Characterization of a Biosynthetic Pathway Yielding Anticancer Natural Products from a Marine Bacterium

Natural products are bioactive secondary metabolites produced by living organisms and are prevalently utilized as pharmaceutical drugs. Marine adapted organisms are a promising source of new natural products possessing unique chemical structures and biological activities. By studying the biosynthetic pathways employed by living organisms to produce natural products, insights into new strategies to generate molecules to combat disease and overcome drug resistance may be gained. This thesis study aimed to uncover the biosynthetic pathway employed by a marine actinomycete, Nocardiopsis sp. CMB-M0232, to catalyze the assembly of the nocardioazines. These molecules are a group of 2,5-diketopiperazine natural products that feature structurally unique functional groups. Nocardioazine A, the hypothesized end product of the nocardioazine biosynthetic pathway, exhibits anticancer activity. Bioinformatics analyses revealed three biosynthetic gene clusters from Nocardiopsis encoding proteins with hypothesized roles in nocardioazine A biosynthesis. Two cyclodipeptide synthases (CDPSs), NozA and NcdA, were biochemically characterized in vivo and in vitro to reveal that both are substrate specific enzymes that utilize tryptophan-charged tRNA substrates to catalyze assembly of cyclo(L-Trp-L-Trp), a proposed precursor of nocardioazines. Fidelity is uncommon amongst characterized CDPSs, making NozA and NcdA important CDPS family additions. This study also aimed to characterize NozD and NozE, two cytochrome P450 homologs with predicted roles as diketopiperazine-tailoring enzymes. Heterologous expression of these enzymes in Streptomyces strains was not able to confirm the functions of NozD and NozE but set the stage for future studies to optimize conditions for probing their roles in nocardioazine A biosynthesis. The results gathered from this study, along with future work to better understand the engineering of unique functional groups from Nocardiopsis may provide opportunities to produce new bioactive molecules

CropPol: A dynamic, open and global database on crop pollination

Seventy five percent of the world's food crops benefit from insect pollination. Hence, there has been increased interest in how global change drivers impact this critical ecosystem service. Because standardized data on crop pollination are rarely available, we are limited in our capacity to understand the variation in pollination benefits to crop yield, as well as to anticipate changes in this service, develop predictions, and inform management actions. Here, we present CropPol, a dynamic, open, and global database on crop pollination. It contains measurements recorded from 202 crop studies, covering 3,394 field observations, 2,552 yield measurements (i.e., berry mass, number of fruits, and fruit density [kg/ha], among others), and 47,752 insect records from 48 commercial crops distributed around the globe. CropPol comprises 32 of the 87 leading global crops and commodities that are pollinator dependent. Malus domestica is the most represented crop (32 studies), followed by Brassica napus (22 studies), Vaccinium corymbosum (13 studies), and Citrullus lanatus (12 studies). The most abundant pollinator guilds recorded are honey bees (34.22% counts), bumblebees (19.19%), flies other than Syrphidae and Bombyliidae (13.18%), other wild bees (13.13%), beetles (10.97%), Syrphidae (4.87%), and Bombyliidae (0.05%). Locations comprise 34 countries distributed among Europe (76 studies), North America (60), Latin America and the Caribbean (29), Asia (20), Oceania (10), and Africa (7). Sampling spans three decades and is concentrated on 2001–2005 (21 studies), 2006–2010 (40), 2011–2015 (88), and 2016–2020 (50). This is the most comprehensive open global data set on measurements of crop flower visitors, crop pollinators and pollination to date, and we encourage researchers to add more datasets to this database in the future. This data set is released for non-commercial use only. Credits should be given to this paper (i.e., proper citation), and the products generated with this database should be shared under the same license terms (CC BY-NC-SA).Fil: Allen Perkins, Alfonso. Universidad Politécnica de Madrid; España. Consejo Superior de Investigaciones Científicas. Estación Biológica de Doñana; EspañaFil: Magrach, Ainhoa. Universidad Politécnica de Madrid; EspañaFil: Dainese, Matteo. Eurac Research. Institute for Alpine Environment; ItaliaFil: Garibaldi, Lucas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Negro; ArgentinaFil: Kleijn, David. Wageningen University & Research; Países BajosFil: Rader, Romina. University of New England; AustraliaFil: Reilly, James R.. Rutgers University; Estados UnidosFil: Winfree, Rachael. Rutgers University; Estados UnidosFil: Lundin, Ola. Swedish University of Agricultural Sciences; SueciaFil: McGrady, Carley M.. North Carolina State University; Estados UnidosFil: Brittain, Claire. University of California at Davis; Estados UnidosFil: Biddinger, David J.. University of California Davis; Estados UnidosFil: Artz, Derek R.. United States Department of Agriculture. Agriculture Research Service; Estados UnidosFil: Elle, Elizabeth. University Fraser Simon; CanadáFil: Hoffman, George. State University of Oregon; Estados UnidosFil: Ellis, James D.. University of Florida; Estados UnidosFil: Daniels, Jaret. University of Florida; Estados Unidos. University Of Florida. Florida Museum Of History; Estados UnidosFil: Gibbs, Jason. University of Manitoba; CanadáFil: Campbell, Joshua W.. University of Florida; Estados Unidos. Usda Ars Northern Plains Agricultural Research Laboratory; Estados UnidosFil: Brokaw, Julia. University of Minnesota; Estados UnidosFil: Wilson, Julianna K.. Michigan State University; Estados UnidosFil: Mason, Keith. Michigan State University; Estados UnidosFil: Ward, Kimiora L.. University of California at Davis; Estados UnidosFil: Gundersen, Knute B.. Michigan State University; Estados UnidosFil: Bobiwash, Kyle. University of Manitoba; Canadá. University Fraser Simon; CanadáFil: Gut, Larry. Michigan State University; Estados UnidosFil: Rowe, Logan M.. Michigan State University; Estados UnidosFil: Boyle, Natalie K.. United States Department of Agriculture. Agriculture Research Service; Estados UnidosFil: Williams, Neal M.. University of California at Davis; Estados UnidosFil: Chacoff, Natacha Paola. Universidad Nacional de Tucumán. Instituto de Ecología Regional. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Ecología Regional; Argentin

CropPol: a dynamic, open and global database on crop pollination

Seventy five percent of the world's food crops benefit from insect pollination. Hence, there has been increased interest in how global change drivers impact this critical ecosystem service. Because standardized data on crop pollination are rarely available, we are limited in our capacity to understand the variation in pollination benefits to crop yield, as well as to anticipate changes in this service, develop predictions, and inform management actions. Here, we present CropPol, a dynamic, open and global database on crop pollination. It contains measurements recorded from 202 crop studies, covering 3,394 field observations, 2,552 yield measurements (i.e. berry weight, number of fruits and kg per hectare, among others), and 47,752 insect records from 48 commercial crops distributed around the globe. CropPol comprises 32 of the 87 leading global crops and commodities that are pollinator dependent. Malus domestica is the most represented crop (32 studies), followed by Brassica napus (22 studies), Vaccinium corymbosum (13 studies), and Citrullus lanatus (12 studies). The most abundant pollinator guilds recorded are honey bees (34.22% counts), bumblebees (19.19%), flies other than Syrphidae and Bombyliidae (13.18%), other wild bees (13.13%), beetles (10.97%), Syrphidae (4.87%), and Bombyliidae (0.05%). Locations comprise 34 countries distributed among Europe (76 studies), Northern America (60), Latin America and the Caribbean (29), Asia (20), Oceania (10), and Africa (7). Sampling spans three decades and is concentrated on 2001-05 (21 studies), 2006-10 (40), 2011-15 (88), and 2016-20 (50). This is the most comprehensive open global data set on measurements of crop flower visitors, crop pollinators and pollination to date, and we encourage researchers to add more datasets to this database in the future. This data set is released for non-commercial use only. Credits should be given to this paper (i.e., proper citation), and the products generated with this database should be shared under the same license terms (CC BY-NC-SA). This article is protected by copyright. All rights reserved

Concussion As a Multi-Scale Complex System: An Interdisciplinary Synthesis of Current Knowledge

Traumatic brain injury (TBI) has been called “the most complicated disease of the most complex organ of the body” and is an increasingly high-profile public health issue. Many patients report long-term impairments following even “mild” injuries, but reliable criteria for diagnosis and prognosis are lacking. Every clinical trial for TBI treatment to date has failed to demonstrate reliable and safe improvement in outcomes, and the existing body of literature is insufficient to support the creation of a new classification system. Concussion, or mild TBI, is a highly heterogeneous phenomenon, and numerous factors interact dynamically to influence an individual’s recovery trajectory. Many of the obstacles faced in research and clinical practice related to TBI and concussion, including observed heterogeneity, arguably stem from the complexity of the condition itself. To improve understanding of this complexity, we review the current state of research through the lens provided by the interdisciplinary field of systems science, which has been increasingly applied to biomedical issues. The review was conducted iteratively, through multiple phases of literature review, expert interviews, and systems diagramming and represents the first phase in an effort to develop systems models of concussion. The primary focus of this work was to examine concepts and ways of thinking about concussion that currently impede research design and block advancements in care of TBI. Results are presented in the form of a multi-scale conceptual framework intended to synthesize knowledge across disciplines, improve research design, and provide a broader, multi-scale model for understanding concussion pathophysiology, classification, and treatment

The Dynamics of Concussion: Mapping Pathophysiology, Persistence, and Recovery with Causal-loop Diagramming

Concussion, also known as mild traumatic brain injury (mTBI),1 is a significant public health issue responsible for a variety of cognitive, emotional, and somatic symptoms and deficits (3). It is unclear why some individuals appear to recover relatively quickly while others suffer prolonged symptoms and impairments (4–7). Robust clinical means of diagnosis, prognosis, and treatment are also lacking (8–11). Research is hindered by an inadequate classification system for traumatic brain injury (TBI) (12), “poor” study quality (13, 14), disagreement about appropriate inclusion and exclusion criteria for concussion (8, 15), and an incomplete understanding of underlying pathophysiology (16–18). The heterogeneity and complexity seen in concussion further complicate research, particularly efforts to individualize treatment (19–22)

Characterization of the Nocardiopsin Biosynthetic Gene Cluster Reveals Similarities to and Differences from the Rapamycin and FK-506 Pathways

Macrolide-pipecolate natural products, such as rapamycin (1) and FK-506 (2), are renowned modulators of FK506-binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB-M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A-D (4-7) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L-lysine into the L-pipecolic acid incorporated into 4 and 5. Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP-binding metabolites and for probing new enzymology in nature\u27s polyketide tailoring arsenal. Expanding nature\u27s macrolide-pipecolate biosynthetic repertoire: Cloning, sequencing, and analysis of the nocardiopsin biosynthetic pathway revealed that Nocardiopsis sp. CMB-M0232 employs a new strategy, featuring both a P450 epoxidase and an epoxide hydrolase, to transform the alkene group of a linear polyketide precursor into the hydroxy group required for macrolactonization

Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis

Marine actinomycete-derived natural products continue to inspire chemical and biological investigations. Nocardioazines A and B (3 and 4), from Nocardiopsis sp. CMB-M0232, are structurally unique alkaloids featuring a 2,5-diketopiperazine (DKP) core functionalized with indole C3-prenyl as well as indole C3- and N-methyl groups. The logic of their assembly remains cryptic. Bioinformatics analyses of the Nocardiopsis sp. CMB-M0232 draft genome afforded the noz cluster, split across two regions of the genome, and encoding putative open reading frames with roles in nocardioazine biosynthesis, including cyclodipeptide synthase (CDPS), prenyltransferase, methyltransferase, and cytochrome P450 homologs. Heterologous expression of a twelve gene contig from the noz cluster in Streptomyces coelicolor resulted in accumulation of cyclo-l-Trp-l-Trp DKP (5). This experimentally connected the noz cluster to indole alkaloid natural product biosynthesis. Results from bioinformatics analyses of the noz pathway along with challenges in actinomycete genetics prompted us to use asymmetric synthesis and mass spectrometry to determine biosynthetic intermediates in the noz pathway. The structures of hypothesized biosynthetic intermediates 5 and 12-17 were firmly established through chemical synthesis. LC-MS and MS-MS comparison of these synthetic compounds with metabolites present in chemical extracts from Nocardiopsis sp. CMB-M0232 revealed which of these hypothesized intermediates were relevant in the nocardioazine biosynthetic pathway. This established the early and mid-stages of the biosynthetic pathway, demonstrating that Nocardiopsis performs indole C3-methylation prior to indole C3-normal prenylation and indole N1′-methylation in nocardioazine B assembly. These results highlight the utility of merging bioinformatics analyses, asymmetric synthetic approaches, and mass spectrometric metabolite profiling in probing natural product biosynthesis

Two Distinct Cyclodipeptide Synthases from a Marine Actinomycete Catalyze Biosynthesis of the Same Diketopiperazine Natural Product

Diketopiperazine natural products are structurally diverse and offer many biological activities. Cyclodipeptide synthases (CDPSs) were recently unveiled as a novel enzyme family that employs aminoacyl-tRNAs as substrates for 2,5-diketopiperazine assembly. Here, the <i>Nocardiopsis</i> sp. CMB-M0232 genome is predicted to encode two CDPSs, NozA and NcdA. Metabolite profiles from <i>E. coli</i> expressing these genes and assays with purified recombinant enzymes revealed that NozA and NcdA catalyze <i>cyclo</i>(l-Trp-l-Trp) (<b>1</b>) biosynthesis from tryptophanyl-tRNA and do not accept other aromatic aminoacyl-tRNA substrates. Fidelity is uncommon among characterized CDPSs, making NozA and NcdA important CDPS family additions. Further, <b>1</b> was previously supported as a biosynthetic precursor of the nocardioazines; the current study suggests that <i>Nocardiopsis</i> sp. may derive this precursor from both NozA and NcdA. This study offers a rare example of a single bacterium encoding multiple phylogenetically distinct enzymes that yield the same secondary metabolite and provides tools for chemoenzymatic syntheses of indole alkaloid diketopiperazines