT, Acyltransferase; CoL, CoA-Ligase; Commodity chemical; DE, Dimerization element; DEBS, 6-deoxyerythronolide B synthase; DH, Dehydratase; ER, Enoylreductase; FAS, Fatty acid synthases; KR, Ketoreductase; KS, Ketosynthase; LM, Loading module; LTTR, LysR-type transcriptional regulator; Metabolic engineering; Natural products; PCC, Propionyl-CoA carboxylase; PDB, Precursor directed biosynthesis; PK, Polyketide; PKS, Polyketide synthase; Polyketide; Polyketide synthase; R, Reductase domain; SARP, Streptomyces antibiotic regulatory protein; SNAC, N-acetylcysteamine; Synthetic biology; TE, Thioesterase; TKL, Triketide lactone.

Fig. 1

Biosynthesis of 6-deoxyerythronolide and examples of both native and engineered polyketide synthases. A) Modular biosynthesis of 6-deoxyerythronolide by the well-studied 6-deoxyerythronolide polyketide synthase. B) The carboxylic acid starter unit promiscuity by the borrelidin PKS was utilized to produce adipic acid (C) from succinic acid and malonic acid using an engineered BorA2 containing a full reductive loop (highlighted in green circle) and a thioesterase. B) The broad starter unit selectivity of the lipomycin PKS was utilized to produce 3-hydroxyacid congeners from branched acids and methylmalonyl-CoA (C). The carbon backbone for both the native and engineered polyketides are represented in bold. Grey domains represent the native pathway and blue domains represent engineered insertions.

Fig. 2

Summary of polyketide synthase engineering strategies highlighted in this review. Starter unit selectivity and incorporation is mediated by either native or non-native swapped LMs. Intermolecular linker regions allow for successful communications between domains. Chemical diversity is further increased by varying the extender building blocks. AT mutagenesis or AT swaps mediate incorporation of various extender units into a polyketide intermediate. Various degrees of reduction at the β-keto position can be accomplished by KR mutagenesis, KR swaps and/or the insertion of full or partially full reductive loops, containing DH and ER domains. Release of the polyketide intermediate is mediated by various releasing domains, which further increase chemical diversity into the final product.

Fig. 3

Examples of loading modules. In type A LMs, the KS^Q decarboxylates malonyl- or methylmalonyl-CoA to yield acetyl- or propionyl starter units, respectively. Type B LMs consist of an AT that selects a CoA-bound priming unit and transfers it to its cognate ACP. Type C LMs consists of a CoL domain located upstream of an ACP. The CoL activates a carboxylic acid substrate in an ATP-dependent fashion in order to load it onto the ACP, either in cis or in trans. It is also common to find other accessory domains within type C LMs, between the CoL and ACP domain (represented by the black line between the cis CoL and ACP).

Fig. 4

Reducing domain(s) product outcomes. Potential stereochemical outcomes of each combination of β-carbon processing domains within a PKS module.

Fig. 5

Offloading domains. Common TE-mediated release include products such as linear acids, lactones, lactams, thiolactones and olefins. The less common R-domains conduct a two-electron reduction to produce aldehyde final products or primary alcohols through a four-electron reduction.

Fig. 6

Examples of precursor-directed biosynthesis altering priming unit. A) Starter unit PDB in the rapamycin PKS. B) Starter unit tolerance in spinosyn biosynthesis. C) Extender unit PDB using a diketide SNAC precursor in the erythromycin PKS. D) Incorporation of fluoromalonyl-CoA through mutasynthesis of the salinosporamide pathway. E) Incorporation of propargyl-, propyl- and allylmalonyl-SNAC into the monensin polyketide backbone. F) Incorporation of propargyl- and allylmalonyl-CoA into the kirromycin polyketide backbone.

Fig. 7

Examples of metabolic engineering for improved precursor pools. A) Overexpression of PCC for the conversion of propionyl-CoA to (2S)-methylmalonyl-CoA allowed for the production of 6-deoxyerythronolide B in bacteria that don't natively produce methylmalonyl-CoA (such as E. coli). B) Expression of the methoxymalonyl-ACP biosynthetic pathway in a platenolide producer generated a platenolide analog containing the methoxy moiety. C) Higher titers of mithramycin were achieved by increasing the precursor supply of malonyl-CoA and glucose-1-phosphate. Green boxes denote upregulated pathways and red cross marks denote downregulation or knockout pathways. D) Post-translational modification of apo-ACPs by PPTases generated active holo-ACPs which can activate FAS biosynthetic pathways in primary metabolism or PKS pathways in secondary metabolism.