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Review
. 2020 May 15:7:87.
doi: 10.3389/fmolb.2020.00087. eCollection 2020.

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

Affiliations
Review

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

Soonkyu Hwang et al. Front Mol Biosci. .

Abstract

In nature, various enzymes govern diverse biochemical reactions through their specific three-dimensional structures, which have been harnessed to produce many useful bioactive compounds including clinical agents and commodity chemicals. Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are particularly unique multifunctional enzymes that display modular organization. Individual modules incorporate their own specific substrates and collaborate to assemble complex polyketides or non-ribosomal polypeptides in a linear fashion. Due to the modular properties of PKSs and NRPSs, they have been attractive rational engineering targets for novel chemical production through the predictable modification of each moiety of the complex chemical through engineering of the cognate module. Thus, individual reactions of each module could be separated as a retro-biosynthetic biopart and repurposed to new biosynthetic pathways for the production of biofuels or commodity chemicals. Despite these potentials, repurposing attempts have often failed owing to impaired catalytic activity or the production of unintended products due to incompatible protein-protein interactions between the modules and structural perturbation of the enzyme. Recent advances in the structural, computational, and synthetic tools provide more opportunities for successful repurposing. In this review, we focused on the representative strategies and examples for the repurposing of modular PKSs and NRPSs, along with their advantages and current limitations. Thereafter, synthetic biology tools and perspectives were suggested for potential further advancement, including the rational and large-scale high-throughput approaches. Ultimately, the potential diverse reactions from modular PKSs and NRPSs would be leveraged to expand the reservoir of useful chemicals.

Keywords: domain; module; non-ribosomal peptide synthetase; polyketide synthase; repurposing.

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Figures

FIGURE 1
FIGURE 1
Domain architectures and mechanisms of polyketide chain extension in modular PKS. (A) Overall flow scheme of polyketide biosynthesis with different domain architectures of modules. Four types of loading modules load the different substrates according to involved domains (chemical examples were indicated). Next, the extender unit is selected and condensed to the growing chain one by one per elongation module for N cycles. Optional reductive domains (dashed circles) reduce the β-carbon group resulting in different X groups (indicated in red). Finally, the growing polyketide chain is cleaved by three different types of offloading domains in termination modules producing different products, including linear carboxylic acids, macrocyclic acids, olefins, aldehydes, and primary alcohols. (B) Mechanism of polyketide chain extension for the elongation modulen. (i) ACPn1 to KSn translocation; the active site cysteine moiety of KSn receives the growing polyketide chain of ACPn1. (ii) ATn acylation; the cognate acyl unit is incorporated into the active site serine moiety of ATn to form the acyl-O-AT intermediate. (iii) ATn to ACPn transacylation; the acyl group of ATn is transacylated to the ACPn. (iv) KSn to ACPn chain elongation; KSn catalyzes a decarboxylative Claisen condensation between the growing polyketide chain and the acyl extender unit of ACPn for the chain extension. (v) Processing; the extender units of ACPn are modified by a reductive loop or other additional domains. ACP, acyl carrier protein; AL, CoA ligase-type domain; AT, acyltransferase; CMT, C-methyltransferase; GNATL, GCN5 N-acetyltransferase-like domain; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; KS, ketosynthase; KSQ, condensation-incompetent ketosynthase; R, reductive domain; ST, sulfotransferase; TE, thioesterase.
FIGURE 2
FIGURE 2
Domain architectures and mechanisms of non-ribosomal peptide chain extension in modular NRPS. (A) Overall flow scheme of non-ribosomal peptide biosynthesis with different domain architectures of modules. Four representative types of loading modules load the different substrates according to the involved domains (chemical examples were indicated). Next, the extender unit is selected and condensed to the growing chain one by one per elongation module for N cycles. An example of optional processing domain is indicated by the dashed circles. Finally, the growing non-ribosomal peptide chain is cleaved by four representative types of offloading domains in termination modules, producing different products including linear peptides, macrocyclic peptides, aldehydes, and tetramate moieties. The terminal X group of the product from terminal R domain includes hydroxyl group (-OH), aldehyde group (-CHO), and other aldehyde derivatives (Barajas et al., 2015; Dan et al., 2019). (B) Mechanism of polyketide chain extension for the elongation modulen. (i) Tn1 to Cn translocation; the growing non-ribosomal peptide chain linked to Ppant arm of Tn1 domain translocates to the solvent channel of Cn domain donor site. (ii) An adenylation; the extender amino acid unit is activated by ATP to form aminoacyl-AMP in An domain. (iii) An to Tn thiolation; the aminoacyl-AMP intermediate of An is transferred to the Ppant arm of Tn domain to form aminoacyl thioester intermediate. (iv) Tn1 to Tn condensation at Cn; the aminoacyl thioester intermediate of Tn domain is translocated to the solvent channel of Cn domain acceptor site, and the peptide bond formation between the growing peptide of Tn1 domain and the amino acid extender unit of Tn domain elongates by adding one amino acid to the growing peptide. (v) Processing; the extender units of ACPn are modified by an epimerase (E) domain or other additional domains. A, adenylation domain; C, condensation domain; CT, terminal condensation domain; F, formylating domain; R, reductive domain; R*, R-like domain; T, thiolation domain; TE, thioesterase.
FIGURE 3
FIGURE 3
Engineering scheme for modular PKS and NRPS. (A) Engineering strategies of PKSs. (B) Engineering strategies of NRPSs. Gray circles and red circles indicate the original and modified domains, respectively. Green, brown, blue, and purple blocks, shaped as lock-and-key models, are the docking domains for (A) and COM domains for (B), respectively. Linkers were indicated as the lines between the domains. In case of (iii) domain and module exchange, the exchangeable units are indicated at the right of the domains. The units indicated as bold characters are currently the best exchangeable units. PDB, precursor-directed biosynthesis.
FIGURE 4
FIGURE 4
Representative repurposing examples of modular PKS and NRPS for de novo biosynthetic pathways. (A) Repurposing the PKS domains and modules for the production of short-chain ketones. Green circles are the domains in module 1 of β-lipomycin PKS (LIPS M1), red circles are the AT domains in module 1 of borrelidin PKS (BORS A1), gray circles with the red crossed line are the inactivated KR domain (KR null), and the blue circles are the TE domain of DEBS PKS. (B) Repurposing the PKS domains and modules for the production of adipic acid. Green circles are the domains in module 1 of borrelidin PKS (BORS M1), red circles are the KR and ER domain in SpnB module of spinosyn PKS (SpnB KR, ER), and the blue circles are the TE domain of DEBS PKS. (C) Repurposing the NRPS module for the production of thiopyrazines. NRPS325 module of ATEG00325 PKS-NRPS hybrid megasynthetase was isolated (red circles) to promote the reaction for the thiopyrazine production itself. (D) Repurposing the NRPS domain for the production of paclitaxel derivatives. The A or A-T didomain in TycA module of tyrocidine A PKS was isolated (red circles) to be repurposed for the production of phenylalanyl-, phenylisoserinyl-, arylisoserinyl-CoAs, which are the precursors of the paclitaxel derivatives; X, NH2 or H; Y, H or OH; Z, NH2 or H.
FIGURE 5
FIGURE 5
Roadmap for repurposing modular PKS and NRPS. Design-build-test-learn cycle with the tools for each step was illustrated.

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