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Nathan Crook

Bio: Nathan Crook is an academic researcher from North Carolina State University. The author has contributed to research in topics: Medicine & Biology. The author has an hindex of 17, co-authored 31 publications receiving 1593 citations. Previous affiliations of Nathan Crook include Washington University in St. Louis & University of Texas at Austin.

Papers
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Journal ArticleDOI
TL;DR: This review summarizes current research on the short-term and long-term consequences of antibiotic use on the human microbiome, from early life to adulthood, and its effect on diseases such as malnutrition, obesity, diabetes, and Clostridium difficile infection.
Abstract: The widespread use of antibiotics in the past 80 years has saved millions of human lives, facilitated technological progress and killed incalculable numbers of microbes, both pathogenic and commensal. Human-associated microbes perform an array of important functions, and we are now just beginning to understand the ways in which antibiotics have reshaped their ecology and the functional consequences of these changes. Mounting evidence shows that antibiotics influence the function of the immune system, our ability to resist infection, and our capacity for processing food. Therefore, it is now more important than ever to revisit how we use antibiotics. This review summarizes current research on the short-term and long-term consequences of antibiotic use on the human microbiome, from early life to adulthood, and its effect on diseases such as malnutrition, obesity, diabetes, and Clostridium difficile infection. Motivated by the consequences of inappropriate antibiotic use, we explore recent progress in the development of antivirulence approaches for resisting infection while minimizing resistance to therapy. We close the article by discussing probiotics and fecal microbiota transplants, which promise to restore the microbiota after damage of the microbiome. Together, the results of studies in this field emphasize the importance of developing a mechanistic understanding of gut ecology to enable the development of new therapeutic strategies and to rationally limit the use of antibiotic compounds.

667 citations

Journal ArticleDOI
TL;DR: The 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.
Abstract: 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

224 citations

Journal ArticleDOI
TL;DR: It is demonstrated that nucleosome architecture can play a role in defining yeast promoter activity and a computationally-guided approach is utilized that can enable both the redesign of endogenous promoter sequences and the de novo design of synthetic promoters.
Abstract: 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.

132 citations

Journal ArticleDOI
TL;DR: This work 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.
Abstract: Directed 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. Directed evolution is a powerful technique for generating improved biological systems through repeated rounds of mutagenesis and selection. Here the authors engineer the yeast retrotransposon Ty1 to enable the creation of large mutant libraries in vivoand use this system to generate improved variants of single enzymes and multigene pathways.

98 citations


Cited by
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01 Jun 2012
TL;DR: SPAdes as mentioned in this paper is a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler and on popular assemblers Velvet and SoapDeNovo (for multicell data).
Abstract: The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

10,124 citations

Journal ArticleDOI
TL;DR: Future studies will focus on understanding the mechanisms underlying the microbiota-gut-brain axis and attempt to elucidate microbial-based intervention and therapeutic strategies for neuropsychiatric disorders.
Abstract: The importance of the gut-brain axis in maintaining homeostasis has long been appreciated. However, the past 15 yr have seen the emergence of the microbiota (the trillions of microorganisms within ...

1,775 citations

Book
01 Jun 2009
TL;DR: The United Nations Children's Fund (UNICEF) as mentioned in this paper was originally created to provide relief for children in countries devastated by the destruction of World War II, and in 1965, it was awarded the Nobel Prize for Peace for its humanitarian efforts.
Abstract: The United Nations Children's Fund, or UNICEF, was originally created to provide relief for children in countries devastated by the destruction of World War II. After 1950, UNICEF turned to focus on general programs for the improvement of children's welfare worldwide, and in 1965, it was awarded the Nobel Prize for Peace for its humanitarian efforts. The organization concentrates on areas in which relatively small expenditures can have a significant impact on the lives of the most disadvantaged children in developing countries, such as the prevention and treatment of disease, child healthcare, malnutrition, illiteracy, and other welfare services.

1,156 citations

Journal ArticleDOI
TL;DR: Directed evolution studies have shown how rapidly some proteins can evolve under strong selection pressures and, because the entire 'fossil record' of evolutionary intermediates is available for detailed study, they have provided new insight into the relationship between sequence and function.
Abstract: Directed evolution circumvents our profound ignorance of how a protein's sequence encodes its function by using iterative rounds of random mutation and artificial selection to discover new and useful proteins. Proteins can be tuned to adapt to new functions or environments by simple adaptive walks involving small numbers of mutations. Directed evolution studies have shown how rapidly some proteins can evolve under strong selection pressures and, because the entire 'fossil record' of evolutionary intermediates is available for detailed study, they have provided new insight into the relationship between sequence and function. Directed evolution has also shown how mutations that are functionally neutral can set the stage for further adaptation.

926 citations

Journal ArticleDOI
TL;DR: Although these proteins have properties that make them particularly attractive for engineering purposes, the large reservoir of P450 enzymes that collectively catalyze an astounding diversity of reactions suggests that P450 catalysis will develop into a highly useful technology.
Abstract: In chemical terms, the regio- and stereoselective hydroxylation of hydrocarbon C-H bonds is a very difficult transformation. Nevertheless, these reactions are deftly catalyzed by a variety of metalloenzymes, among which the most diverse are the many members of the cytochrome P450 family. Cytochrome P450 enzymes are found in most classes of organisms, including bacteria, fungi, plants, insects, and mammals. Thousands of such proteins are now known (http://drnelson.utmem.edu/cytochromeP450.html), including 57 in the human genome (1), 20 in Mycobacterium tuberculosis (2), 272 in Arabidopsis (3), and the amazing number of 457 in rice (4). The nomenclature for these enzymes is based on their sequence similarity when appropriately aligned, a somewhat arbitrary similarity cutoff (approximately >40% identity) being used to define members of a family and a higher cutoff (approximately >55% identity) members of a subfamily (5). Thus CYP3A4 corresponds to the fourth enzyme in family 3, subfamily A. This nomenclature allows the naming of enzymes without regard to their origin or specific properties. The mammalian, plant, and fungal proteins are commonly membrane bound and are relatively difficult to manipulate, but the bacterial proteins are usually soluble, monomeric proteins. For that reason, much of the early research on mechanisms of cytochrome P450 enzymes was carried out with bacterial enzymes, particularly with the prototypical enzyme CYP101 (P450cam) from Pseudomonas putida (6, 7). From a chemist's point of view, there is a particular interest in the thermophilic enzymes, which currently include CYP119 (8-10), P450st (11), CYP174A1 (12), and CYP231A2 (13). The thermal stability of these enzymes makes them attractive starting points for the development of industrially useful catalysts. In this context, particular attention has also focused on CYP102 (P450BM3), a self-sufficient enzyme from Bacillus megaterium in which the flavoprotein protein required for transfer of electrons from NADPH is fused to the hemoprotein (14). The resulting simplicity and high catalytic rate have led to extensive efforts to engineer this protein for practical catalytic purposes (15-19). Although these proteins have properties that make them particularly attractive for engineering purposes, the large reservoir of P450 enzymes that collectively catalyze an astounding diversity of reactions suggests that P450 catalysis will develop into a highly useful technology. The cytochrome P450 enzymes are defined by the presence in the proteins of a heme (iron protoporphyrin IX) prosthetic group coordinated on the proximal side by a thiolate ion (20, 21). This feature gives rise to the spectroscopic signature that defines these enzymes, as the thiolate-ligated ferrous-CO complex is characterized by a Soret absorption maximum at ∼450 nm (21). A thiolate-coordinated heme group is present in all P450 enzymes, although not all proteins with such coordination are members of this superfamily. One obvious exception, for example, is chloroperoxidase, which has a thiolate-coordinated heme group but normally catalyzes a very different reaction than the P450 enzymes (21-23). Although there are unusual P450 enzymes, such as the thromboxane and prostacyclin synthases (24), or CYP152 from Sphingomonas paucimobilis or Bacillus subtilis (25, 26), that normally utilize peroxides as substrates, the defining reaction for P450 enzymes is the reductive activation of molecular oxygen. In this reaction, one of the oxygen atoms of molecular oxygen is inserted into the substrate and the other oxygen atom is reduced to a molecule of water. With one exception to date (27, 28), the electrons required for this reduction of molecular oxygen derive from reduced pyridine nucleotides (NADH or NADPH). The overall equation for the reaction thus adheres to the formula: RH + NAD(P)H + O2 + H+ -> ROH + NAD(P)+ + H2O, where RH stands for a substrate with a hydroxylatable site. P450 enzymes therefore belong to the monooxygenase class of enzymes that only insert one of the oxygen atoms of molecular oxygen into their substrates. However, under appropriate circumstances or with specific substrates, other P450-catalyzed reactions can be observed, including desaturation, carbon-carbon bond scission, and carbon-carbon bond formation (29, 30). This review specifically focuses on P450-catalyzed hydrocarbon hydroxylation, the reaction that is most characteristic of P450 enzymes and that has been most extensively investigated. However, the principles that apply in these reactions also apply to other hydroxylation reactions, including those that occur on carbons adjacent to nitrogen, sulfur, or oxygen.

880 citations