GNPS - A simple and versatile cell-free expression method for producing secondary metabolites

Secondary metabolites are a major source of natural products with industrially relevant bioactivities. Lysate-based cell-free expression (CFE) is an emerging platform for accelerating the discovery and engineering of these natural products. While Escherichia coli cell extracts are widely used for CFE, Streptomyces extracts are believed to offer a more biochemically compatible environment for natural product synthesis. However, current Streptomyces-based CFE systems remain underdeveloped, with protocols that are either strain-specific or not readily scalable. To address these limitations and enable broader access to cell-free natural product biosynthesis, we present a generalizable and simple set of reaction conditions that support high-yield protein expression (180–230?µg/mL) in lysates derived from Streptomyces venezuelae NRRL B-65422 and Streptomyces lividans TK24. Like Escherichia coli-based systems, these extracts enable iterative and pathway-level biosynthesis, as demonstrated by the production of the polyketide flaviolin and the cyclic dipeptide albonoursin. Notably, the S. lividans lysate outperforms E. coli systems by also supporting the expression and catalytic activity of a (~250?kDa) type I polyketide synthase (T1PKS), producing its corresponding ethyl ketone product, 2-methyl-3-pentanone, without the need for precursor or post-translational modification supplements. To our knowledge, this represents the first demonstration coupling both expression and catalysis of a megasynthase in a Streptomyces-based system, and of a T1PKS in any bacterial extract. By addressing key challenges in the generalizability and scalability of prior Streptomyces CFE, we establish a protocol that enables parallelized evaluation of diverse lysate systems and provide a foundation for high-throughput T1PKS engineering in vitro.

The work (proposal:https://doi.org/10.46936/10.25585/60008760) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231.