Biology subjects

Moore, S.

Publications and source records attributed to Moore, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

VISTA-induced tumor suppression by a four amino acid intracellular motif

VISTA is a key immune checkpoint receptor under investigation for cancer immunotherapy; however, its signaling mechanisms remain unclear. Here we identify a conserved four amino acid (NPGF) intracellular motif in VISTA that suppresses cell proliferation by constraining cell-intrinsic growth receptor signaling. The NPGF motif binds to the adapter protein NUMB and recruits Rab11 endosomal recycling machinery. We identify and characterize a class of triple-negative breast cancers with high VISTA expression and low proliferative index. In tumor cells with high VISTA levels, the NPGF motif sequesters NUMB at endosomes, which interferes with epidermal growth factor receptor (EGFR) trafficking and signaling to suppress tumor growth. These effects do not require canonical VISTA ligands, nor a functioning immune system. As a consequence of VISTA expression, EGFR receptor remains abnormally phosphorylated and cannot propagate ligand-induced signaling. Mutation of the VISTA NPGF domain reverts VISTA-induced growth suppression in multiple breast cancer mouse models. These results define a mechanism by which VISTA represses NUMB to control malignant epithelial cell growth and signaling. They also define distinct intracellular residues that are critical for VISTA-induced cell-intrinsic signaling that could be exploited to improve immunotherapy.

cancer biology↗

bioRxiv2024

Dynamics of gut colonization by commensal and pathogenic bacteria that adhere to the intestinal epithelium

Maintaining a healthy gut microbiome plays a critical role in avoiding gut-related pathologies. Bacterial adherence to the intestinal epithelium plays a vital role in niche establishment in the gut, as in vitro experiments and mathematical modeling suggest that adherence provides a strong competitive advantage over free-floating planktonic microbes. Currently, we lack the ability to study gut microbiome adherence in an in vivo model system. Through sampling natural populations of Caenorhabditis, we discovered three bacterial species that adhere to the intestinal epithelium of several wild Caenorhabditis isolates. When transferred to C. elegans, all three bacterial species colonized the entire anterior to posterior length of the intestine lumen. We isolated these bacterial species via in vitro growth or selective enrichment in the nematode gut and identified them as Lelliottia jeotgali, Candidatus Lumenectis limosiae, and Candidatus Enterosymbion pterelaium, the latter two representing new species. Adherent Ca. L. limosiae negatively affects host fitness, while Lelliottia jeotgali and Ca. E. pterelaium exhibited a neutral effect in our assays. We demonstrated that two of these species can actively proliferate in the intestine throughout the host lifespan, with Lelliottia jeotgali colonizing throughout the lumen simultaneously and Candidatus Lumenectis limosiae showing anterior-to-posterior directionality. In competition assays, animals pre-colonized with L. jeotgali significantly reduced colonization by pathogenic Ca. L. limosiae, but this effect was not seen when animals were colonized by both bacteria simultaneously. Strikingly, regardless of the colonization paradigm, populations exposed to both bacteria showed a near-identical mitigation of the pathogenic effects of Ca. L. limosiae. Altogether, these strains illustrate the capacity of microbiome bacteria to adhere, replicate, and establish a niche across the entire intestinal lumen in C. elegans and they present an opportunity to study bacterial adherence in the context of a whole, intact and transparent animal.

microbiology↗

bioRxiv2024

Early events in midzone formation stabilize nascent bipolar spindles

The formation of a stable mitotic spindle is critical to the accurate partitioning of the chromosomes during mitosis. Interpolar microtubules of the spindle midzone consist of antiparallel microtubules crosslinked by kinesin-5 and stabilize the spindle by opposing forces produced when sister chromatids are attached to microtubules and under tension. Despite the importance of the interpolar microtubules, how and when they form and what determines their number remain unknown. In this study, we report that a {gamma}-tubulin mutation ({gamma}tub-Y445D) disrupts the localization of kinesin-5 and the formation of the interpolar microtubules, resulting in spindle instability. We find that kinesin-5 crosslinking is intact in this mutant, but that it is incapable of the subsequent kinesin-5 microtubule sliding needed to stabilize the nascent spindle. Early activation of the PRC1 homolog Ase1 restores nascent spindle stability to the {gamma}tub-Y445D mutant but cannot stabilize spindles during centromere attachment. Our work shows that midzone assembly begins with the formation of interpolar microtubule precursors in monopolar spindles that persist until early metaphase and limit the formation of kinetochore attachments in new spindles.

cell biology↗

bioRxiv2024

Biosynthesis of Arcyriaflavin F from Streptomyces venezuelae ATCC 10712

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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/589956v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@c0c397org.highwire.dtl.DTLVardef@16915dforg.highwire.dtl.DTLVardef@1ae538org.highwire.dtl.DTLVardef@1bef4ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Loss of prohibitin 2 in Schwann cells dysregulates key transcription factors controlling developmental myelination

Schwann cells are critical for the proper development and function of the peripheral nervous system, where they form a mutually beneficial relationship with axons. Past studies have highlighted that a pair of proteins called the prohibitins play major roles in Schwann cell biology. Prohibitins are ubiquitously expressed and versatile proteins. We have previously shown that while prohibitins play a crucial role in Schwann cell mitochondria for long-term myelin maintenance and axon health, they may also be present at the Schwann cell-axon interface during development. Here, we expand on this work, showing that drug-mediated modulation of prohibitins in vitro disrupts myelination and confirming that Schwann cell-specific ablation of prohibitin 2 (Phb2) in vivo results in early and severe defects in peripheral nerve development. Using a proteomic approach in vitro, we identify a pool of candidate PHB2 interactors that change their interaction with PHB2 depending on the presence of axonal signals. Furthermore, we show in vivo that loss of Phb2 in mouse Schwann cells causes ineffective proliferation and dysregulation of transcription factors EGR2 (KROX20), POU3F1 (OCT6) and POU3F2 (BRN2) that are necessary for proper Schwann cell maturation. Schwann cell-specific deletion of Jun, a transcription factor associated with negative regulation of myelination, confers partial rescue of the development defect seen in mice lacking Schwann cell Phb2. This work develops our understanding of Schwann cell biology, revealing that Phb2 may directly or indirectly modulate the timely expression of transcription factors necessary for proper peripheral nervous system development, and proposing candidates that may play a role in PHB2-mediated integration of axon signals in the Schwann cell.

neuroscience↗

bioRxiv2024

Why models underestimate tropical forest productivity: a case study in West Africa

Tropical forests dominate terrestrial photosynthesis, yet there are major contradictions in our understanding due to a lack of field studies, especially outside the tropical Americas. A recent field study indicated that West African forests have among the highest forests gross primary productivity (GPP) yet observed, contradicting models that rank them lower than Amazonian forests. Here, we explore possible reasons for this data-model mismatch. We found the in situ GPP measurements higher than multiple global GPP products at the studied sites in Ghana. The underestimation of GPP by models largely disappears when a standard photosynthesis model is informed by local field-measured values of (a) fractional absorbed photosynthetic radiation (fAPAR), and (b) photosynthetic traits. Satellites systematically underestimate fAPAR in the tropics due to cloud contamination issues. The study highlights the potential widespread underestimation of tropical forests GPP and carbon cycling and hints at the ways forward for model and input data improvement. Related manuscriptThe recent field study mentioned above is a manuscript currently accepted by Nature Communications (manuscript id NCOMMS-23-37419), which is available as a preprint https://www.researchsquare.com/article/rs-3136892/v1 Codes and data availabilityAll data and codes underlying the study are currently shared via Github (link here) which will be made available through Zenodo upon acceptance.

ecology↗

bioRxiv2024

Re-activation of neurogenic niches in aging brain

Recent studies proposing induced glia-to-neuron conversion raised the potential for generating new neurons to replace those lost due to injury, aging or neurodegenerative diseases. Here, single-cell spatial transcriptomics [Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH)] is used to construct a spatial cell atlas of the subventricular and dentate gyrus neurogenic niches of young and aged adult murine brain. RNAs that encode the RNA binding protein Polypyrimidine Tract-Binding Protein (PTBP1) in the aged murine brain are determined to be highest in glia that line previously active neurogenic niches. A glial cell population with ependymal character within an initially quiescent subventricular neurogenic niche in the aged murine brain is identified that upon transient suppression of PTBP1 reenters the cell cycle, replicates DNA, and converts into neurons through a canonical adult neurogenesis pathway. Glia-derived neurons migrate from this niche, with some neurons transiting to the striatum and acquiring a transcriptome characteristic of GABAergic inhibitory neurons. Similar PTBP1 expressing quiescent glia are identified in the corresponding neurogenic niche of aged human brain. Thus, transient reduction of PTBP1 holds potential for inducing the generation of new neurons in quiescent neurogenic niches of the aged nervous system, thereby offering promising therapeutic applications. Bullet point summary1) Single-cell spatial transcriptomics is used to validate active neurogenesis in the two neurogenic niches of the young adult murine brain, determine that those niches are quiescent in the aging adult brain of mice, and demonstrate the absence of neurogenesis in the aging human brain. 2) The RNA binding protein PTBP1 is determined to be most highly expressed within glia that line the aged murine and human neurogenic niches, with its transient reduction sufficient in mice to activate/re-activate expression of genes characteristic of immature neurons. 3) Suppression of PTBP1 using a single intra-cerebral-ventricular injection of PTBP1-targeting antisense oligonucleotide (ASO) induces generation of new immature neurons in the neurogenic niches of the aged mouse brain via a canonical adult neurogenesis pathway. 4) Single-cell RNA signature tracing is used to identify a) a subclass of ependymal cells in a previously quiescent neurogenic niche of the aged mouse brain that convert into GABAergic inhibitory neurons following transient suppression of PTBP1, and b) the molecular steps in the conversion process including cell cycle re-entry, DNA replication, and transcriptome changes that mimic canonical neurogenesis. 5) A similar class of PTBP1-expressing ependymal cells lining the ventricle of the aging non-human primate and human brains is identified, suggesting the promise of re-activation of neurogenesis as a therapeutic approach in humans.

neuroscience↗

bioRxiv2023

Dynamics of odor-source localization: Insights from real-time odor plume recordings and head-motion tracking in freely moving mice

Animals navigating turbulent odor plumes exhibit a rich variety of behaviors, and employ efficient strategies to locate odor sources. A growing body of literature has started to probe this complex task of localizing airborne odor sources in walking mammals to further our understanding of neural encoding and decoding of naturalistic sensory stimuli. However, correlating the intermittent olfactory information with behavior has remained a long-standing challenge due to the stochastic nature of the odor stimulus. We recently reported a method to record real-time olfactory information available to freely moving mice during odor-guided navigation, hence overcoming that challenge. Here we combine our odor-recording method with head-motion tracking to establish correlations between plume encounters and head movements. We show that mice exhibit robust head-pitch motions in the 5-14Hz range during an odor-guided navigation task, and that these head motions are modulated by plume encounters. Furthermore, mice reduce their angles with respect to the source upon plume contact. Head motions may thus be an important part of the sensorimotor behavioral repertoire during naturalistic odor-source localization.

neuroscience↗

bioRxiv2023

Revealing abrupt transitions from goal-directed to habitual behavior

A fundamental tenet of animal behavior is that decision-making involves multiple controllers. Initially, behavior is goal-directed, driven by desired outcomes, shifting later to habitual control, where cues trigger actions independent of motivational state. Clark Hulls question from 1943 still resonates today: "Is this transition abrupt, or is it gradual and progressive?"1 Despite a century-long belief in gradual transitions, this question remains unanswered2,3 as current methods cannot disambiguate goal-directed versus habitual control in real-time. Here, we introduce a novel volitional engagement approach, motivating animals by palatability rather than biological need. Offering less palatable water in the home cage4,5 reduced motivation to work for plain water in an auditory discrimination task when compared to water-restricted animals. Using quantitative behavior and computational modeling6, we found that palatability-driven animals learned to discriminate as quickly as water-restricted animals but exhibited state-like fluctuations when responding to the reward-predicting cue--reflecting goal-directed behavior. These fluctuations spontaneously and abruptly ceased after thousands of trials, with animals now always responding to the reward-predicting cue. In line with habitual control, post-transition behavior displayed motor automaticity, decreased error sensitivity (assessed via pupillary responses), and insensitivity to outcome devaluation. Bilateral lesions of the habit-related dorsolateral striatum7 blocked transitions to habitual behavior. Thus, volitional engagement reveals spontaneous and abrupt transitions from goal-directed to habitual behavior, suggesting the involvement of a higher-level process that arbitrates between the two.

neuroscience↗

bioRxiv2023

Contrasting carbon cycle along tropical forest aridity gradients in W Africa and Amazonia

2Tropical forests cover large areas of equatorial Africa and play a significant role in the global carbon cycle. However, there has been a lack of in-situ measurements to understand the forests gross and net primary productivity (GPP and NPP) and their allocation. Here we present the first detailed field assessment of the carbon budget of multiple forest sites in Africa, by monitoring 14 one-hectare plots along an aridity gradient in Ghana. When compared with an equivalent aridity gradient in Amazonia using the same measurement protocol, the studied West African forests generally had higher GPP and NPP and lower carbon use efficiency (CUE). The West African aridity gradient consistently shows the highest NPP, CUE, GPP, and autotrophic respiration at a medium-aridity site, Bobiri. Notably, NPP and GPP of the site are the highest yet reported anywhere in the tropics using similar methods. Widely used data products (MODIS and FLUXCOM) substantially underestimate productivity when compared to in situ measurements, in Amazonia and especially in Africa. Our analysis suggests that the high productivity of the African forests is linked to their large GPP allocation to canopy and semi-deciduous characteristics, which may be a result of a seasonal climate coupled with high soil fertility.

ecology↗

bioRxiv2023

Fitting high-resolution electron density maps from atomic models to solution scattering data

Solution scattering techniques, such as small and wide-angle X-ray scattering (SWAXS), provide valuable insights into the structure and dynamics of biological macromolecules in solution. In this study, we present an approach to accurately predict solution X-ray scattering profiles at wide angles from atomic models by generating high-resolution electron density maps. Our method accounts for the excluded volume of bulk solvent by calculating unique adjusted atomic volumes directly from the atomic coordinates. This approach eliminates the need for a free fitting parameter commonly used in existing algorithms, resulting in improved accuracy of the calculated SWAXS profile. An implicit model of the hydration shell is generated which uses the form factor of water. Two parameters, namely the bulk solvent density and the mean hydration shell contrast, are adjusted to best fit the data. Results using eight publicly available SWAXS profiles show high quality fits to the data. In each case, the optimized parameter values show small adjustments demonstrating that the default values are close to the true solution. Disabling parameter optimization results in a significant improvement of the calculated scattering profiles compared to the leading software. The algorithm is computationally efficient, showing more than tenfold reduction in execution time compared to the leading software. The algorithm is encoded in a command line script called denss.pdb2mrc.py and is available open source as part of the DENSS v1.7.0 software package (https://github.com/tdgrant1/denss). In addition to improving the ability to compare atomic models to experimental SWAXS data, these developments pave the way for increasing the accuracy of modeling algorithms utilizing SWAXS data while decreasing the risk of overfitting. Statement of SignificanceAccurate calculation of small and wide-angle scattering (SWAXS) profiles from atomic models is useful for studying the solution state and conformational dynamics of biological macromolecules in solution. Here we present a new approach to calculating SWAXS profiles from atomic models using high resolution real space density maps. This approach includes novel calculations of solvent contributions that remove a significant fitting parameter. The algorithm is tested on multiple high quality experimental SWAXS datasets, showing improved accuracy compared to leading software. The algorithm is computationally efficient and robust to overfitting, paving the way for increasing the accuracy and resolution of modeling algorithms utilizing experimental SWAXS data.

biophysics↗

bioRxiv2023

Automatic Abdominal Multi Organ Segmentationusing Residual UNet

Automated segmentation of abdominal organs plays an important role in supporting computer-assisted diagnosis, radiotherapy, biomarker extraction, surgery navigation, and treatment planning. Segmenting multiple abdominal organs using a single algorithm would improve model development efficiency and accelerate model deployment into clinical workflows. To achieve broadly generalized performance, we trained a residual UNet using 500 CT/MRI scans collected from multi-center, multi-vendor, multi-phase, multi-disease patients, each with voxel-level annotation of 15 abdominal organs. Using the model trained on multimodality (CT/MRI), we achieved an average dice of 0.8990 in the held-out test dataset with only CT scans (N=100). An average dice of 0.8948 was achieved in the held-out test dataset with both CT and MRI scans (N=120. Our results demonstrate broad generalization of the model.

bioengineering↗

bioRxiv2023

Automatic Whole Body FDG PET/CT Lesion Segmentation using Residual UNet and Adaptive Ensemble

Multimodal Positron Emission Tomography/Computed Tomography (PET/CT) plays a key role in the diagnosis, staging, restaging, treatment response assessment, and radiotherapy planning of malignant tumors. The complementary nature of high-resolution anatomic CT and high sensitivity/specificity molecular PET imaging provides accurate assessment of disease status [14] In oncology, 18-fluorodeoxyglucose (FDG) PET/CT is the most widely used method to identify and analyze metabolically active tumors. In particular, FDG uptake allows for more accurate detection of both nodal and distant forms of metastatic disease. Accurate quantification and staging of tumors is the most important prognostic factor for predicting the survival of patients and for designing personalized patient management plans. [8,3] Analyzing PET/CT quantitatively by experienced medical imaging experts/radiologists is timeconsuming and error-prone. Automated quantitative analysis by deep learning algorithms to segment tumor lesions will enable accurate feature extraction, tumor staging, radiotherapy planning, and treatment response assessment. The AutoPET Challenge 2022 provided an opensource platform to develop and benchmark deep learning models for automated PET lesion segmentation by providing large open-source wholebody FDG-PET/CT data. Using the multimodal PET/CT data from 900 subjects with 1014 studies provided by the AutoPET MICCAI 2022 Challenge, we applied fivefold cross-validation on residual UNETs to automatically segment lesions. We then utilized the output from adaptive ensemble highly contributive models as the final segmentation. Our method achieved a 10th ranking with a dice score of 0.5541 in the heldout test dataset (N=150 studies).

bioengineering↗

bioRxiv2022

High emotional reactivity is associated with activation of a molecularly distinct hippocampal-amygdala circuit modulated by the glucocorticoid receptor

Emotions are characterized not only by their valence but also by whether they are stable or labile. Yet, we do not understand the molecular or circuit mechanisms that control the dynamic nature of emotionality. We have shown that glucocorticoid receptor overexpression in the forebrain (GRov) leads to a highly reactive mouse with increased anxiety behavior coupled with greater swings in emotional responses. This phenotype is established early in development and persists into adulthood. However, the neural circuitry mediating this lifelong emotional lability remains unknown. In the present study, optogenetic stimulation in ventral dentate gyrus (vDG) of GRov mice led to a greater range and a prolonged duration of anxiety behavior. cFos expression showed that the amplified behavioral response to vDG activation in GRov mice is coupled to increased neuronal activity in specific brain regions. Relative to wild type mice, GRov mice displayed glutamatergic/GABAergic activation imbalance in ventral CA1 (vCA1) and selectively increased glutamatergic activation in the basal posterior amygdaloid complex. Fluorescence in situ hybridization chain reaction studies showed that forebrain GR overexpression led to increased activation of molecularly distinct subpopulations of neurons within the hippocampus and the posterior basolateral amygdala (pBLA). Increased cFos labeling was observed in the Calbindin 1 (Calbn1+) glutamatergic neurons in vCA1 and in the DARPP-32/Ppp1r1b+ glutamatergic neurons in pBLA. We propose that a molecularly distinct hippocampal-amygdala circuit is shaped by stress early in life and tunes the dynamics of emotional responses.

neuroscience↗

bioRxiv2022

The genome of tropically adapted Brahman cattle (Bos taurus indicus) reveals novel genome variation in production animals

Recent advances in sequencing technology have revolutionised access to large scale genomic data that can be assembled into a platinum quality genome. Here we present a high quality genome assembly with less than 300 gaps of a Brahman cow (B. taurus indicus). The assembly was generated using 195GB of PacBio and 169GB of Oxford Nanopore Technologies sequence data. The high quality genome assembly allows us to identify substantial GC content variation that is positively associated with gene rich islands, and negatively associated with genetic variation in the form of structural variants. In addition, 92371 structural variants that are segregating in the brahman population were identified. Gene ontology analysis revealed that genes with varying copy numbers were enriched for gene ontology terms related to immune function. This analysis has revealed the complex structure of the mammalian genome of an outbred species, and identifies the ability of long read data from diploid species can be used to not only assemble a high quality genome, but also discover novel genetic variation within that genome.

genomics↗

bioRxiv2021

Compositional analysis of Sindbis virus ribonucleoproteins reveals an extensive co-opting of key nuclear RNA-binding proteins

RNA is a central molecule for RNA viruses, acting as mRNA and genome. However, the interactions that viral (v)RNA establishes with the host cell is only starting to be elucidated. Here, we determine with unprecedented depth the composition of the ribonucleoproteins (RNPs) of the prototypical arthropod-borne Sindbis virus (SINV) using viral RNA interactome capture. We show that SINV RNAs engage with hundreds of cellular proteins and pathways, including a group of nuclear RNA-binding proteins (RBPs) with unknown roles in infection. Combining subcellular fractionation and proteomics with several orthogonal approaches, we demonstrate that these nuclear RBPs are selectively redistributed to the cytoplasm after infection, where they associate with the viral replication organelles. These nuclear RBPs potently supress viral gene expression, with activities spanning viral species and families. Our study provides a comprehensive and systematic analysis of SINV RNP composition, revealing a network of nuclear RBPs with moonlighting antiviral function. Research highlightsO_LISINV RNAs interact with over four hundred cellular RBPs C_LIO_LISINV induces selective cytoplasmic translocation of a subset of nuclear RBPs C_LIO_LIThese nuclear RBPs display potent antiviral effects C_LIO_LIThe SF3B complex binds to SINV RNA and supresses infection in a splicing-independent manner C_LI

microbiology↗

bioRxiv2021

Investigating Regional Specificity of Alzheimers Disease Pathology in the 5xFAD Mouse Model

Alzheimers disease (AD) is a neurodegenerative disease that affects cognition and memory. Mouse models such as 5xFAD, incorporate mutations found in familial or early-onset (EOAD) AD; these mutations increase the production of the toxic AB species that contributes to plaque formation in the brain. However, not all brain regions in the 5xAD animals accumulate plaques to the same degree. For example, the cerebellum appears to be resistant to AB plaque formation, while other regions such as the cortex and subiculum develop copious plaques. The mechanism(s) underlying this regional specificity remains unclear. Thus, we used a fluorescent antibody to APP/AB to quantify the amount of these proteins in several brain regions of interest, including subiculum, cortex, and cerebellum. We found that the cerebellum had less APP/AB than the other regions quantified, which demonstrates a correlation between the levels of APP/AB and plaque formation. Taken together, this suggests that the regional specificity of AD pathology may be a result of different levels of protein expression.

neuroscience↗

bioRxiv2020

A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test-tube. The key advantages of this approach are the reduced experimental timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters, with strong protein production and well-characterised synthetic biology tools, is essential. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool and an active background metabolism. However, our initial yields were low (36 g/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 g/mL of expressed recombinant protein. To complement this, we rapidly characterise a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes and demonstrate an initial proof of concept for combined transcription, translation and biosynthesis of Streptomyces metabolic pathways in a single one-pot reaction.

synthetic biology↗