@article{vento_durmusoglu_li_patinios_sullivan_ttofali_schaik_yu_wang_barquist_et al._2024, title={A cell-free transcription-translation pipeline for recreating methylation patterns boosts DNA transformation in bacteria}, volume={84}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2024.06.003}, abstractNote={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.}, number={14}, journal={MOLECULAR CELL}, author={Vento, Justin M. and Durmusoglu, Deniz and Li, Tianyu and Patinios, Constantinos and Sullivan, Sean and Ttofali, Fani and Schaik, John and Yu, Yanying and Wang, Yanyan and Barquist, Lars and et al.}, year={2024}, month={Jul} }
 @article{li_crook_2024, title={Chips, guts, and gas: unraveling volatile microbial mysteries in real time!}, volume={42}, ISSN={["1879-3096"]}, url={https://doi.org/10.1016/j.tibtech.2023.12.009}, DOI={10.1016/j.tibtech.2023.12.009}, abstractNote={<h2>Abstract</h2> Exploring the gastrointestinal role of hydrogen sulfide (H<sub>2</sub>S) is difficult because of its volatility and the absence of a precisely controllable model system for manipulating the gut environment. Hayes et al. address this issue by engineering <i>Escherichia coli</i> to titrate H<sub>2</sub>S levels in a gas-impermeable gut-on-chip device.}, number={2}, journal={TRENDS IN BIOTECHNOLOGY}, author={Li, Zidan and Crook, Nathan C.}, year={2024}, month={Feb}, pages={144–146} }
 @article{durmusoglu_haller_al’abri_day_sands_clark_san-miguel_vazquez-uribe_sommer_crook_2024, title={Programming Probiotics: Diet-Responsive Gene Expression and Colonization Control in Engineered S. boulardii}, url={https://doi.org/10.1021/acssynbio.4c00145}, DOI={10.1021/acssynbio.4c00145}, abstractNote={(}, journal={ACS Synthetic Biology}, author={Durmusoglu, Deniz and Haller, Daniel J. and Al’Abri, Ibrahim S. and Day, Katie and Sands, Carmen and Clark, Andrew and San-Miguel, Adriana and Vazquez-Uribe, Ruben and Sommer, Morten O. A. and Crook, Nathan C.}, year={2024}, month={Jun} }
 @article{eroglu_wang_crook_bohn_2024, title={The Carotenoids}, volume={15}, ISSN={["2156-5376"]}, url={https://doi.org/10.1016/j.advnut.2024.100304}, DOI={10.1016/j.advnut.2024.100304}, number={11}, journal={ADVANCES IN NUTRITION}, author={Eroglu, Abdulkerim and Wang, Genan and Crook, Nathan and Bohn, Torsten}, year={2024}, month={Nov} }
 @article{gates_crook_2024, title={The biochemical mechanisms of plastic biodegradation}, url={https://doi.org/10.1093/femsre/fuae027}, DOI={10.1093/femsre/fuae027}, journal={FEMS Microbiology Reviews}, author={Gates, Ethan and Crook, Nathan}, year={2024}, month={Nov} }
 @article{bang_bergman_li_mukherjee_alshehri_abbott_crook_velev_hall_you_2023, title={An integrated chemical engineering approach to understanding microplastics}, volume={1}, ISSN={["1547-5905"]}, DOI={10.1002/aic.18020}, abstractNote={AbstractEnvironmental and health risks posed by microplastics (MPs) have spurred numerous studies to better understand MPs' properties and behavior. Yet, we still lack a comprehensive understanding due to MP's heterogeneity in properties and complexity of plastic property evolution during aging processes. There is an urgent need to thoroughly understand the properties and behavior of MPs as there is increasing evidence of MPs' adverse health and environmental effects. In this perspective, we propose an integrated chemical engineering approach to improve our understanding of MPs. The approach merges artificial intelligence, theoretical methods, and experimental techniques to integrate existing data into models of MPs, investigate unknown features of MPs, and identify future areas of research. The breadth of chemical engineering, which spans biological, computational, and materials sciences, makes it well‐suited to comprehensively characterize MPs. Ultimately, this perspective charts a path for cross‐disciplinary collaborative research in chemical engineering to address the issue of MP pollution.}, journal={AICHE JOURNAL}, author={Bang, Rachel S. and Bergman, Michael and Li, Tianyu and Mukherjee, Fiona and Alshehri, Abdulelah S. and Abbott, Nicholas L. and Crook, Nathan C. and Velev, Orlin D. and Hall, Carol K. and You, Fengqi}, year={2023}, month={Jan} }
 @article{li_menegatti_crook_2023, title={Breakdown of polyethylene therepthalate microplastics under saltwater conditions using engineered Vibrio natriegens}, volume={9}, ISSN={["1547-5905"]}, url={https://doi.org/10.1002/aic.18228}, DOI={10.1002/aic.18228}, abstractNote={AbstractPoly(ethylene terephthalate) (PET) is a highly recyclable plastic that has been extensively used and manufactured. Like other plastics, PET resists natural degradation, thus accumulating in the environment. Several recycling strategies have been applied to PET, but these tend to result in downcycled products that eventually end up in landfills. This accumulation of landfilled PET waste contributes to the formation of microplastics, which pose a serious threat to marine life and ecosystems, and potentially to human health. To address this issue, our project leveraged synthetic biology to develop a whole‐cell biocatalyst capable of depolymerizing PET in seawater environments by using the fast‐growing, nonpathogenic, moderate halophile Vibrio natriegens. By leveraging a two‐enzyme system—comprising a chimera of IsPETase and IsMHETase from Ideonella sakaiensis—displayed on V. natriegens, we constructed whole‐cell catalysts that depolymerize PET and convert it into its monomers in salt‐containing media and at a temperature of 30°C.}, journal={AICHE JOURNAL}, author={Li, Tianyu and Menegatti, Stefano and Crook, Nathan}, year={2023}, month={Sep} }
 @misc{bohn_balbuena_ulus_iddir_wang_crook_eroglu_2023, title={Carotenoids in Health as Studied by Omics-Related Endpoints}, volume={14}, ISSN={["2156-5376"]}, url={https://doi.org/10.1016/j.advnut.2023.09.002}, DOI={10.1016/j.advnut.2023.09.002}, abstractNote={Carotenoids have been associated with risk reduction for several chronic diseases, including the association of their dietary intake/circulating levels with reduced incidence of obesity, type 2 diabetes, certain types of cancer, and even lower total mortality. In addition to some carotenoids constituting vitamin A precursors, they are implicated in potential antioxidant effects and pathways related to inflammation and oxidative stress, including transcription factors such as nuclear factor κB and nuclear factor erythroid 2-related factor 2. Carotenoids and metabolites may also interact with nuclear receptors, mainly retinoic acid receptor/retinoid X receptor and peroxisome proliferator-activated receptors, which play a role in the immune system and cellular differentiation. Therefore, a large number of downstream targets are likely influenced by carotenoids, including but not limited to genes and proteins implicated in oxidative stress and inflammation, antioxidation, and cellular differentiation processes. Furthermore, recent studies also propose an association between carotenoid intake and gut microbiota. While all these endpoints could be individually assessed, a more complete/integrative way to determine a multitude of health-related aspects of carotenoids includes (multi)omics-related techniques, especially transcriptomics, proteomics, lipidomics, and metabolomics, as well as metagenomics, measured in a variety of biospecimens including plasma, urine, stool, white blood cells, or other tissue cellular extracts. In this review, we highlight the use of omics technologies to assess health-related effects of carotenoids in mammalian organisms and models.}, number={6}, journal={ADVANCES IN NUTRITION}, author={Bohn, Torsten and Balbuena, Emilio and Ulus, Hande and Iddir, Mohammed and Wang, Genan and Crook, Nathan and Eroglu, Abdulkerim}, year={2023}, month={Nov}, pages={1538–1578} }
 @article{sohail_pirzada_guenther_barbieri_sit_menegatti_crook_opperman_khan_2023, title={Cellulose Acetate-Stabilized Pickering Emulsions: Preparation, Rheology, and Incorporation of Agricultural Active Ingredients}, volume={11}, ISSN={["2168-0485"]}, url={https://doi.org/10.1021/acssuschemeng.3c02428}, DOI={10.1021/acssuschemeng.3c02428}, abstractNote={We report the use of cellulose acetate (CA) nanoparticles (NPs) to produce oil in water Pickering emulsions. The CA NP can emulsify various oils and form stable emulsions at concentrations as low as 0.5 wt %. Rheological and microscopic analyses show evidence of interconnected NP aggregate networks between droplets. Yield stress measurements display evidence of "double" yielding. We postulate that the presence of the NP aggregates provides a secondary network between droplet clusters resulting in such behavior. We demonstrate the suitability of the emulsions as agriculture formulations by incorporating an agrochemical, abamectin (Abm), and a plant-growth-promoting microbe (PGPM) in the emulsions. Release assays exhibit sustained Abm release, promising higher efficacy at lower usage volumes. Incorporation of nonsporulating PGPM Pseudomonas simiae in the emulsions shows significantly higher microbe viability compared to controls after 70 days of storage. By demonstrating the application of CA NPs as a sustainable Pickering emulsifier, this study introduces the use of CA as a platform technology for the delivery of diverse agriculture cargos. A comprehensive evaluation of the system is articulated in a fundamental microstructure analysis and a demonstration of practical on-site attributes, including shelf-life stability and functional performance, verified through bioassays and plant growth studies.}, number={42}, journal={ACS SUSTAINABLE CHEMISTRY & ENGINEERING}, author={Sohail, Mariam and Pirzada, Tahira and Guenther, Richard and Barbieri, Eduardo and Sit, Tim and Menegatti, Stefano and Crook, Nathan and Opperman, Charles H. and Khan, Saad A.}, year={2023}, month={Sep}, pages={15178–15191} }
 @book{sarma_catella_pedro_xiao_durmusoglu_menegatti_crook_magness_hall_2023, title={Design of 8-mer Peptides that Block Clostridioides difficile Toxin A in Intestinal Cells}, url={https://doi.org/10.1101/2023.01.10.523493}, DOI={10.1101/2023.01.10.523493}, abstractNote={AbstractClostridioides difficile(C. diff.) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity ofC. diff. infection is derived from two major toxins, toxins A (TcdA) and B (TcdB). Peptide inhibitors that can be delivered to the gut to inactivate these toxins are an attractive therapeutic strategy. In this work, we present a new approach that combines apeptidebindingdesign algorithm (PepBD), molecular-level simulations, rapid screening of candidate peptides for toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block the glucosyltransferase activity of TcdA by targeting its glucosyltransferase domain (GTD). Using PepBD and explicit-solvent molecular dynamics simulations, we identified seven candidate peptides, SA1-SA7. These peptides were selected for specific TcdA GTD binding through a custom solid-phase peptide screening system, which eliminated the weaker inhibitors SA5-SA7. The efficacies of SA1-SA4 were then tested using a trans-epithelial electrical resistance (TEER) assay on monolayers of the human gut epithelial culture model. One peptide, SA1, was found to block TcdA toxicity in primary-derived human jejunum (small intestinal) and colon (large intestinal) epithelial cells. SA1 bound TcdA with a KDof 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR).Significance StatementInfections byClostridioides difficile, a bacterium that targets the large intestine (colon), impact a significant number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can inhibit the biocatalytic activity of these toxins represent a promising strategy to prevent and treatC. diff. infection. We describe an approach that combines aPeptide BindingDesign (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 small intestinal and colon epithelial cells. Importantly, our designed peptide, SA1, bound toxin A with nanomolar affinity and blocked toxicity in colon cells.}, author={Sarma, S. and Catella, C.M. and Pedro, E.T.San and Xiao, X. and Durmusoglu, D. and Menegatti, S. and Crook, N. and Magness, S.T. and Hall, C.K.}, year={2023} }
 @article{sarma_catella_san pedro_xiao_durmusoglu_menegatti_crook_magness_hall_2023, title={Design of 8-mer peptides that block <i>Clostridioides difficile</i> toxin A in intestinal cells}, volume={6}, ISSN={["2399-3642"]}, url={https://doi.org/10.1038/s42003-023-05242-x}, DOI={10.1038/s42003-023-05242-x}, abstractNote={AbstractInfections 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 KD of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR).}, number={1}, journal={COMMUNICATIONS BIOLOGY}, author={Sarma, Sudeep and Catella, Carly M. and San Pedro, Ellyce T. and Xiao, Xingqing and Durmusoglu, Deniz and Menegatti, Stefano and Crook, Nathan and Magness, Scott T. and Hall, Carol K.}, year={2023}, month={Aug} }
 @article{schaik_li_cheadle_crook_2023, title={Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for Studying Plant-Microbe Interactions}, volume={9}, ISSN={["2161-5063"]}, url={https://doi.org/10.1021/acssynbio.3c00371}, DOI={10.1021/acssynbio.3c00371}, abstractNote={Sustainably enhancing crop production is a global necessity to meet the escalating demand for staple crops while sustainably managing their associated carbon/nitrogen inputs. Leveraging plant-associated microbiomes is a promising avenue for addressing this demand. However, studying these communities and engineering them for sustainable enhancement of crop production have remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root Microbiome ToolKit (MRMTK), a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 h to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida strain 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 (107-109 transformants/μg of DNA) 1 for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous Expression Sequences (ES; promoter and ribosomal binding sites) for each strain via genomic promoter trapping. Overall, MRMTK is a scalable and adaptable platform that expands the genetic engineering toolbox while providing a standardized, high-efficiency transformation method 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.}, journal={ACS SYNTHETIC BIOLOGY}, author={Schaik, John and Li, Zidan and Cheadle, John and Crook, Nathan}, year={2023}, month={Sep} }
 @article{schaik_li_cheadle_crook_2023, title={Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for studying Plant-Microbe Interactions}, url={https://doi.org/10.1101/2023.06.05.543168}, DOI={10.1101/2023.06.05.543168}, abstractNote={ABSTRACTSustainably 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 inEnterobacter ludwigiistrain AA4 (Elu),Pseudomonas putidaAA7 (Ppu),Herbaspirillum robiniaestrain AA6 (Hro),Stenotrophomonas maltophiliastrain AA1 (Sma) andBrucella pituitosastrain 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/µg 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.}, author={Schaik, John and Li, Zidan and Cheadle, John and Crook, Nathan}, year={2023}, month={Jun} }
 @article{durmusoglu_al'abri_li_islam williams_collins_martinez_crook_2023, title={Improving therapeutic protein secretion in the probiotic yeast Saccharomyces boulardii using a multifactorial engineering approach}, volume={22}, ISSN={["1475-2859"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85161032784&partnerID=MN8TOARS}, DOI={10.1186/s12934-023-02117-y}, abstractNote={AbstractThe probiotic yeastSaccharomyces boulardii(Sb) is a promising chassis to deliver therapeutic proteins to the gut due toSb’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 engineerSbstrains with enhanced levels of protein secretion. In this work, we explored genetic modifications in bothcis-(i.e. to the expression cassette of the secreted protein) andtrans-(i.e. to theSbgenome) that enhanceSb’s ability to secrete proteins, taking aClostridioides difficileToxin 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 ofS. 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-deficientSbstrain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-typeSb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion inSband 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 ofSbto deliver therapeutics to the gut and other settings to which it is adapted.}, number={1}, journal={MICROBIAL CELL FACTORIES}, author={Durmusoglu, Deniz and Al'Abri, Ibrahim and Li, Zidan and Islam Williams, Taufika and Collins, Leonard B. and Martinez, Jose L. and Crook, Nathan}, year={2023}, month={Jun} }
 @article{durmusoglu_haller_al’abri_day_sands_clark_san-miguel_vazquez-uribe_sommer_crook_2023, title={Programming Probiotics: Diet-responsive gene expression and colonization control in engineeredS. boulardii}, url={https://doi.org/10.1101/2023.11.17.567539}, DOI={10.1101/2023.11.17.567539}, abstractNote={AbstractSaccharomyces 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 inSbhave remained challenging. To address this, we developed five ligand-responsive gene expression systems and repaired galactose metabolism inSb, 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 expandedSb’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 bySbvia control over itsin vivogene expression levels and localization within the gastrointestinal tract.}, author={Durmusoglu, Deniz and Haller, Daniel J. and Al’Abri, Ibrahim S. and Day, Katie and Sands, Carmen and Clark, Andrew and San-Miguel, Adriana and Vazquez-Uribe, Ruben and Sommer, Morten O. A. and Crook, Nathan C.}, year={2023}, month={Nov} }
 @article{al'abri_li_haller_crook_2022, title={A Novel Method of Inducible Directed Evolution to Evolve Complex Phenotypes}, volume={12}, ISSN={["2331-8325"]}, DOI={10.21769/BioProtoc.4535}, abstractNote={Directed evolution is a powerful technique for identifying beneficial mutations in defined DNA sequences with the goal of improving desired phenotypes. Recent methodological advances have made the evolution of short DNA sequences quick and easy. However, the evolution of DNA sequences >5kb in length, notably gene clusters, is still a challenge for most existing methods. Since many important microbial phenotypes are encoded by multigene pathways, they are usually improved via adaptive laboratory evolution (ALE), which while straightforward to implement can suffer from off-target and hitchhiker mutations that can adversely affect the fitness of the evolved strain. We have therefore developed a new directed evolution method (Inducible Directed Evolution, IDE) that combines the specificity and throughput of recent continuous directed evolution methods with the ease of ALE. Here, we present detailed methods for operating Inducible Directed Evolution (IDE), which enables long (up to 85kb) DNA sequences to be mutated in a high throughput manner via a simple series of incubation steps. In IDE, an intracellular mutagenesis plasmid (MP) tunably mutagenizes the pathway of interest, located on the phagemid (PM). MP contains a mutagenic operon ( danQ926, dam, seqA, emrR, ugi , and cda1 ) that can be expressed via the addition of a chemical inducer. Expression of the mutagenic operon during a cell cycle represses DNA repair mechanisms such as proofreading, translesion synthesis, mismatch repair, and base excision and selection, which leads to a higher mutation rate. Induction of the P1 lytic cycle results in packaging of the mutagenized phagemid, and the pathway-bearing phage particles infect naïve cells, generating a mutant library that can be screened or selected for improved variants. Successive rounds of IDE enable optimization of complex phenotypes encoded by large pathways (as of this writing up to 36 kb), without requiring inefficient transformation steps. Additionally, IDE avoids off-target genomic mutations and enables decoupling of mutagenesis and screening steps, establishing it as a powerful tool for optimizing complex phenotypes in E. coli .}, number={20}, journal={BIO-PROTOCOL}, author={Al'Abri, Ibrahim S. and Li, Zidan and Haller, Daniel J. and Crook, Nathan}, year={2022}, month={Oct} }
 @misc{eroglu_al'abri_kopec_crook_bohn_2023, title={Carotenoids and Their Health Benefits as Derived via Their Interactions with Gut Microbiota}, volume={14}, ISSN={["2156-5376"]}, DOI={10.1016/j.advnut.2022.10.007}, abstractNote={Carotenoids have been related to a number of health benefits. Their dietary intake and circulating levels have been associated with a reduced incidence of obesity, diabetes, certain types of cancer, and even lower total mortality. Their potential interaction with the gut microbiota (GM) has been generally overlooked but may be of relevance, as carotenoids largely bypass absorption in the small intestine and are passed on to the colon, where they appear to be in part degraded into unknown metabolites. These may include apo-carotenoids that may have biological effects because of higher aqueous solubility and higher electrophilicity that could better target transcription factors, i.e., NF-κB, PPARγ, and RAR/RXRs. If absorbed in the colon, they could have both local and systemic effects. Certain microbes that may be supplemented were also reported to produce carotenoids in the colon. Although some bactericidal aspects of carotenoids have been shown in vitro, a few studies have also demonstrated a prebiotic-like effect, resulting in bacterial shifts with health-associated properties. Also, stimulation of IgA could play a role in this respect. Carotenoids may further contribute to mucosal and gut barrier health, such as stabilizing tight junctions. This review highlights potential gut-related health-beneficial effects of carotenoids and emphasizes the current research gaps regarding carotenoid-GM interactions.}, number={2}, journal={ADVANCES IN NUTRITION}, author={Eroglu, Abdulkerim and Al'Abri, Ibrahim S. and Kopec, Rachel E. and Crook, Nathan and Bohn, Torsten}, year={2023}, month={Mar}, pages={238–255} }
 @misc{heavey_durmusoglu_crook_anselmo_2022, title={Discovery and delivery strategies for engineered live biotherapeutic products}, volume={40}, ISSN={["1879-3096"]}, DOI={10.1016/j.tibtech.2021.08.002}, abstractNote={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.}, number={3}, journal={TRENDS IN BIOTECHNOLOGY}, author={Heavey, Mairead K. and Durmusoglu, Deniz and Crook, Nathan and Anselmo, Aaron C.}, year={2022}, month={Mar}, pages={354–369} }
 @article{jensen_deichmann_ma_vilandt_schiesaro_rojek_lengger_eliasson_vento_durmusoglu_et al._2022, title={Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts}, volume={13}, ISSN={["2041-1723"]}, DOI={10.1038/s41467-022-33961-y}, abstractNote={AbstractG 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.}, number={1}, journal={NATURE COMMUNICATIONS}, author={Jensen, Emil D. and Deichmann, Marcus and Ma, Xin and Vilandt, Rikke U. and Schiesaro, Giovanni and Rojek, Marie B. and Lengger, Bettina and Eliasson, Line and Vento, Justin M. and Durmusoglu, Deniz and et al.}, year={2022}, month={Oct} }
 @article{kwak_crook_yoneda_ahn_ning_cheng_dantas_2022, title={Functional mining of novel terpene synthases from metagenomes}, volume={15}, ISSN={["2731-3654"]}, DOI={10.1186/s13068-022-02189-9}, abstractNote={Abstract
                Background
                Terpenes are one of the most diverse and abundant classes of natural biomolecules, collectively enabling a variety of therapeutic, energy, and cosmetic applications. Recent genomics investigations have predicted a large untapped reservoir of bacterial terpene synthases residing in the genomes of uncultivated organisms living in the soil, indicating a vast array of putative terpenoids waiting to be discovered.
              
                Results
                We aimed to develop a high-throughput functional metagenomic screening system for identifying novel terpene synthases from bacterial metagenomes by relieving the toxicity of terpene biosynthesis precursors to the Escherichia coli host. The precursor toxicity was achieved using an inducible operon encoding the prenyl pyrophosphate synthetic pathway and supplementation of the mevalonate precursor. Host strain and screening procedures were finely optimized to minimize false positives arising from spontaneous mutations, which avoid the precursor toxicity. Our functional metagenomic screening of human fecal metagenomes yielded a novel β-farnesene synthase, which does not show amino acid sequence similarity to known β-farnesene synthases. Engineered S. cerevisiae expressing the screened β-farnesene synthase produced 120 mg/L β-farnesene from glucose (2.86 mg/g glucose) with a productivity of 0.721 g/L∙h.
              
                Conclusions
                A unique functional metagenomic screening procedure was established for screening terpene synthases from metagenomic libraries. This research proves the potential of functional metagenomics as a sequence-independent avenue for isolating targeted enzymes from uncultivated organisms in various environmental habitats.
              }, number={1}, journal={BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS}, author={Kwak, Suryang and Crook, Nathan and Yoneda, Aki and Ahn, Naomi and Ning, Jie and Cheng, Jiye and Dantas, Gautam}, year={2022}, month={Oct} }
 @article{durmusoglu_al’abri_williams_collins_martínez_crook_2022, title={Improving Therapeutic Protein Secretion in the Probiotic YeastSaccharomyces boulardiiusing a Multifactorial Engineering Approach}, url={https://doi.org/10.1101/2022.12.30.522352}, DOI={10.1101/2022.12.30.522352}, abstractNote={AbstractThe probiotic yeastSaccharomyces boulardii(Sb) is a promising chassis to deliver therapeutic proteins to the gut due toSb’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 engineerSbstrains with enhanced levels of protein secretion. In this work, we explored genetic modifications in bothcis- (i.e., to the expression cassette of the secreted protein) andtrans- (i.e., to theSbgenome) that enhanceSb’s ability to secrete proteins, taking aClostridioides difficileToxin 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 ofS. 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-deficientSbstrain that produces 5045 mg/L secretory NPA, an improvement of >10-fold over wild-typeSb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion inSband 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 ofSbto deliver therapeutics to the gut and other settings to which it is adapted.}, journal={bioRxiv}, author={Durmusoglu, Deniz and Al’Abri, Ibrahim and Williams, Taufika Islam and Collins, Leonard B. and Martínez, José L. and Crook, Nathan}, year={2022}, month={Dec} }
 @article{al'abri_haller_li_crook_2022, title={Inducible directed evolution of complex phenotypes in bacteria}, volume={2}, ISSN={["1362-4962"]}, url={https://doi.org/10.1093/nar/gkac094}, DOI={10.1093/nar/gkac094}, abstractNote={Abstract
               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 >5–10 kb 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. Next, we evolved a 15.4 kb, 10-gene pathway from Bifidobacterium breve UC2003 that aids E. coli’s utilization of melezitose. After three rounds of IDE, we isolated evolved pathways that both reduced lag time by more than 2-fold and enabled 150% higher final optical density. Taken together, this work enhances the capacity and utility of a whole pathway directed evolution approach in E. coli.}, journal={NUCLEIC ACIDS RESEARCH}, publisher={Oxford University Press (OUP)}, author={Al'Abri, Ibrahim S. and Haller, Daniel J. and Li, Zidan and Crook, Nathan}, year={2022}, month={Feb} }
 @article{kwon_cheeseman_frias‐de‐diego_hong_yang_jung_yin_murdoch_scholle_crook_et al._2021, title={A Liquid Metal Mediated Metallic Coating for Antimicrobial and Antiviral Fabrics}, volume={33}, ISSN={0935-9648 1521-4095}, url={http://dx.doi.org/10.1002/adma.202104298}, DOI={10.1002/adma.202104298}, abstractNote={AbstractFabrics are widely used in hospitals and many other settings for bedding, clothing, and face masks; however, microbial pathogens can survive on surfaces for a long time, leading to microbial transmission. Coatings of metallic particles on fabrics have been widely used to eradicate pathogens. However, current metal particle coating technologies encounter numerous issues such as nonuniformity, processing complexity, and poor adhesion. To overcome these issues, an easy‐to‐control and straightforward method is reported to coat a wide range of fabrics by using gallium liquid metal (LM) particles to facilitate the deposition of liquid metal copper alloy (LMCu) particles. Gallium particles coated on the fabric provide nucleation sites for forming LMCu particles at room temperature via galvanic replacement of Cu2+ ions. The LM helps promote strong adhesion of the particles to the fabric. The presence of the LMCu particles can eradicate over 99% of pathogens (including bacteria, fungi, and viruses) within 5 min, which is significantly more effective than control samples coated with only Cu. The coating remains effective over multiple usages and against contaminated droplets and aerosols, such as those encountered in facemasks. This facile coating method is promising for generating robust antibacterial, antifungal, and antiviral fabrics and surfaces.}, number={45}, journal={Advanced Materials}, publisher={Wiley}, author={Kwon, Ki Yoon and Cheeseman, Samuel and Frias‐De‐Diego, Alba and Hong, Haeleen and Yang, Jiayi and Jung, Woojin and Yin, Hong and Murdoch, Billy J. and Scholle, Frank and Crook, Nathan and et al.}, year={2021}, month={Sep}, pages={2104298} }
 @article{durmusoglu_catella_purnell_menegatti_crook_2021, title={Design and in situ biosynthesis of precision therapies against gastrointestinal pathogens}, volume={23}, ISSN={["2468-8673"]}, DOI={10.1016/j.cophys.2021.06.007}, abstractNote={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.}, journal={CURRENT OPINION IN PHYSIOLOGY}, author={Durmusoglu, Deniz and Catella, Carly M. and Purnell, Ethan F. and Menegatti, Stefano and Crook, Nathan C.}, year={2021}, month={Oct} }
 @article{xiao_sarma_menegatti_crook_magness_hall_2021, title={In Silico Identification and Experimental Validation of Peptide-Based Inhibitors Targeting Clostridium difficile Toxin A}, volume={17}, ISSN={["1554-8937"]}, url={https://doi.org/10.1021/acschembio.1c00743}, DOI={10.1021/acschembio.1c00743}, abstractNote={Clostridium difficile infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of C. difficile infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three in silico discovered peptides, viz. the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, viz. the reference peptide (RP). These peptides may serve as potential inhibitors against C. difficile infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in vitro in human jejunum cells. Both phage-display peptide RP and in silico peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the in silico peptide NPB demonstrates a relatively low efficacy against TcdA.}, number={1}, journal={ACS CHEMICAL BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Xiao, Xingqing and Sarma, Sudeep and Menegatti, Stefano and Crook, Nathan and Magness, Scott T. and Hall, Carol K.}, year={2021}, month={Dec} }
 @article{durmusoglu_al'abri_collins_cheng_eroglu_beisel_crook_2021, title={In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii}, volume={10}, ISSN={["2161-5063"]}, url={https://doi.org/10.1021/acssynbio.0c00562}, DOI={10.1021/acssynbio.0c00562}, abstractNote={Saccharomyces boulardii 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 S. boulardii 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 S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.}, number={5}, journal={ACS SYNTHETIC BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Durmusoglu, Deniz and Al'Abri, Ibrahim S. and Collins, Scott P. and Cheng, Junrui and Eroglu, Abdulkerim and Beisel, Chase L. and Crook, Nathan}, year={2021}, month={May}, pages={1039–1052} }
 @article{al'abri_durmusoglu_crook_2021, title={What E. coli knows about your 1-year-old infant: Antibiotic use, lifestyle, birth mode, and siblings}, volume={29}, ISSN={["1934-6069"]}, DOI={10.1016/j.chom.2021.05.006}, abstractNote={The infant gut microbiota is shaped by diverse environmental exposures that alter its composition and can enrich antimicrobial resistance genes (ARGs). In this issue of Cell Host & Microbe, Li et al. (2021) studied the causes, spread, and dynamics of ARGs and their relationship with asthma-associated microbiota in Danish children. The infant gut microbiota is shaped by diverse environmental exposures that alter its composition and can enrich antimicrobial resistance genes (ARGs). In this issue of Cell Host & Microbe, Li et al. (2021) studied the causes, spread, and dynamics of ARGs and their relationship with asthma-associated microbiota in Danish children. The gut microbiota is fundamental to human health, but we lack a clear understanding of how it matures and how environmental factors modulate this process (Robertson et al., 2019Robertson R.C. Manges A.R. Finlay B.B. Prendergast A.J. The Human Microbiome and Child Growth - First 1000 Days and Beyond.Trends Microbiol. 2019; 27: 131-147Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). In the last decade, research has focused on understanding how the development of the gut microbiota changes in response to perturbations, especially in early life. We have learned that factors such as delivery mode, sex, exposure to antibiotics, rural versus urban lifestyle, hospitalization, and age significantly influence microbiota assembly, and consequently, human health (Stokholm et al., 2018Stokholm J. Blaser M.J. Thorsen J. Rasmussen M.A. Waage J. Vinding R.K. Schoos A.-M.M. Kunøe A. Fink N.R. Chawes B.L. et al.Maturation of the gut microbiome and risk of asthma in childhood.Nat. Commun. 2018; 9: 141Crossref PubMed Scopus (220) Google Scholar). However, the impact of these perturbations on the gut resistome is less well understood. The resistome is the collection of antibiotic resistance genes (ARGs) present in a microbial community. The overuse of antibiotics is associated with a low microbiota maturity, which can lead to metabolic disorders, malnutrition, infections, and even colon cancer (Langdon et al., 2016Langdon A. Crook N. Dantas G. The effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation.Genome Med. 2016; 8: 39Crossref PubMed Scopus (437) Google Scholar). In addition, antibiotic treatment can lead to an increased abundance of microbes containing ARGs, facilitating future transfer of ARGs to pathogens and making treatment of infections more difficult (Ferreiro et al., 2018Ferreiro A. Crook N. Gasparrini A.J. Dantas G. Multiscale Evolutionary Dynamics of Host-Associated Microbiomes.Cell. 2018; 172: 1216-1227Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The high density of microbes in the gut enables horizontal gene transfer and makes the study of the gut resistome particularly important. Although we know that factors such as birth mode, breastfeeding, and antibiotic use impact resistome development, we still don't fully understand how important other environmental factors are and how they are interconnected (Stokholm et al., 2020Stokholm J. Thorsen J. Blaser M.J. Rasmussen M.A. Hjelmsø M. Shah S. Christensen E.D. Chawes B.L. Bønnelykke K. Brix S. et al.Delivery mode and gut microbial changes correlate with an increased risk of childhood asthma.Sci. Transl. Med. 2020; 12: eaax9929Crossref PubMed Scopus (29) Google Scholar). In particular, there exists a gap in our understanding of resistome development between very early life (<1 year) (Gasparrini et al., 2016Gasparrini A.J. Crofts T.S. Gibson M.K. Tarr P.I. Warner B.B. Dantas G. Antibiotic perturbation of the preterm infant gut microbiome and resistome.Gut Microbes. 2016; 7: 443-449Crossref PubMed Scopus (57) Google Scholar, Gasparrini et al., 2019Gasparrini A.J. Wang B. Sun X. Kennedy E.A. Hernandez-Leyva A. Ndao I.M. Tarr P.I. Warner B.B. Dantas G. Persistent metagenomic signatures of early-life hospitalization and antibiotic treatment in the infant gut microbiota and resistome.Nat. Microbiol. 2019; 4: 2285-2297Crossref PubMed Scopus (81) Google Scholar) and adulthood (Schwartz et al., 2020Schwartz D.J. Langdon A.E. Dantas G. Understanding the impact of antibiotic perturbation on the human microbiome.Genome Med. 2020; 12: 82Crossref PubMed Scopus (48) Google Scholar), especially in healthy individuals. To fill this gap, Li et al., 2021Li X. Stokholm J. Brejnrod A. Vestergaard G.A. Russel J. Trivedi U. Thorsen J. Gupta S. Hjelmsø M.H. Shah S.A. et al.The infant gut resistome associates with E. coli, environmental exposures, gut microbiome maturity, and asthma-associated bacterial composition.Cell Host Microbe. 2021; 29 (this issue): 975-987Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar explored how diverse environmental factors shaped the resistomes of 662 1-year-old Danish children and the relationship of their resistomes to longer-term health impacts (Li et al., 2021Li X. Stokholm J. Brejnrod A. Vestergaard G.A. Russel J. Trivedi U. Thorsen J. Gupta S. Hjelmsø M.H. Shah S.A. et al.The infant gut resistome associates with E. coli, environmental exposures, gut microbiome maturity, and asthma-associated bacterial composition.Cell Host Microbe. 2021; 29 (this issue): 975-987Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The authors identified 409 ARGs in this cohort, with 167 of them conferring resistance to multiple antibiotics. Interestingly, ARG abundance was bimodal, with one cluster of children exhibiting more abundant and diverse ARGs in their gut microbiome than the other (Figure 1). To understand the drivers of this phenomenon, the authors investigated the species that comprised each cluster. This analysis revealed that the abundance of E. coli determined cluster membership, with individuals in the "ARG high" group having a much greater abundance of intestinal E. coli. This finding agrees with previous studies that found that the microbiotas of antibiotic-treated preterm infants are dominated by Gammaproteobacteria (Gasparrini et al., 2016Gasparrini A.J. Crofts T.S. Gibson M.K. Tarr P.I. Warner B.B. Dantas G. Antibiotic perturbation of the preterm infant gut microbiome and resistome.Gut Microbes. 2016; 7: 443-449Crossref PubMed Scopus (57) Google Scholar, Gasparrini et al., 2019Gasparrini A.J. Wang B. Sun X. Kennedy E.A. Hernandez-Leyva A. Ndao I.M. Tarr P.I. Warner B.B. Dantas G. Persistent metagenomic signatures of early-life hospitalization and antibiotic treatment in the infant gut microbiota and resistome.Nat. Microbiol. 2019; 4: 2285-2297Crossref PubMed Scopus (81) Google Scholar). Removing E. coli from the analyzed samples resulted in a 10-fold reduction in ARG richness in the "ARG high" group. This work therefore prioritizes further study on the ability of intestinal E. coli to mobilize ARGs to other members of the microbiota, perhaps serving as an important "node" for ARG "traffic" in the large intestine. The authors then wondered whether the "E. coli-high" cluster was persistent across age. However, when they measured the abundance of E. coli in samples from the same individuals collected at 1 week, 1 month, 4 years, 5 years, or 6 years of age, they found no difference in E. coli levels between each cluster. This indicates that the abundance of E. coli, and perhaps ARG abundance as a consequence, is mainly influenced by transient factors such as seasons, antibiotic exposure, or ecological dynamics in the microbiota. To understand how these ARGs were acquired, the authors then examined the correlation between ARG abundance and patient metadata. Interestingly, even infants that were not treated with any antibiotics had ARGs in their microbiomes, which led the researchers to investigate whether other environmental exposures had an impact. They found that the most prominent factors in shaping the distribution of ARGs were the presence of older siblings, living environment, mode of delivery, time since antibiotic treatment, and antibiotic use 40 days before childbirth. Other factors, such antibiotic usage frequency, living in an apartment, and usage of antibiotics by pregnant mothers in the first two trimesters had a smaller role. Comparing the effects of antibiotics to other factors revealed that while all factors affect the distribution of ARGs, antibiotic use was unique because it did not change the microbiota composition. Potential explanations for this counterintuitive finding include horizontal transfer of ARGs among gut microbes in response to antibiotic exposure, or the presence of antibiotic-resistant subpopulations with the same taxonomic assignments. It is noteworthy that not all environmental exposures propel the gut microbiota to inherit the same ARGs or at the same rate. Therefore, it is possible that minimizing childhood exposure into the most influential factors may protect against ARG proliferation in the gut microbiome. Finally, the authors investigated the relationship between ARG carriage, microbiota composition, and asthma, finding that high ARG abundance was associated with a microbiota composition that increases the risk of later asthma. Additionally, samples with abundant ARGs also had lower microbiota maturity, which is a risk factor for a variety of metabolic and immunological disorders. Although more studies are needed to fully elucidate the associations between ARGs, microbiota composition, asthma, and other diseases, this study supports a microbial route to identify asthma-prone individuals. Li et al., 2021Li X. Stokholm J. Brejnrod A. Vestergaard G.A. Russel J. Trivedi U. Thorsen J. Gupta S. Hjelmsø M.H. Shah S.A. et al.The infant gut resistome associates with E. coli, environmental exposures, gut microbiome maturity, and asthma-associated bacterial composition.Cell Host Microbe. 2021; 29 (this issue): 975-987Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar shed light on ARGs in the healthy developing gut in remarkable detail, finding that distribution of ARGs in the gut is strongly bimodal and largely based on the abundance of E.coli. It would be very fascinating to determine whether this clustering occurs in other parts of the world, or in other age groups. Additionally, the authors showed that different environmental factors can have different effects on the acquisition of ARGs, thereby setting priorities for future ARG mitigation. Interestingly, high ARG richness was correlated with low microbiota maturity and a high risk of asthma later in life, agreeing with prior studies in the nasopharyngeal microbiome (Teo et al., 2015Teo S.M. Mok D. Pham K. Kusel M. Serralha M. Troy N. Holt B.J. Hales B.J. Walker M.L. Hollams E. et al.The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development.Cell Host Microbe. 2015; 17: 704-715Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). By elucidating the risk factors for high rates of ARG acquisition during development, this study facilitates efforts to combat the rise of antimicrobial-resistant pathogens and provides a roadmap to studying these important questions in other cohorts. The infant gut resistome associates with E. coli, environmental exposures, gut microbiome maturity, and asthma-associated bacterial compositionLi et al.Cell Host & MicrobeApril 21, 2021In BriefIn this comprehensive analysis of antibiotic resistance genes (ARGs) distribution in the infant gut, Li et al. show that E. coli is an extremely important reservoir of ARGs. They also reveal associations between infant gut resistome and environmental factors, gut microbiome maturation, and bacteria associated with later development of asthma. Full-Text PDF Open Archive}, number={6}, journal={CELL HOST & MICROBE}, author={Al'Abri, Ibrahim S. and Durmusoglu, Deniz and Crook, Nathan}, year={2021}, month={Jun}, pages={854–855} }
 @article{durmusoglu_al’abri_collins_beisel_crook_2020, title={Establishing ProbioticSaccharomyces boulardiias a Model Organism for Synthesis and Delivery of Biomolecules}, url={https://doi.org/10.1101/2020.01.22.915389}, DOI={10.1101/2020.01.22.915389}, abstractNote={AbstractSaccharomyces 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. boulardii’s 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.}, author={Durmusoglu, Deniz and Al’Abri, Ibrahim and Collins, Scott P. and Beisel, Chase and Crook, Nathan}, year={2020}, month={Jan} }
 @article{al’abri_haller_crook_2020, title={Inducible Directed Evolution of Complex Phenotypes in Bacteria}, volume={10}, url={https://doi.org/10.1101/2020.10.30.362871}, DOI={10.1101/2020.10.30.362871}, abstractNote={AbstractDirected 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.}, publisher={Cold Spring Harbor Laboratory}, author={Al’Abri, Ibrahim S. and Haller, Daniel J. and Crook, Nathan}, year={2020}, month={Oct} }
 @article{crook_ferreiro_condiotte_dantas_2020, title={Transcript Barcoding Illuminates the Expression Level of Synthetic Constructs in E. coli Nissle Residing in the Mammalian Gut}, volume={9}, ISSN={["2161-5063"]}, url={https://doi.org/10.1021/acssynbio.0c00040}, DOI={10.1021/acssynbio.0c00040}, abstractNote={The development of robust engineered probiotic therapies demands accurate knowledge of genetic construct expression in the gut. However, the monetary and ethical costs of testing engineered strains in vertebrate hosts are incompatible with current high-throughput design-build-test cycles. To enable parallel measurement of multiple construct designs, we placed unique DNA barcodes in engineered transcripts and measured barcode abundances via sequencing. In standard curve experiments the barcode sequences exhibited consistent relationships between input and measured abundances, which allowed us to use transcript barcoding to measure expression levels of 30 GFP-expressing strains of E. coli Nissle in parallel. Applying this technology in culture and in the mouse gut, we found GFP expression in the gut could often be predicted from expression levels in culture, but several strains exhibited gut-specific expression. This work establishes the experimental design parameters and advantages of transcript barcoding to measure the performance of many engineered probiotic designs in mammalian hosts.}, number={5}, journal={ACS SYNTHETIC BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Crook, Nathan and Ferreiro, Aura and Condiotte, Zevin and Dantas, Gautam}, year={2020}, month={May}, pages={1010–1021} }
 @article{crook_ferreiro_gasparrini_pesesky_gibson_wang_sun_condiotte_dobrowolski_peterson_et al._2019, title={Adaptive Strategies of the Candidate Probiotic E. coli Nissle in the Mammalian Gut}, volume={25}, ISSN={["1934-6069"]}, url={https://doi.org/10.1016/j.chom.2019.02.005}, DOI={10.1016/j.chom.2019.02.005}, abstractNote={Probiotics are living microorganisms that are increasingly used as gastrointestinal therapeutics by virtue of their innate or engineered genetic function. Unlike abiotic therapeutics, probiotics can replicate in their intended site, subjecting their genomes and therapeutic properties to natural selection. We exposed the candidate probiotic E. coli Nissle (EcN) to the mouse gastrointestinal tract over several weeks, systematically altering the diet and background microbiota complexity. In-transit EcN accumulates genetic mutations that modulate carbohydrate utilization, stress response, and adhesion to gain competitive fitness, while previous exposure to antibiotics reveals an acquisition of resistance. We then leveraged these insights to generate an EcN strain that shows therapeutic efficacy in a mouse model of phenylketonuria and found that it was genetically stable over 1 week, thereby validating EcN’s utility as a chassis for engineering. Collectively, we demonstrate a generalizable pipeline that can be applied to other probiotics to better understand their safety and engineering potential.}, number={4}, journal={CELL HOST & MICROBE}, publisher={Elsevier BV}, author={Crook, Nathan and Ferreiro, Aura and Gasparrini, Andrew J. and Pesesky, Mitchell W. and Gibson, Molly K. and Wang, Bin and Sun, Xiaoqing and Condiotte, Zevin and Dobrowolski, Stephen and Peterson, Daniel and et al.}, year={2019}, month={Apr}, pages={499-+} }
 @misc{vento_crook_beisel_2019, title={Barriers to genome editing with CRISPR in bacteria}, volume={46}, ISSN={["1476-5535"]}, url={https://doi.org/10.1007/s10295-019-02195-1}, DOI={10.1007/s10295-019-02195-1}, abstractNote={Abstract
               Genome editing is essential for probing genotype–phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains.}, number={9-10}, journal={JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY}, publisher={Springer Science and Business Media LLC}, author={Vento, Justin M. and Crook, Nathan and Beisel, Chase L.}, year={2019}, month={Oct}, pages={1327–1341} }
 @article{crook_ferreiro_gasparrini_pesesky_gibson_wang_sun_condiotte_dobrowolski_peterson_et al._2018, title={Adaptive strategies of the candidate probiotic E. coli Nissle in the mammalian gut}, volume={7}, url={https://doi.org/10.1101/364505}, DOI={10.1101/364505}, abstractNote={SummaryProbiotics are living microorganisms that are increasingly used as gastrointestinal therapeutics by virtue of their innate or engineered genetic function. Unlike abiotic therapeutics, probiotics can replicate in their intended site, subjecting their genomes and therapeutic properties to natural selection. By exposing the candidate probioticE. coliNissle (EcN) to the mouse gastrointestinal tract over several weeks, we uncovered the consequences of gut transit, inter-species competition, antibiotic pressure, and engineered genetic function on the processes under selective pressure during both within-genome and horizontal evolutionary modes. We then show the utility of EcN as a chassis for engineered function by achieving the highest reported reduction in serum phenylalanine levels in a mouse model of phenylketonuria using an engineered probiotic. Collectively, we demonstrate a generalizable pipeline which can be applied to other probiotic strains to better understand their safety and engineering potential.}, publisher={Cold Spring Harbor Laboratory}, author={Crook, Nathan and Ferreiro, Aura and Gasparrini, Andrew J. and Pesesky, Mitchell and Gibson, Molly K. and Wang, Bin and Sun, Xiaoqing and Condiotte, Zevin and Dobrowolski, Stephen and Peterson, Daniel and et al.}, year={2018}, month={Jul} }
 @article{crook_abatemarco_sun_wagner_schmitz_alper_2016, title={In vivo continuous evolution of genes and pathways in yeast}, url={https://doi.org/10.1038/ncomms13051}, DOI={10.1038/ncomms13051}, abstractNote={AbstractDirected evolution remains a powerful, highly generalizable approach for improving the performance of biological systems. However, implementations in eukaryotes rely either on in vitro diversity generation or limited mutational capacities. Here we synthetically optimize the retrotransposon Ty1 to enable in vivo generation of mutant libraries up to 1.6 × 107 l−1 per round, which is the highest of any in vivo mutational generation approach in yeast. We demonstrate this approach by using in vivo-generated libraries to evolve single enzymes, global transcriptional regulators and multi-gene pathways. When coupled to growth selection, this approach enables in vivo continuous evolution (ICE) of genes and pathways. Through a head-to-head comparison, we find that ICE libraries yield higher-performing variants faster than error-prone PCR-derived libraries. Finally, we demonstrate transferability of ICE to divergent yeasts, including Kluyveromyces lactis and alternative S. cerevisiae strains. Collectively, this work establishes a generic platform for rapid eukaryotic-directed evolution across an array of target cargo.}, journal={Nature Communications}, author={Crook, Nathan and Abatemarco, Joseph and Sun, Jie and Wagner, James M. and Schmitz, Alexander and Alper, Hal S.}, year={2016}, month={Oct} }
 @article{curran_crook_karim_gupta_wagman_alper_2014, title={Design of synthetic yeast promoters via tuning of nucleosome architecture}, volume={5}, DOI={10.1038/ncomms5002}, abstractNote={Model-based design of biological parts is a critical goal of synthetic biology, especially for eukaryotes. Here we demonstrate that nucleosome architecture can have a role in defining yeast promoter activity and utilize a computationally-guided approach that can enable both the redesign of endogenous promoter sequences and the de novo design of synthetic promoters. Initially, we use our approach to reprogram native promoters for increased expression and evaluate their performance in various genetic contexts. Increases in expression ranging from 1.5- to nearly 6-fold in a plasmid-based system and up to 16-fold in a genomic context were obtained. Next, we demonstrate that, in a single design cycle, it is possible to create functional, purely synthetic yeast promoters that achieve substantial expression levels (within the top sixth percentile among native yeast promoters). In doing so, this work establishes a unique DNA-level specification of promoter activity and demonstrates predictive design of synthetic parts. Model-based part design is a key step in synthetic biology. Here, the authors report a method for tuning nucleosome architecture in order to strengthen native promoters and facilitate synthetic promoter design in yeast.}, journal={Nat Comms}, publisher={Nature Publishing Group}, author={Curran, Kathleen A. and Crook, Nathan C. and Karim, Ashty S. and Gupta, Akash and Wagman, Allison M. and Alper, Hal S.}, year={2014}, month={May} }
 @article{crook_schmitz_alper_2014, title={Optimization of a Yeast RNA Interference System for Controlling Gene Expression and Enabling Rapid Metabolic Engineering}, volume={3}, DOI={10.1021/sb4001432}, abstractNote={Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.}, number={5}, journal={ACS Synth. Biol.}, publisher={American Chemical Society (\lbraceACS\rbrace)}, author={Crook, Nathan C. and Schmitz, Alexander C. and Alper, Hal S.}, year={2014}, month={May}, pages={307–313} }
 @article{crook_alper_2012, title={Classical Strain Improvement}, DOI={10.1002/9781118433034.ch1}, abstractNote={Improving complex phenotypes, which are typically multigenic in nature, has been a long standing goal of the food and biotechnology industry well before the advent of recombinant DNA technology and the genomics revolution. For thousands of years, humans have (whether intentionally or not) placed selective pressure on plants, animals, and microorganisms, resulting in improvements to desired phenotypes. Clear evidence of these efforts can be seen from the dramatic morphological changes to food crops since domestication (1) . These improvements have been predominantly achieved through a “ classical ” approach to strain engineering, whereby phenotypic improvements are made by screening and mutagenesis of strains that use methods naive of genome sequences or the resulting genetic changes. This approach is well suited for strain optimization in industrial microbiology, which commonly exploits complex phenotypes in organisms with poorly defi ned or monitored genetics. As a recognition of importance, Arnold Demain and Julian Davies begin their Handbook of Industrial Microbiology and Biotechnology with “ Almost all industrial microbiology processes require the initial isolation of cultures from nature, followed by small scale cultivations and optimization, before large scale production can become a reality ” (2) . The classical approach is concerned}, journal={Engineering Complex Phenotypes in Industrial Strains}, publisher={John Wiley & Sons, Inc.}, author={Crook, Nathan and Alper, Hal S.}, year={2012}, month={Oct}, pages={1–33} }
 @article{lanza_crook_alper_2012, title={Innovation at the intersection of synthetic and systems biology}, volume={23}, DOI={10.1016/j.copbio.2011.12.026}, abstractNote={The promises of modern biotechnology hinge upon the hope that we can understand microscopic cellular complexity and in doing so create novel function. In this regard, the fields of systems and synthetic biology are important for accelerating both our understanding of biological systems and our ability to quantitatively engineer cells. At the nexus of these two fields is a unique synergy that can help attain these goals. Thus, the next greatest advances in biology and biotechnology are arising at the intersection of the top-down systems approach and the bottom-up synthetic approach. Collectively, these developments enable the precise control of cellular state for systems studies and the discovery of novel parts, control strategies, and interactions for the design of robust synthetic function. This review seeks to highlight this activity as well as provide a perspective for future directions. Combining these efforts can provide novel insights into cellular function and lead to robust, novel synthetic design.}, number={5}, journal={Current Opinion in Biotechnology}, publisher={Elsevier \lbraceBV\rbrace}, author={Lanza, Amanda M and Crook, Nathan C and Alper, Hal S}, year={2012}, month={Oct}, pages={712–717} }
 @article{lanza_blazeck_crook_alper_2012, title={Linking Yeast Gcn5p Catalytic Function and Gene Regulation Using a Quantitative, Graded Dominant Mutant Approach}, volume={7}, DOI={10.1371/journal.pone.0036193}, abstractNote={Establishing causative links between protein functional domains and global gene regulation is critical for advancements in genetics, biotechnology, disease treatment, and systems biology. This task is challenging for multifunctional proteins when relying on traditional approaches such as gene deletions since they remove all domains simultaneously. Here, we describe a novel approach to extract quantitative, causative links by modulating the expression of a dominant mutant allele to create a function-specific competitive inhibition. Using the yeast histone acetyltransferase Gcn5p as a case study, we demonstrate the utility of this approach and (1) find evidence that Gcn5p is more involved in cell-wide gene repression, instead of the accepted gene activation associated with HATs, (2) identify previously unknown gene targets and interactions for Gcn5p-based acetylation, (3) quantify the strength of some Gcn5p-DNA associations, (4) demonstrate that this approach can be used to correctly identify canonical chromatin modifications, (5) establish the role of acetyltransferase activity on synthetic lethal interactions, and (6) identify new functional classes of genes regulated by Gcn5p acetyltransferase activity—all six of these major conclusions were unattainable by using standard gene knockout studies alone. We recommend that a graded dominant mutant approach be utilized in conjunction with a traditional knockout to study multifunctional proteins and generate higher-resolution data that more accurately probes protein domain function and influence.}, number={4}, journal={PLoS ONE}, publisher={Public Library of Science (\lbracePLoS\rbrace)}, author={Lanza, Amanda M. and Blazeck, John J. and Crook, Nathan C. and Alper, Hal S.}, editor={Chan, ChristinaEditor}, year={2012}, month={Apr}, pages={e36193} }
 @article{crook_alper_2013, title={Model-based design of synthetic, biological systems}, volume={103}, DOI={10.1016/j.ces.2012.12.022}, abstractNote={Synthetic biology brings engineering tools and perspectives to the design of living systems. In contrast to classical cell engineering approaches, synthetic biology enables cellular networks to be understood as a combination of modular elements in much the same way as unit operations combine to describe a chemical plant. Consequently, models for the behavior of these designed systems are inspired by frameworks developed for traditional chemical engineering design. There are direct analogies between cellular metabolism and reaction networks in a chemical process. As examples, thermodynamic and kinetic models of chemical reaction networks have been used to simulate fluxes within living systems and predict the performance of synthetic parts. Concepts from process control have been brought to bear on the design of transcriptional and translational regulatory networks. Such engineering frameworks have greatly aided the design and understanding of living systems and have enabled the design of cells exhibiting complex dynamic behavior and high productivity of desirable compounds. This review summarizes efforts to quantitatively model cellular behavior (both endogenous and synthetic), especially as related to the design of living systems.}, journal={Chemical Engineering Science}, publisher={Elsevier \lbraceBV\rbrace}, author={Crook, Nathan and Alper, Hal S.}, year={2013}, month={Nov}, pages={2–11} }
 @article{brustad_lelyveld_snow_crook_jung_martinez_scholl_jasanoff_arnold_2012, title={Structure-Guided Directed Evolution of Highly Selective P450-Based Magnetic Resonance Imaging Sensors for Dopamine and Serotonin}, volume={422}, DOI={10.1016/j.jmb.2012.05.029}, abstractNote={New tools that allow dynamic visualization of molecular neural events are important for studying the basis of brain activity and disease. Sensors that permit ligand-sensitive magnetic resonance imaging (MRI) are useful reagents due to the noninvasive nature and good temporal and spatial resolution of MR methods. Paramagnetic metalloproteins can be effective MRI sensors due to the selectivity imparted by the protein active site and the ability to tune protein properties using techniques such as directed evolution. Here, we show that structure-guided directed evolution of the active site of the cytochrome P450‐BM3 heme domain produces highly selective MRI probes with submicromolar affinities for small molecules. We report a new, high‐affinity dopamine sensor as well as the first MRI reporter for serotonin, with which we demonstrate quantification of neurotransmitter release in vitro. We also present a detailed structural analysis of evolved cytochrome P450‐BM3 heme domain lineages to systematically dissect the molecular basis of neurotransmitter binding affinity, selectivity, and enhanced MRI contrast activity in these engineered proteins.}, number={2}, journal={Journal of Molecular Biology}, publisher={Elsevier \lbraceBV\rbrace}, author={Brustad, Eric M. and Lelyveld, Victor S. and Snow, Christopher D. and Crook, Nathan and Jung, Sang Taek and Martinez, Francisco M. and Scholl, Timothy J. and Jasanoff, Alan and Arnold, Frances H.}, year={2012}, month={Sep}, pages={245–262} }
 @article{crook_freeman_alper_2011, title={Re-engineering multicloning sites for function and convenience}, volume={39}, DOI={10.1093/nar/gkr346}, abstractNote={Multicloning sites (MCSs) in standard expression vectors are widely used and thought to be benign, non-interacting elements that exist for mere convenience. However, MCSs impose a necessary distance between promoter elements and genes of interest. As a result, the choice of cloning site defines the genetic context and may introduce significant mRNA secondary structure in the 5′-untranslated region leading to strong translation inhibition. Here, we demonstrate the first performance-based assessment of MCSs in yeast, showing that commonly used MCSs can induce dramatic reductions in protein expression, and that this inhibition is highly promoter and gene dependent. In response, we develop and apply a novel predictive model of structure-based translation inhibition to design improved MCSs for significantly higher and more consistent protein expression. In doing so, we were able to minimize the inhibitory effects of MCSs with the yeast TEF, CYC and GPD promoters. These results highlight the non-interchangeable nature of biological parts and represent the first complete, global redesign of a genetic circuit of such widespread importance as a multicloning site. The improved translational control offered by these designed MCSs is paramount to obtaining high titers of heterologous proteins in eukaryotes and to enabling precise control of genetic circuits.}, number={14}, journal={Nucleic Acids Research}, publisher={Oxford University Press (\lbraceOUP\rbrace)}, author={Crook, N. C. and Freeman, E. S. and Alper, H. S.}, year={2011}, month={May}, pages={e92–e92} }
 @article{curran_crook_alper_2011, title={Using Flux Balance Analysis to Guide Microbial Metabolic Engineering}, DOI={10.1007/978-1-61779-483-4_13}, abstractNote={Metabolic engineers modify biological systems through the use of modern molecular biology tools in order to obtain desired phenotypes. However, due to the extreme complexity and interconnectedness of metabolism in all organisms, it is often difficult to a priori predict which changes will yield the optimal results. Flux balance analysis (FBA) is a mathematical approach that uses a genomic-scale metabolic network models to afford in silico prediction and optimization of metabolic changes. In particular, a genome-scale approach can help select gene targets for knockout and overexpression. This approach can be used to help expedite the strain engineering process. Here, we give an introduction to the use of FBA and provide details for its implementation in a microbial metabolic engineering context.}, journal={Microbial Metabolic Engineering}, publisher={Springer Science \mathplus Business Media}, author={Curran, Kathleen A. and Crook, Nathan C. and Alper, Hal S.}, year={2011}, month={Nov}, pages={197–216} }
 @article{fasan_crook_peters_meinhold_buelter_landwehr_cirino_arnold_2010, title={Improved product-per-glucose yields in P450-dependent propane biotransformations using engineered Escherichia coli}, volume={108}, DOI={10.1002/bit.22984}, abstractNote={AbstractP450‐dependent biotransformations in Escherichia coli are attractive for the selective oxidation of organic molecules using mild and sustainable procedures. The overall efficiency of these processes, however, relies on how effectively the NAD(P)H cofactors derived from oxidation of the carbon source are utilized inside the cell to support the heterologous P450‐catalyzed reaction. In this work, we investigate the use of metabolic and protein engineering to enhance the product‐per‐glucose yield (YPPG) in whole‐cell reactions involving a proficient NADPH‐dependent P450 propane monooxygenase prepared by directed evolution [P450PMOR2; Fasan et al. (2007); Angew Chem Int Ed 46:8414–8418]. Our studies revealed that the metabolism of E. coli (W3110) is able to support only a modest propanol:glucose molar ratio (YPPG ∼ 0.5) under aerobic, non‐growing conditions. By altering key processes involved in NAD(P)H metabolism of the host, considerable improvements of this ratio could be achieved. A metabolically engineered E. coli strain featuring partial inactivation of the endogenous respiratory chain (Δndh) combined with removal of two fermentation pathways (ΔadhE, Δldh) provided the highest YPPG (1.71) among the strains investigated, enabling a 230% more efficient utilization of the energy source (glucose) in the propane biotransformation compared to the native E. coli strain. Using an engineered P450PMOR2 variant which can utilize NADPH and NADH with equal efficiency, we also established that dual cofactor specificity of the P450 enzyme can provide an appreciable improvement in YPPG. Kinetic analyses suggest, however, that much more favorable parameters (KM, kcat) for the NADH‐driven reaction are required to effectively compete with the host's endogenous NADH‐utilizing enzymes. Overall, the metabolic/protein engineering strategies described here can be of general value for improving the performance of NAD(P)H‐dependent whole‐cell biotransformations in E. coli. Biotechnol. Bioeng. 2011; 108:500–510. © 2010 Wiley Periodicals, Inc.}, number={3}, journal={Biotechnol. Bioeng.}, publisher={Wiley-Blackwell}, author={Fasan, Rudi and Crook, Nathan C. and Peters, Matthew W. and Meinhold, Peter and Buelter, Thomas and Landwehr, Marco and Cirino, Patrick C. and Arnold, Frances H.}, year={2010}, month={Nov}, pages={500–510} }
 @article{fasan_chen_crook_arnold_2007, title={Engineered Alkane-Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties}, volume={119}, DOI={10.1002/ange.200702616}, abstractNote={Eine Domänenstrategie (siehe Schema) führte zu einer hoch effizienten Katalyse der Propanhydroxylierung in einem Mehrdomänen-Cytochrom-P450-Enzym. Die entwickelten Enzyme zeichnen sich durch hohe Gesamtaktivitäten bei der Biotransformation von Propan zu Propanol in ganzen Zellen unter milden Bedingungen aus, wobei Luft als Oxidans wirkt.}, number={44}, journal={Angew. Chem.}, publisher={Wiley-Blackwell}, author={Fasan, Rudi and Chen, Mike M. and Crook, Nathan C. and Arnold, Frances H.}, year={2007}, month={Nov}, pages={8566–8570} }
 @article{fasan_chen_crook_arnold_2007, title={Engineered Alkane-Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties}, volume={46}, DOI={10.1002/anie.200702616}, abstractNote={Cytochrome P450 enzymes (P450s) are exceptional oxygenating catalysts with enormous potential in drug discovery, chemical synthesis, bioremediation, and biotechnology. Compared to their natural counterparts, however, engineered P450s often exhibit poor catalytic and cofactor coupling efficiencies. Obtaining native-like catalytic proficiencies is a mandatory first step towards utilizing the power of these versatile oxygenases in chemical synthesis. Cytochrome P450BM3 (119 kDa, B. megaterium) catalyzes the subterminal hydroxylation of long-chain (C12–C20) fatty acids. Its high activity and catalytic self-sufficiency (heme and diflavin reductase domains are fused in a single polypeptide chain) 5] make P450BM3 an excellent platform for biocatalysis. However, despite numerous reports of the heme domain being engineered to accept nonnative substrates, including short-chain fatty acids, aromatic compounds, alkanes, and alkenes, reports of preparative-scale applications of P450BM3 remain scarce. [9] P450BM3 function is finely regulated through conformational rearrangements in the heme and reductase domains and possibly also through hinged domain motions. 10] Hydroxylation of fatty acids occurs almost fully coupled to cofactor (NADPH) utilization (93–96% depending on the substrate). In the presence of nonnative substrates or when amino acid substitutions are introduced, the mechanisms controlling efficient catalysis in P450s are disrupted, leading to the formation of reactive oxygen species and rapid enzyme inactivation. High coupling efficiencies on substrates whose physicochemical properties are substantially different from the native substrates have not been achieved, and coupling efficiencies ranging from less than 1% to 30– 40% are typical. 8] Strategies for addressing this “coupling problem” are needed in order to take engineered P450s to larger-scale applications. Selective hydroxylation of short alkanes is a long-standing problem, for which no practical catalysts are available. In an effort to produce P450BM3-based biocatalysts for selective hydroxylation of small alkanes, we previously engineered this enzyme to accept propane and ethane (35E11 variant). Despite greater than 5000 total turnover (TTN) supported in vitro, the utility of this catalyst remained limited because of its poor in vivo performance (see below), which was mostly due to the low efficiencies for coupling the product formation to cofactor consumption (17.4% for propane and 0.01% for ethane oxidation). Our goal was to engineer a P450BM3 variant with nativelike activity and coupling efficiency towards a structurally challenging, nonnative substrate (propane) and evaluate the impact of these features on performance in preparative-scale biotransformations. To this end, we used a domain-based protein-engineering strategy, in which the heme, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) domains of the 35E11 variant were evolved separately in the context of the holoenzyme, and beneficial mutations were recombined in a final step (Figure 1). Previous work suggested that mutations in the reductase and linker regions can affect catalytic properties. However, no systematic engineering efforts had been undertaken to engineer the complete 1048 amino acid holoenzyme. Holoenzyme libraries outlined in Figure 1 were created using random, saturation, and site-directed mutagenesis and}, number={44}, journal={Angew. Chem. Int. Ed.}, publisher={Wiley-Blackwell}, author={Fasan, Rudi and Chen, Mike M. and Crook, Nathan C. and Arnold, Frances H.}, year={2007}, month={Nov}, pages={8414–8418} }