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 <i>Escherichia coli</i> cell extracts are widely used for CFE, <i>Streptomyces</i> extracts are likely to offer a more biochemically compatible environment for their expression. However, current <i>Streptomyces</i>-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 <i>Streptomyces venezuelae</i> NRRL B-65422 and <i>Streptomyces lividans</i> TK24. Like <i>E. coli</i>-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 <i>S. lividans</i> lysate outperforms the <i>E. coli</i> 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 <i>Streptomyces</i>-based system, and of a T1PKS in any bacterial extract. By addressing key challenges in the generalizability and scalability of prior <i>Streptomyces</i> CFE, we establish a protocol that enables parallelized evaluation of diverse lysate systems and provides a foundation for high-throughput T1PKS engineering <i>in vitro</i>.