Simon J. Moore

Indolocarbazoles are π-conjugated heterocycles with rich optoelectronic and redox properties, offering potential as sustainable redox active materials. Here, we report the electrochemical characterisation of arcyriaflavin A (Arc A), the simplest fully conjugated indolocarbazole scaffold derived by recombinant biosynthesis. Cyclic voltammetry in non-aqueous media revealed two irreversible one-electron reduction events at –1.90 and –2.42 V vs Fc/Fc⁺, consistent with an ECEC-type mechanism and a diffusion constant of 0.81 x 10-6 cm2 s-1. Kinetic studies of the first reduction step confirmed a second-order rate constant of 7.3 × 10⁴ M⁻¹ s⁻¹, analogous to the phthalimide reduction mechanism. Half-cell charge/discharge experiments and complementary spectroelectrochemical UV–vis and ex situ NMR measurements suggested irreversible degradation of the electroactive species via proton-transfer steps and radical recombination reactions involving the phthalimide ring. TD-DFT simulations supported the proposed degradation mechanisms by qualitatively reproducing the observed electronic transitions and NMR shifts. Based on these results, N-alkylation of the phthalimide ring was proposed as a strategy to stabilise the reduced forms and enhance solubility, providing a molecular design route toward robust, biologically derived redox-active materials for sustainable energy storage.

Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterising emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression (CFE) systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimising the pre-incubation time of ARGs and dispensing protocol, expression of aac(3)-IIa increased the half-maximal inhibition concentration (IC50) of gentamicin by over 150-fold, while arr-3 increased the IC50 of rifampicin by approximately 20-fold compared to controls. Future work could extend this platform to characterise novel ARGs identified through genomic surveillance or rapidly profile activity of new or derivative antibiotics.

Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterizing emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimizing the pre-incubation time of ARGs and dispensing protocol, expression of aac(3)-IIa increased the half-maximal inhibition concentration (IC50) of gentamicin by over 150-fold, whilst arr-3 increased the IC50 of rifampicin by ~20-fold compared to controls. This methodology for rapid, semi-automated ARG characterization offers a strategy to combat AMR by assessing novel ARGs identified through genomic surveillance or profiling activity of new or derivative antibiotics.

Natural products have applications as biopharmaceuticals, agrochemicals, and other high-value chemicals. However, there are challenges in isolating natural products from their native producers (e.g. bacteria, fungi, plants). In many cases, synthetic chemistry or heterologous expression must be used to access these important molecules. The biosynthetic machinery to generate these compounds is found within biosynthetic gene clusters, primarily consisting of the enzymes that biosynthesise a range of natural product classes (including, but not limited to ribosomal and nonribosomal peptides, polyketides, and terpenoids). Cell-free synthetic biology has emerged in recent years as a bottom-up technology applied towards both prototyping pathways and producing molecules. Recently, it has been applied to natural products, both to characterise biosynthetic pathways and produce new metabolites. This review discusses the core biochemistry of cell-free synthetic biology applied to metabolite production and critiques its advantages and disadvantages compared to whole cell and/or chemical production routes. Specifically, we review the advances in cell-free biosynthesis of ribosomal peptides, analyse the rapid prototyping of natural product biosynthetic enzymes and pathways, highlight advances in novel antimicrobial discovery, and discuss the rising use of cell-free technologies in industrial biotechnology and synthetic biology.

Indolocarbazoles are natural products with a broad spectrum of bioactivity. A distinct feature of indolocarbazole biosynthesis is the modification of the indole and maleimide rings by regioselective tailoring enzymes. Here, we study a new indolocarbazole variant, which is encoded by the acfXODCP genes from Streptomyces venezuelae ATCC 10712. We characterise the pathway by expressing the acfXODCP genes in Streptomyces coelicolor, which led to the production of a C‐5/C‐5’‐dihydroxylated indolocarbazole, which we assign as arcyriaflavin F. We also show that a flavin‐dependent monooxygenase AcfX catalyses the C‐5/C‐5’ dihydroxylation of the unsubstituted arcyriaflavin A into arcyriaflavin F. Interestingly, AcfX shares homology to EspX from erdasporine A biosynthesis, which instead catalyses a single C‐6 indolocarbazole hydroxylation. In summary, we report a new indolocarbazole biosynthetic pathway and a regioselective C‐5 indole ring tailoring enzyme AcfX.

Indolocarbazoles are natural products with a broad spectrum of reported bioactivities. A distinct feature of indolocarbazole biosynthesis is the modification of the indole and maleimide rings by regioselective tailoring enzymes. Here, we study a new indolocarbazole variant, which is encoded by the acfXODCP genes from Streptomyces venezuelae ATCC 10712. First, we characterise this pathway by expressing the acfXODCP genes in Streptomyces coelicolor , which led to the production of a C-5/C-5’-dihydroxylated indolocarbazole. We name this new product arcyriaflavin F. Second, we demonstrate the flavin-dependent monooxygenase AcfX catalyses the C-5/C-5’ dihydroxylation of the unsubstituted arcyriaflavin A into arcyriaflavin F. Interestingly, AcfX shares homology to EspX from erdasporine A biosynthesis, which instead catalyses a single C-6 indolocarbazole hydroxylation. In summary, we report a new indolocarbazole biosynthetic pathway and a regioselective C-5 indole ring tailoring enzyme AcfX.

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.

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.

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 a Klebsiella pneumoniae cell-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 the K. pneumoniae CFE 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 evolved K. pneumoniae strains 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 resistant Mycobacterium 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 clinical K. pneumoniae strains 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.

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.