G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. ...GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker's yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.
The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and ...high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.
Infections by Clostridioides difficile, a bacterium that targets the large intestine (colon), impact a large number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A ...and B. Short peptides that can be delivered to the gut and inhibit the biocatalytic activity of these toxins represent a promising therapeutic strategy to prevent and treat C. diff. infection. We describe an approach that combines a Peptide Binding Design (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in colon epithelial cells. One peptide, SA1, is found to block TcdA toxicity in primary-derived human colon (large intestinal) epithelial cells. SA1 binds TcdA with a K
of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR).
Genetically engineered microbes that secrete therapeutics, sense and respond to external environments, and/or target specific sites in the gut fall under an emergent class of therapeutics, called ...live biotherapeutic products (LBPs). As live organisms that require symbiotic host interactions, LBPs offer unique therapeutic opportunities, but also face distinct challenges in the gut microenvironment. In this review, we describe recent approaches (often demonstrated using traditional probiotic microorganisms) to discover LBP chassis and genetic parts utilizing omics-based methods and highlight LBP delivery strategies, with a focus on addressing physiological challenges that LBPs encounter after oral administration. Finally, we share our perspective on the opportunity to apply an integrated approach, wherein discovery and delivery strategies are utilized synergistically, towards tailoring and optimizing LBP efficacy.
The physiological microenvironment of the gut influences the efficacy of live biotherapeutic products (LBPs) and both discovery and delivery strategies can be used to overcome physiological challenges in the gut.Multi-omics illuminates colonization mechanisms of nonengineered LBPs to inspire engineering strategies.Functional genomics generates and tests engineered LBPs in a high-throughput manner to provide improved strains.Pharmaceutical formulations can be used to control the interactions between LBPs and their physiological microenvironment, creating modular technologies and approaches that can be applied to all LBPs.Genetic engineering approaches can improve LBP delivery through overcoming physiological challenges, enabling molecular interactions with host surfaces, controlling therapeutic functions in response to local physiological cues.
Engineered probiotics are genetically modified microorganisms to perform sophisticated diagnostic, preventative and/or therapeutic functions in the gut. Up to date, majority of this technology has ...been established in bacterial probiotics species, while not noticing the potential of fungal hosts for engineered probiotics technologies and microbiome studies. During my PhD, we focused on developing tools and workflows to establish the only fungal probiotic generally regarded as safe (GRAS) by the FDA, Saccharomyces boulardii (Sb), as an in situ biomanufacturing platform, delivering a range of biomolecules to into the mammalian gut through tunable intracellular and extracellular expression vectors that are constitutive or inducible. In chapter 1, we covered the literature behind molecular and synthetic biology methods that are used to establish new strategies to design engineered probiotics. We covered these strategies under the perspectives of improving chassis discovery and chassis delivery. In chapter 2, we applied combinatorial design rules to synthesize and deliver small molecules into the mammalian gut by engineered Sb. We first characterized the effect of constitutive expression parts on heterologous gene expression in Sb, in both plasmidic and genomic contexts. Then, we created a library of Sb strains that can synthesize high titers of β-carotene and violacein, through combinatorial in vivo cloning strategies. From this strain library, we chose the Sb strain with highest β-carotene production performance to investigate colonization of and small molecule delivery kinetics of engineered Sb’s in germ-free mice model. In addition, we investigated the effect of gut microbiota on Sb’s colonization kinetics in specific-pathogen free mice models. In chapter 3, we developed inducible expression systems to achieve further precise tuning of engineered functions in Sb. We modified the galactose metabolism in Sb to further improve the application of inducible systems in Sb. We characterized the growth profiles of the Sb strain with modified galactose metabolism with various carbon sources. We identified the effect of galactose metabolism modification in Sb on its colonization kinetics and biodistribution profile using specific-pathogen free mice model. We also characterized the activity of these small molecule inducible systems under aerobic and anaerobic environments via use of multiple reporter proteins expressed intracellularly. We further expanded the application inducible systems via achieving inducible yeast surface display platform in Sb. In chapter 4, we investigated recombinant protein secretion kinetics in Sb. We developed a library of Sb strains secreting a heterologous peptide targeting Clostridioides difficile Toxin A. Using this library, we identified the engineering bottlenecks to achieve and improve secretory production of recombinant proteins in Sb. We improved recombinant peptide secretion by effective vector design and secretory pathway optimization through combinatorial engineering guided by proteomics studies. In chapter 5, we covered the literature on precision therapies targeting gastrointestinal pathogens. We discussed the mode of actions of these therapies in the context of molecule biochemical structure and characteristics of cellular functions integrated into the engineered microbial delivery systems. Overall, the work presented in this thesis provides a range of engineering considerations to adapt in Sb as a promising host for in situ biomanufacturing applications.
The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has ...faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium’s restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium’s DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.
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•IMPRINT allows the rapid recreation of DNA methylation patterns with cell-free TXTL•Enhanced transformation with IMPRINT in Salmonella and Bifidobacterium strains•HT-IMPRINT identifies the best methyltransferase combinations in one experiment•Enhanced transformation by IMPRINT allows screening of a large RBS library
Vento et al. report IMPRINT for recreating a host bacterium’s DNA methylation pattern by expressing its unique set of DNA methyltransferases with cell-free transcription-translation. Subsequently methylating DNA with these expressed methyltransferases boosts its transformation by circumventing the host’s restriction-modification defense systems.
is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by ...multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (β-carotene) and drug (violacein) titers. We found that
colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled β-carotene synthesis (194 μg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional β-carotene recovered in feces was 56-fold higher than the β-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by
living in the mammalian gut and provides a set of tools for modulating these titers.
Saccharomyces boulardii (Sb) is an emerging probiotic chassis for delivering biomolecules to the mammalian gut, offering unique advantages as the only eukaryotic probiotic. However, precise control ...over gene expression and gut residence time in Sb have remained challenging. To address this, we developed five ligand-responsive gene expression systems and repaired galactose metabolism in Sb, enabling inducible gene expression in this strain. Engineering these systems allowed us to construct AND logic gates, control the surface display of proteins, and turn on protein production in the mouse gut in response to dietary sugar. Additionally, repairing galactose metabolism expanded Sb’s habitat within the intestines and resulted in galactose-responsive control over gut residence time. This work opens new avenues for precise dosing of therapeutics by Sb via control over its in vivo gene expression levels and localization within the gastrointestinal tract.
Gastrointestinal pathogens employ a variety of mechanisms to damage host tissue, acquire nutrients, and evade treatment. To supplement broad-spectrum antimicrobials, there has been increasing ...interest in designing molecules that target specific taxa and virulence processes. Excitingly, these antivirulence therapies may be able to be synthesized by gut-resident microbes, thereby enabling delivery of these drugs directly to the spatial and temporal site of infection. In this review, we highlight recent progress in our understanding of small molecules that inhibit specific virulence mechanisms. We additionally discuss emerging methods to discover pathogen-specific and mechanism-specific peptides and small proteins. Finally, we cover recent demonstrations of probiotics engineered to produce antimicrobials in response to pathogen-specific cues in the gut. Collectively, these advances point to an emerging integrative approach to treatment of gastrointestinal diseases, comprising microbiologists, peptide chemists, and synthetic biologists.
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