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 likely to offer a more biochemically compatible environment for their expression. 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 E. 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 the 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 provides a foundation for high-throughput T1PKS engineering in vitro.