Reinvigorating natural product combinatorial biosynthesis with synthetic biology

Abstract

Natural products continue to play a pivotal role in drug-discovery efforts and in the understanding if human health. The ability to extend nature's chemistry through combinatorial biosynthesis--altering functional groups, regiochemistry and scaffold backbones through the manipulation of biosynthetic enzymes--offers unique opportunities to create natural product analogs. Incorporating emerging synthetic biology techniques has the potential to further accelerate the refinement of combinatorial biosynthesis as a robust platform for the diversification of natural chemical drug leads. Two decades after the field originated, we discuss the current limitations, the realities and the state of the art of combinatorial biosynthesis, including the engineering of substrate specificity of biosynthetic enzymes and the development of heterologous expression systems for biosynthetic pathways. We also propose a new perspective for the combinatorial biosynthesis of natural products that could reinvigorate drug discovery by using synthetic biology in combination with synthetic chemistry.

Figure 1

Structures of novel natural products generated by engineering the substrate specificity of biosynthetic enzymes. Modified structures from natural products are highlighted in blue.

Figure 2

General strategies for the combinatorial biosynthesis of PKs and NRPs. (a) Through domain truncation and AT domain exchange, the type I aureotin PKS was morphed to produce its homologue luteoreticulin (3) in the heterologous host S. albus. (b) The tetracycline SF2575 biosynthetic pathway harboring a type II PKS and modifying enzymes was reconstituted in the heterologous host S. lividans. Subsequent inactivation of the methyltransferase gene generated tetracycline analogue (4). (c) Novel lipopeptide (5) was produced by replacement of module 11 of daptomycin NRPS (DptBC) with the corresponding module from related A15454 NRPS (LptC) in the native daptomycin-producing S. roseosporus. Modified structures from natural products are highlighted in blue. AT, acyltransferase domain, ACP, acyl carrier protein; DH, dehydratase domain; ER, enoyl reductase domain, KS, ketosynthase domain; KR, ketoreductase domain; TE, thioesterase domain. A, adenylation domain; C, condensation domain; T, thiolation domain; E, epimerization domain.

Figure 3

Approaches to assemble natural product biosynthetic pathways. See main text for details of each approach. LCHR, linear plus circular homologous recombination; LLHR, linear plus linear homologous recombination; SLIC, sequence and ligation independent cloning; TAR, transformation-associated recombination.

Figure 4

The structures of novel or cryptic natural products produced by heterologous expression or chemobiosynthesis. Modified structures from natural products are highlighted in blue.

Figure 5

Representative synthetic biology tools for optimization of the expression of combinatorially assembled biosynthetic machineries. See main text for details of each approach. RBS, ribosome-binding site; T, terminator.

Figure 6

Representative synthetic biology tools for optimization of producing hosts. See main text for details of each approach. asRNA, antisense RNA; CRISPR/Cas, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein; MAGE, multiplex automated genome engineering; PAM, protospacer-adjacent motif; sgRNA, single-guide RNA; sRNA, small regulatory RNA.