Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters

Abstract

18A variety of chemicals can be produced in a living host cell via optimized and engineered 19 biosynthetic pathways. Despite the successes, pathway engineering remains demanding 20 and partly impossible owing to the lack of specific functions or substrates in the host 21 cell, its sensitivity in vital physiological processes to the heterologous components, or 22 constrained mass transfer across the membrane. In this study, we demonstrate that cell-23 free systems can be useful in driving the characterization and engineering of 24 biosynthetic pathways. We show that complex multidomain proteins involved in natural 25 compound biosynthesis can be produced from encoding DNA in vitro in a minimal 26 complex PURE system to directly run multistep reactions. We prove the concept of this 27 approach on the direct synthesis of indigoidine and rhabdopeptides with the in vitro 28 produced multidomain megasynthases BpsA and KJ12ABC. The in vitro produced 29 proteins are analyzed in detail, i.e., in yield, quality, post-translational modification and 30 specific activity, and compared to recombinantly produced proteins. Our study 31 highlights cell-free PURE systems as suitable setting for the rapid engineering of 32 biosynthetic pathways. 33 34 Keywords: cell-free protein synthesis, PURE system, natural products, biosynthetic 35 pathways, non-ribosomal peptide synthetase, polyketide synthase

AStreptomyces venezuelaeCell-Free Toolkit for Synthetic Biology

“…Our study complements a recent surge in interest in the use of cell-free systems for the study of biosynthetic pathways (2, 4, 23, 42). Here we wanted to expand the palette of plasmid tools for the further development of S. venezuelae as a synthetic biology chassis by developing an optimised streptomyces TX-TL toolkit (5, 20, 21).…”

“…For protein synthesis, while we could detect HemB (35 kDa) and HemC (38 kDa), the fusion protein HemD/CysG A was less clear, with other major bands at the expected mass – 58.3 kDa (Figure S6). To verify pathway function, we ran a semi-continuous reaction (42) to facilitate purification by separating the haem intermediates from the cell-extract proteins (inset image in Figure 4C). Interestingly, LC-MS analysis detected the air-oxidised product of the HemD enzyme (uroporphyrinogen III - 837 m/z), observed as a 6-electron oxidised uroporphyrin III (red fluorescent) intermediate at 831 m/z; typical for these air-sensitive intermediates (Figure S8).…”

Cell‐Free Exploration of the Natural Product Chemical Space

“…[47] A separate study described similar syntheses of NRPs indigoidine and rhabdopeptide using the commercially available PURExpress system. [48] The authors were also able to use CFE to synthesize several other megasynthases from additional NRPs, fatty acid, and polyketide pathways although their native activity was not demonstrated. A series of indole alkaloids was produced using cell-free technology.…”

“…While such enzymes have been synthesized in CFE systems, there are still relatively few examples. [46,48] Several strategies could be used to address this. First, pre-enriched lysates from traditional heterologous expression might provide one current alternative strategy to access larger constructs.…”

Cell-free protein synthesis for nonribosomal peptide synthetic biology

“…Thus, this is additionally appealing for NRPS characterization and engineering because it can accelerate designbuild-test-learn (DBTL) cycles. To these ends, NRPSs have recently been expressed in both lysate-based (Goering et al, 2017;Zhuang et al, 2020;Moore et al, 2021;Dinglasan et al, 2023) and PURExpress (Siebels et al, 2020a) cell-free systems. This review highlights examples of NRPS natural products that are expressed in CFE systems in synergy with different transcriptional and translational machineries.…”