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.