@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={Exploring the gastrointestinal role of hydrogen sulfide (H2S) 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 Escherichia coli to titrate H2S 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{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{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={The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.}, 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{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} }
 @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={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} }