<i>Streptomyces</i> cell-free systems for natural product discovery and engineering

Lai, Hung-En; Li, Jian; Freemont, Paul S.

doi:10.1039/d2np00057a

Cited by 26 publications

(19 citation statements)

References 53 publications

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“…Depending on the source of cell extracts, various cell-free expression systems have been developed. ,− Among them, the cell-free system derived from Escherichia coli is commonly used to study natural product biosynthetic pathways due to its simple preparation, high yield, short production time, and low cost. ,, Here, we selected the strain E. coli BL21 Star (DE3), which is a robust chassis for high-level expression of heterologous proteins , and has been widely used to establish cell-free protein synthesis (CFPS). ,, To express BesA-E, we constructed expression plasmids by inserting each codon-optimized gene into a pET28a vector.…”

Section: Resultsmentioning

confidence: 99%

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups

et al. 2023

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Crude extract-based cell-free expression systems have been used to produce natural products by reconstitution of their biosynthetic pathways in vitro. However, the chemical scope of cell-free synthesized natural compounds is still limited, which is partially due to the length of biosynthetic gene clusters. To expand the product scope, here, we report cell-free biosynthesis of several lysine-derived unnatural amino acids with functional moieties such as chloro, alkene, and alkyne groups. Specifically, five related enzymes (i.e., halogenase, oxidase, lyase, ligase, and hydroxylase) involved in β-ethynylserine biosynthesis are selected for cell-free expression. These enzymes can be expressed in single, in pairs, or in trios to synthesize different compounds, including, for instance, 4-Cl−L-lysine, 4-Cl−allyl-L-glycine, and L-propargylglycine. The final product of γ-L-glutamyl−L-β-ethynylserine (a dipeptide with an alkyne group) can also be synthesized by cell-free expression of the full biosynthetic pathway (i.e., five enzymes). Our results demonstrate the flexibility of cell-free systems, enabling easy regulation and rational optimization for target compound formation. Overall, this work expands not only the type of enzymes (e.g., halogenase) but also the scope of natural products (e.g., terminal-alkyne amino acid) that can be rapidly produced in cell-free systems. With the development of cell-free biotechnology, we envision that cell-free strategies will create a new frontier for natural product biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups

et al. 2023

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“…However, with the exception of a bespoke S. pneumoniae CFE system 17 , early IVT systems generally use established models such as E. coli and B. subtilis 13,14 . Recent advances in cell-free systems provide new opportunities to study almost any cell type that can be grown in the laboratory including from prokaryotes [19][20][21][22][23][24] and eukaryotes [59][60][61] . After the initiation of our study, a K. pneumoniae CFE system was recently released 62 , but alternatively this study focused on the potential of this cell-free system for biotechnology and protein production.…”

Section: Discussionmentioning

confidence: 99%

“…A range of CFE systems have recently emerged from diverse bacterial genera, providing a fresh approach to study non-model microbes [19][20][21][22][23][24] . To make a highly active CFE system, this requires the isolation of a cell extract enriched with active ribosomes from rapidly dividing cells.…”

Section: Establishing a Rapid And Safe K Pneumoniae Cfe Platform From...mentioning

confidence: 99%

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

Chengan

Hind²,

Nagappa

et al. 2023

Preprint

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Antimicrobial resistance (AMR) is a pandemic spread across multiple priority infectious disease threats. While the cell envelope plays a key role in AMR, this also makes it challenging to study how antibiotics function inside the cell. Herein, we present aKlebsiella pneumoniaecell-free gene expression (CFE) platform for the rapid profiling of intracellular antibiotic sensitivity and resistance. This cell-free approach provides the unique macromolecular and metabolite components from this microbe, which include multiple antibiotic targets from transcription, translation, and metabolic processes. First, we compare theK. pneumoniaeCFE system to whole cell antimicrobial assays. We find that several antibiotic classes show higher sensitivity in the CFE system, suggesting limitations in antibiotic transport in the whole cell assay. Next, we evolvedK. pneumoniaestrains with resistance to specific antibiotics and use whole genome sequencing analysis for genotyping. As an exemplary case, we show that a single RNA polymerase beta subunit variant H526L (also frequently found in multidrug resistantMycobacterium tuberculosis) confers a 58-fold increase in CFE resistance to rifampicin. Overall, we describe a safe (i.e., non-living, non-pathogenic) platform suitable for studying an infectious disease model in a Containment Level 1 laboratory. Our CFE strategy is generalisable to laboratory and clinicalK. pneumoniaestrains and provides a new experimental tool to profile intracellular AMR variants. In conclusion, our CFE tool provides a significant advance towards understanding AMR and complements wider infectious disease studies.

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“…Recently, we and others have developed a wide range of cell-free gene expression systems (CFE) -also referred to as cell-free protein synthesis (CFPS) -from both Gram-negative and Gram-positive bacterial cell types [19][20][21][22][23][24] . This cell-free synthetic biology strategy provides an alternative approach to investigate established areas of research.…”

Section: Introductionmentioning

confidence: 99%

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

Chengan,

Hind,

Nagappa

et al. 2023

Preprint

View full text Add to dashboard Cite

Antimicrobial resistance (AMR) is a pandemic spread across multiple priority infectious disease threats. Herein, we present a Klebsiella pneumoniae cell-free gene expression (CFE) system for the rapid profiling of intracellular antibiotic sensitivity and resistance. Specifically, this cell-free approach provides multiple antibiotic targets, which we exploit to identify which antibiotics are potent inside the cell but have limited transport. We also derive CFE systems from a selection of laboratory-adapted K. pneumoniae AMR variants, and as an exemplary case, show that a single RNA polymerase beta subunit variant H526L confers a 58-fold increase in CFE resistance to rifampicin. Overall, our cell-free model provides a safe (i.e., non-living) and rapid platform suitable for studying AMR variants in a Containment Level 1 laboratory. The approach is generalisable to laboratory and clinical strains and provides a new experimental tool that will significantly advance the understanding of antibiotic transport, AMR and provides a new infectious disease model.

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Streptomyces cell-free systems for natural product discovery and engineering

Abstract: Streptomyces cell-free gene expression (CFE) systems for natural product discovery. The CFE system simply requires DNA, a cell-extract, amino acids, and a metabolite solution to study transcription, translation, and biosynthesis in a single reaction.

Cited by 26 publications

References 53 publications

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups

Cell-Free Biosynthesis of Lysine-Derived Unnatural Amino Acids with Chloro, Alkene, and Alkyne Groups

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

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

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