SAINT LOUIS, MO, United States
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Patent
Intact Genomics, Inc. | Date: 2016-04-29

Fungal artificial chromosome (FAC) vectors are disclosed. A vector can be replicated in a bacterial or a fungal host, and can comprise an insert of heterologous DNA up to about 500 kilobases. A vector can be used for cloning and expressing a secondary metabolite (SM) gene cluster. An insert sequence can be modified by homologous recombination. A vector can be a plasmid comprising bacterial and fungal origins of replication, as well as bacterial and fungal selection marker genes. Also disclosed are vectors that can be integrated into a fungal genome, and dual function vectors which can be replicated in a bacterial or a fungal host and can also be integrated into a fungal genome. Also disclosed are methods of generating plasmid libraries including vectors comprising intact SM gene clusters.


Alexeyenko A.,KTH Royal Institute of Technology | Alexeyenko A.,Karolinska Institutet | Nystedt B.,University of Stockholm | Nystedt B.,Uppsala University | And 10 more authors.
BMC Genomics | Year: 2014

Background: Sampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality.Results: In order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with ~40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS.Conclusions: By exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process.We have made public the input data (FASTQ format) for the set of pools used in this study:. ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/. © 2014 Alexeyenko et al.; licensee BioMed Central Ltd.


Bok J.W.,University of Wisconsin - Madison | Ye R.,Intact Genomics, Inc. | Ye R.,Lucigen Corporation | Clevenger K.D.,Northwestern University | And 10 more authors.
BMC Genomics | Year: 2015

Background: With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80 kb in size, breakthrough techniques are needed to characterize this SM wealth. Results: Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery. Conclusion: The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery. © 2015 Bok et al.; licensee BioMed Central.


PubMed | Lucigen Corporation, Intact Genomics, Inc., University of Wisconsin - Madison and Northwestern University
Type: | Journal: BMC genomics | Year: 2015

With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80kb in size, breakthrough techniques are needed to characterize this SM wealth.Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery.The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.


Liu Y.-H.,Texas A&M University | Zhang M.,Jilin Agricultural University | Wu C.,Texas A&M University | Wu C.,Intact Genomics, Inc. | And 2 more authors.
Genome | Year: 2014

Knowledge of how a genome is structured and organized from its constituent elements is crucial to understanding its biology and evolution. Here, we report the genome structuring and organization pattern as revealed by systems analysis of the sequences of three model species, Arabidopsis, rice and yeast, at the whole-genome and chromosome levels. We found that all fundamental function elements (FFE) constituting the genomes, including genes (GEN), DNA transposable elements (DTE), retrotransposable elements (RTE), simple sequence repeats (SSR), and (or) low complexity repeats (LCR), are structured in a nonrandom and correlative manner, thus leading to a hypothesis that the DNA of the species is structured as a linear "jigsaw puzzle". Furthermore, we showed that different FFE differ in their importance in the formation and evolution of the DNA jigsaw puzzle structure between species. DTE and RTE play more important roles than GEN, LCR, and SSR in Arabidopsis, whereas GEN and RTE play more important roles than LCR, SSR, and DTE in rice. The genes having multiple recognized functions play more important roles than those having single functions. These results provide useful knowledge necessary for better understanding genome biology and evolution of the species and for effective molecular breeding of rice. © 2013 Published by NRC Research Press.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 900.55K | Year: 2016

PROJECT SUMMARY According to a CDC report antibiotic resistant infections are associated with deaths and million illnesses in the United States each year The estimated annual impact of antibiotic resistant infections on the national economy is $ billion in excess direct health care costs and in lost productivity from hospitalizations and sick days The emergence of drug resistant microbes the diminishing supply of novel classes of antibiotics and the dramatic reduction in discovery and development of anti infective anti proliferation and anti inflammation agents have further amplified public health concerns Fungi are prolific producers of anti microbial secondary metabolites SM and since the turn of the century have provided of bioactive molecules from all microbial sources However fungal SM pathways remain largely untapped due to difficulties in efficiently handling and expressing these SM pathways This research proposal is to advance the science of functional SM metagenomics to further advance our newly developed fungal artificial chromosome FAC technology precisely engineer and activate large intact silent SM pathways containing FAC clones and to discover new antibiotics for pharmaceutical and clinical development Ongoing research from Dr Wu s team at Intact Genomics and scientists at the University of Wisconsin Madison and Northwestern University applied numerous key technological breakthroughs that resulted in the next generation fungal SM discovery platform This discovery technology combined an improved methodology for the isolation and purification of high molecular weight genomic DNA from fungi a new E coli Aspergillus shuttle or FAC vector and an A nidulans host for enhanced expression of cloned large DNAs a random shear BAC FAC cloning method to produce unbiased very large insert sizes andgt kb for covering the entire set of intact SM pathways of a fungal genome one FAC clone one intact SM pathway precisely engineering and activating large intact silent SM gene clusters FACs by Red ET techniques and a rapid and improved small molecule identification method to identify unique compounds In Phase I research we tested FAC engineering by precisely cutting FAC sizes deleting individual SM genes and or inserting a strong promoter and generated more than engineered FAC constructs from the large SM FAC clones with a successful rate We have discovered the benzomalvin compounds and established the biosynthesis of this NRPS metabolites that has long eluded the field The FAC SM gene deletants not only allow us to see loss of their corresponding gene products but also accumulation of biosynthetic precursors Moreover the engineered silent SM gene clusters show at least a fold increase in expression in response to the strong promoter and analysis We believe that we are the first group to develop a superior BAC recombineering and transgenic animal system for fungal functional SM study We propose in Phase II study to engineered andgt silent SM pathways from sequenced fungi SM pathways which will be extensively screened for small molecule compounds and antibiotics We expect to uncover andgt novel chemical entities using this approach and lead candidates with high potency against multiple drug resistance bacterial and fungal pathogens These technologies represent an important advancement for the science of natural product discovery in general and antibiotic discovery in particular In addition the FACs produced from this research are a valuable genomic resource that may be screened for other bioactive compounds such as antiviral anticancer and anti inflammatory activities PROJECT NARRATIVE We are losing the battle against infectious diseases as evidenced by the alarming increase number of multi drug resistance microbes and our inability to find antibiotics with novel acting mechanisms The loss of life and the burden of treatment is a significant public health threat to American citizens The proposed research utilizes a new methodology advancing fungal artificial chromosome FAC technology and tools for drug discovery This novel technology permits access to the majority of fungal LARGE INTACT and SILENT natural product pathways in a sequenced fungal genome with the aim to eventually activate all silent and cryptic pathways for pharmaceutical discovery Our functional metagenomic approach will be used to identify and characterize novel antibiotic compounds to combat the threat of bacterial and fungal pathogens


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 211.45K | Year: 2015

DESCRIPTION provided by applicant There is societal need for new therapeutic agents in our arsenal of defenses against bacterial and fungal pathogens many of which are increasingly resistant to existing antibiotics Filamentous fungi are considered promising resources for the development of novel bioactive compounds because of their great potential to produce various kinds of secondary metabolites SM however antibiotic discovery and production in fungi lags far behind bacteria This research proposal advances sciences of fungal functional genomics to activate fungal silent SM clusters by using the newly developed fungal artificial chromosomes FACs Our purpose is to discover novel antibiotics and identify the best lead candidates for clinical development Scientists at Intact Genomics Inc and the University of Wisconsin at Madison will develop utilize and combine three aspects of novel technology innovation and genomic tools to enable therapeutic agent discovery in fungi Specifically the proposed research will identify antibiotic compounds using genetically enhanced A nidulans strains in vitro BAC FAC engineering and culture conditions with epigenetic modifications and bacterial co culture The primary objectives are to activate at least of silent and or cryptic SM gene clusters FACs of A terreus for proof of concept using the above technologies and to screen these activated FACs against bacterial and fungal tester strains to discover novel antibacterial and antifungal properties Our long term goals are to develop a high through put small molecule discovery platform in fungi in order to discover novel natural products from at least silent SM pathways from completely sequenced fungal genomes Moreover we will characterize identified antimicrobial agents to determine the best lead candidates for clinical development Lead candidates will have novel chemical structures high potency against bacterial and or fungal pathogens and minimal toxicity for eukaryotic cells Each of the different technologies necessary for the proposed research has been proven effective separately therefore the combination of these different techniques has a high probability of success and also represents a significant advancement for the science of antibiotic discovery In addition the activated silent SM clusters and their metabolites produced from this research are a valuable resource that may be screened for other bioactive compounds e g with anticancer or antiviral activities in subsequent research PUBLIC HEALTH RELEVANCE The need for new therapeutic agents has reached an intensity not experienced since the commercialization of antibiotics in the s but many traditionally fruitful sources of chemistry have ceased to yield new compounds This research will develop utilize and combine three aspects of novel technology innovation and genomic tools to enable therapeutic agent discovery in the successfully engineered fungal host Aspergillus nidulans by activating and expressing fungal silent secondary metabolic pathways directly without the need to cultivate and engineer the different fungi in a laboratory This technology will access the nove small molecules produced by a great diversity of filamentous fungi many of which are unknown to science and will identify the best novel therapeutic compounds for use in treating bacterial and fungal diseases


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.87K | Year: 2016

DESCRIPTION provided by applicant There is societal need for new therapeutic agents in our arsenal of defenses against bacterial and fungal pathogens many superbugs of which are increasingly resistant to existing antibiotics Filamentous fungi are considered promising resources for the development of novel bioactive compounds because of their great potential to produce various kinds of secondary metabolites SM however natural product NP discovery and production in fungi lags far behind plants and bacteria This research proposal advances sciences of fungal functional genomics to develop a robust heterologous expression system for intact SM gene clusters in the filamentous fungus Aspergillus nidulans by using the newly developed fungal artificial chromosomes FACs Our purpose is to discover novel antibiotics and identify the best lead candidates for clinical development Scientists at Intact Genomics Inc University of Wisconsin at Madison Donald Danforth Plant Science Center and Northwestern University will develop utilize and combine at least aspects of novel technology innovation and genomic tools to enable NP discovery in fungi Specifically the proposed research will identify NP compounds using i the unbiased large insert Random Shear Shuttle BAC libraries as FACs ii more than large intact SM gene clusters about kb in the completely sequenced genome of fungal strains iii the knowledge of regulatory elements strong promotors for high heterologous expression of SM gene clusters in Aspergillus iv the successfully engineered fungal host A nidulans to provide a robust background in which to search for new metabolites v in vitro BAC FAC engineering vi advanced LC MS analysis The primary objectives of Phase I research are to develop simple FAC transformation method in an optimized A nidulans strain to activate at least of silent and or cryptic SM gene clusters FACs for proof of concept using the above technologies to discover novel NP compounds Our long term goals are to develop a high through put small molecule discovery platform in fungi in order to discover novel natural products from at least fungal intact SM pathways from completely sequenced fungal genomes Moreover we will characterize identified antimicrobial agents to determine the best lead candidates for clinical development Lead candidates will have novel chemical structures high potency against bacterial and or fungal pathogens and minimal toxicity for eukaryotic cells The combination of these novel technological innovations has a high probability of success and also represents a significant advancement for the science of natural product discovery In addition the novel SM clusters and their metabolites produced from this research are a valuable resource that may be screened for other bioactive compounds e g with anticancer or antiviral activities in subsequent research PUBLIC HEALTH RELEVANCE The need of new natural products for therapeutic agents has reached an intensity not experienced since the commercialization of antibiotics in the s but many traditionally fruitful sources of chemistry have ceased to yield new compounds This research will develop utilize and combine at least aspects of novel technological innovations and genomic tools to achieve a robust fungal artificial chromosome heterologous expression system for natural product production in Aspergillus nidulans without the need to cultivate and engineer the different fungi in a laboratory This technology will access the novel small molecules produced by a great diversity of filamentous fungi many of which are unknown to science and will identify the best novel therapeutic compounds for use in treating bacterial and fungal diseases


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Intact Genomics, Inc. | Entity website

Intact Genomics was selected for a showcase booth and presented at theTechConnect World Innovation Summit & Expo,co-located with the National Innovation Summit and National SBIR/STTR Conference on May 22-25, 2016 in Washington, D.C ...

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