Biology subjects

Crook, N. C.

Publications and source records attributed to Crook, N. C..

7 recordsLinked to original sources

bioRxiv2025

Construction of a Heterologous Pathway in Escherichia coli for Terephthalate Assimilation

Polyethylene terephthalate (PET) is extensively used in products such as packaging and textiles, and it remains a significant contributor to plastic and microplastic pollution. Effective valorization of PET waste requires not only depolymerizing it into its monomers but also utilizing the resulting products, ethylene glycol (EG) and terephthalic acid (TPA). While prior research has demonstrated that Escherichia coli can convert TPA into value-added compounds for PET upcycling, limited work has focused on its assimilation for cell growth. This study focuses on constructing a complete TPA metabolic pathway in E. coli comprising TPA uptake and its conversion to protocatechuate (PCA). We engineered E. coli JME3, which already possesses PCA catabolism, to utilize TPA as a sole carbon source by introducing a heterologous transporter TpaK and a PCA-synthesizing enzyme cascade. The strain next underwent adaptive laboratory evolution (ALE) and expression tuning to improve growth performance. The final strain achieved a maximum growth rate of 0.25 h-{superscript 1}, demonstrating efficient TPA assimilation. These results expand the range of value-added compounds derived from PET upcycling and establish E. coli as a platform for coupling PET depolymerization with microbial growth, enabling high-throughput optimization of enzyme activity and PET biodegradation conditions.

bioengineering↗

bioRxiv2023

Programming Probiotics: Diet-responsive gene expression and colonization control in engineered S. boulardii

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 a dietary sugar. Additionally, repairing galactose metabolism expanded Sbs 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.

bioengineering↗

bioRxiv2023

Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for studying Plant-Microbe Interactions

Sustainably enhancing crop production is a necessity given the increasing demands for staple crops and their associated carbon/nitrogen inputs. Plant-associated microbiomes offer one avenue for addressing this demand; however, studying these communities and engineering them has remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root ToolKit (MRTK); a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 hours to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida AA7 (Ppu), Herbaspirillum robiniae strain AA6 (Hro), Stenotrophomonas maltophilia strain AA1 (Sma) and Brucella pituitosa strain AA2 (Bpi) which comprise a model maize root synthetic community (SynCom). In addition to these genetic tools, we describe a highly efficient transformation protocol (10^7-10^9 transformants/{micro}g of DNA) for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous expression sequences for each strain (ES; promoter and ribosomal binding sites) via genomic promoter trapping. Overall, the MRTK is a scalable platform that expands the genetic engineering toolbox while providing a standardized, high efficiency transformation method that can be implemented across a diverse group of root commensals. These results unlock the ability to elucidate and engineer plant-microbe interactions promoting plant growth for each of the 5 bacterial strains in this study.

synthetic biology↗

bioRxiv2022

Improving Therapeutic Protein Secretion in the Probiotic Yeast Saccharomyces boulardii using a Multifactorial Engineering Approach

The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sbs 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 Sbs 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 6-fold (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 - 463 mg/L. Then, guided by prior knowledge of S. cerevisiaes 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 >10-fold 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.

synthetic biology↗

bioRxiv2022

Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts

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 bakers 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. One-sentence summaryEnabling sensing in probiotic yeast and synthetic yeast mating with human and fungal G protein-coupled receptors.

cell biology↗

bioRxiv2020

Inducible Directed Evolution of Complex Phenotypes in Bacteria

Directed evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5-10kb in length, elimination of genomic hitchhiker mutations, and decoupling of diversification and screening steps. To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to package large plasmids and transfer them to naive cells after intracellular mutagenesis. To demonstrate IDE, we evolved a 5-gene pathway from Bacillus licheniformis that accelerates tagatose catabolism in Escherichia coli, resulting in clones with 65% shorter lag times during growth on tagatose after only two rounds of evolution.

synthetic biology↗

bioRxiv2020

Establishing Probiotic Saccharomyces boulardii as a Model Organism for Synthesis and Delivery of Biomolecules

Saccharomyces boulardii is a widely used yeast probiotic which can counteract various gastrointestinal disorders1. As a relative of Saccharomyces cerevisiae, S. boulardii exhibits rapid growth and is easy to transform2 and thus represents a promising chassis for the engineered secretion of biomolecules. To establish S. boulardii as a platform for delivery of biomolecules to the mammalian gut, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements in this organism. These genetic elements were characterized in plasmidic and genomic contexts, revealing strategies for tunable control of gene expression and CRISPR-mediated genome editing in this strain. We then leveraged this set of genetic parts to combinatorially assemble pathways enabling a wide range of drug and vitamin titers. Finally, we measured S. boulardiis residence time in the gastrointestinal tracts of germ-free and antibiotic-treated mice, revealing the relationships between dosing strategy and colonization level. This work establishes S. boulardii as a genetically tractable commensal fungus and provides a set of strategies for engineering S. boulardii to synthesize and deliver biomolecules during gut colonization.

synthetic biology↗