A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emergingas a powerful tool to examine natural product biosynthetic pathways in a test-tube.The key advantages of this approach are the reduced experimental timescales andcontrolled reaction conditions. To realise this potential, it is essential to developspecialised cell-free systems in organisms enriched for biosynthetic gene clusters.This requires strong protein production and well-characterised synthetic biology tools.The Streptomyces genus is a major source of natural products. To study enzymes andpathways from Streptomyces, we originally developed a homologous Streptomycescell-free system to provide a native protein folding environment, a high G+C (%) tRNApool and an active background metabolism. However, our initial yields were low (36μg/mL) and showed a high level of batch-to-batch variation. Here, we present anupdated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of266 μg/mL of expressed recombinant protein. To complement this, we rapidlycharacterise a range of DNA parts with different reporters, express high G+C (%)biosynthetic genes and demonstrate an initial proof of concept for combinedtranscription, translation, and biosynthesis of Streptomyces metabolic pathways in asingle ‘one-pot’ reaction

DataSheet1_A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems.pdf

Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.</p

A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance

Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease-causing microbes. To provide a host-specific tool to study antibiotic susceptibility and resistance, here we develop Klebsiella pneumoniae cell-free gene expression (CFE) systems from laboratory and clinical isolates. Using proteomics, we identify relative differences and unique proteins for these new CFE systems in comparison to an Escherichia coli MG1655 CFE model. Then we profile antimicrobial susceptibility in parallel with whole cells to quantify CFE antibiotic potency. Finally, we apply this native CFE tool to study AMR variants at a proof-of-concept level. Definably we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin—a genotype observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. Overall, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial extract processing requires Biosafety Level 2, the CFE system is non-living, suitable for long-term storage and study in a Biosafety Level 1 lab. We anticipate the K. pneumoniae CFE bioassay is advantageous for host-specific antimicrobial testing, the characterisation of intracellular AMR variants and potentially structure-activity relationship studies