[1S05e] 13. Microbial Engineering for Natural Products Synthesis - Part 3

5:45 PM - 6:00 PM JST(8:45 AM - 9:00 AM UTC)

[1MENP-17] Simple and rapid production of secondary metabolites by cell-free expression

○Hiroshi Otani¹, Jaime Lorenzo Dinglasan¹, Nigel J. Mouncey¹ (1. DOE Joint Genome Institute (USA))

Keywords:

Secondary metabolite,Cell-free expression,Actinomycetes,Polyketide,Cyclodipeptide

Secondary metabolites are a major source of natural products with industrially relevant bioactivities. Despite their proven ability for biotechnological applications, there remains a huge and untapped reservoir of such metabolites to be discovered and engineered. Production of the vast majority of such metabolites is conditional owing to often complex and unknown regulatory networks. Engineering metabolites involves testing manipulated biosynthetic pathways in production hosts, which is lengthy and cumbersome. At the DOE Joint Genome Institute, we are developing and applying various functional genomics capabilities to accelerate such processes. As a part of this effort, we established a simple and versatile lysate-based cell-free expression (CFE) method that enables rapid production of secondary metabolites. In addition to Escherichia coli, which is widely used for CFE, we optimised CFE procedures using cell lysates from various actinomycetes including streptomycetes. Since actinomycetes are the large sources of industrial chemicals, their cell extracts are expected to offer a more biochemically compatible environment for secondary metabolite biosynthesis. Our optimised and simple procedure allows high yield protein production using diverse actinomycetes. We subsequently applied this CFE method to various types of secondary metabolite biosynthetic pathways, notably, type I polyketide synthase (PKS), type III PKS and cyclodipeptide synthase (CDPS) pathways. While both E. coli- and Streptomyces-based CFE reactions were able to produce the products of the type III PKS and CDPS pathways tested, only the Streptomyces cell lysate was able to catalyse the biosynthesis of the product of the type I PKS pathway involving 250kDa megasynthase. These results highlight the versatility of the Streptomyces CFE platform for secondary metabolite biosynthesis. As the CFE-based metabolite synthesis completes in less than a day and involves no microbial transformation and cultivation, our platform is more amenable to multiplexed biosynthetic reactions, high throughput screening and automation, and should enable more efficient discovery and engineering of bioactive compounds.

Dinglasan, J.L.N., Lee, N., Pham, N.N., Faltane, M., Lynde, M., Louie, K.B., Nath, S., Keasling, J.D., Otani, H,, and Mouncey, N.J. ACS Synth. Biol. 2026, 15, 1, 99–113