Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq.

Abstract:

:Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii. Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low- to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data.

journal_name

ACS Synth Biol

journal_title

ACS synthetic biology

authors

Brady JR,Tan MC,Whittaker CA,Colant NA,Dalvie NC,Love KR,Love JC

doi

10.1021/acssynbio.0c00299

subject

Has Abstract

pub_date

2020-09-18 00:00:00

pages

2515-2524

issue

9

issn

2161-5063

journal_volume

9

pub_type

杂志文章
  • Incorporation of a Synthetic Amino Acid into dCas9 Improves Control of Gene Silencing.

    abstract::The CRISPR-Cas9 nuclease has been repurposed as a tool for gene repression (CRISPRi). This catalytically dead Cas9 (dCas9) variant inhibits transcription by blocking either initiation or elongation by the RNA polymerase complex. Conditional control of dCas9-mediated repression has been achieved with inducible promoter...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.8b00347

    authors: Koopal B,Kruis AJ,Claassens NJ,Nobrega FL,van der Oost J

    更新日期:2019-02-15 00:00:00

  • Development of High-Performance Whole Cell Biosensors Aided by Statistical Modeling.

    abstract::Whole cell biosensors are genetic systems that link the presence of a chemical, or other stimulus, to a user-defined gene expression output for applications in sensing and control. However, the gene expression level of biosensor regulatory components required for optimal performance is nonintuitive, and classical iter...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00448

    authors: Berepiki A,Kent R,Machado LFM,Dixon N

    更新日期:2020-03-20 00:00:00

  • Biosynthetic Routes for Producing Various Fucosyl-Oligosaccharides.

    abstract::Fucosyl-oligosaccharides (FOSs) play physiologically important roles as prebiotics, neuronal growth factors, and inhibitors of enteropathogens. However, challenges in designed synthesis and mass production of FOSs hamper their industrial applications. Here, we report flexible biosynthetic routes to produce various FOS...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.8b00436

    authors: Yun EJ,Liu JJ,Lee JW,Kwak S,Yu S,Kim KH,Jin YS

    更新日期:2019-02-15 00:00:00

  • RNA-Catalyzed Polymerization of Deoxyribose, Threose, and Arabinose Nucleic Acids.

    abstract::An RNA-dependent RNA polymerase ribozyme that was highly optimized through in vitro evolution for the ability to copy a broad range of template sequences exhibits promiscuity toward other nucleic acids and nucleic acid analogues, including DNA, threose nucleic acid (TNA), and arabinose nucleic acid (ANA). By operating...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00044

    authors: Horning DP,Bala S,Chaput JC,Joyce GF

    更新日期:2019-05-17 00:00:00

  • "Site and Mutation"-Specific Predictions Enable Minimal Directed Evolution Libraries.

    abstract::Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of benef...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.7b00359

    authors: Moore JC,Rodriguez-Granillo A,Crespo A,Govindarajan S,Welch M,Hiraga K,Lexa K,Marshall N,Truppo MD

    更新日期:2018-07-20 00:00:00

  • Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection.

    abstract::Dissimilatory metal-reducing bacteria, particularly those from the genus Shewanella, are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulat...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00188

    authors: Corts AD,Thomason LC,Gill RT,Gralnick JA

    更新日期:2019-08-16 00:00:00

  • Synthetic tunable amplifying buffer circuit in E. coli.

    abstract::While predictable design of a genetic circuit's output is a major goal of synthetic biology, it remains a significant challenge because DNA binding sites in the cell affect the concentration of available transcription factors (TF). To mitigate this problem, we propose to use a TF that results from the (reversible) pho...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/sb5002533

    authors: Nilgiriwala KS,Jiménez J,Rivera PM,Del Vecchio D

    更新日期:2015-05-15 00:00:00

  • Exploring Chemical Biosynthetic Design Space with Transform-MinER.

    abstract::Transform-MinER (Transforming Molecules in Enzyme Reactions) is a web application facilitating the exploration of chemical biosynthetic space, guiding the user toward promising start points for enzyme design projects or directed evolution experiments. Two types of search are possible: Molecule Search allows a user to ...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00105

    authors: Tyzack JD,Ribeiro AJM,Borkakoti N,Thornton JM

    更新日期:2019-11-15 00:00:00

  • An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics.

    abstract::While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tiss...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00175

    authors: Allen ME,Zhou W,Thangaraj J,Kyriakakis P,Wu Y,Huang Z,Ho P,Pan Y,Limsakul P,Xu X,Wang Y

    更新日期:2019-10-18 00:00:00

  • Genetically Encodable Bacterial Flavin Transferase for Fluorogenic Protein Modification in Mammalian Cells.

    abstract::A bacterial flavin transferase (ApbE) was recently employed for flavin mononucleotide (FMN) modification on the Na+-translocating NADH:quinone oxidoreductase C (NqrC) protein in the pathogenic Gram-negative bacterium Vibrio cholerae. We employed this unique post-translational modification in mammalian cells and found ...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.6b00284

    authors: Kang MG,Park J,Balboni G,Lim MH,Lee C,Rhee HW

    更新日期:2017-04-21 00:00:00

  • Synthesis and Characterization of Heterodimers and Fluorescent Nisin Species by Incorporation of Methionine Analogues and Subsequent Click Chemistry.

    abstract::Noncanonical amino acids form a highly diverse pool of building blocks that can render unique physicochemical properties to peptides and proteins. Here, four methionine analogues with unsaturated and varying side chain lengths were successfully incorporated at four different positions in nisin in Lactococcus lactis th...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.0c00308

    authors: Deng J,Viel JH,Chen J,Kuipers OP

    更新日期:2020-09-18 00:00:00

  • GeneORator: An Effective Strategy for Navigating Protein Sequence Space More Efficiently through Boolean OR-Type DNA Libraries.

    abstract::Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional site-directed methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of ge...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00063

    authors: Currin A,Kwok J,Sadler JC,Bell EL,Swainston N,Ababi M,Day P,Turner NJ,Kell DB

    更新日期:2019-06-21 00:00:00

  • A Lambda Red and FLP/FRT-Mediated Site-Specific Recombination System in Komagataeibacter xylinus and Its Application to Enhance the Productivity of Bacterial Cellulose.

    abstract::Komagataeibacter xylinus has received increasing attention as an important microorganism for the conversion of several carbon sources to bacterial cellulose (BC). However, BC productivity has been impeded by the lack of efficient genetic engineering techniques. In this study, a lambda Red and FLP/FRT-mediated site-spe...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.0c00450

    authors: Liu LP,Yang X,Zhao XJ,Zhang KY,Li WC,Xie YY,Jia SR,Zhong C

    更新日期:2020-11-20 00:00:00

  • Analog Computation by DNA Strand Displacement Circuits.

    abstract::DNA circuits have been widely used to develop biological computing devices because of their high programmability and versatility. Here, we propose an architecture for the systematic construction of DNA circuits for analog computation based on DNA strand displacement. The elementary gates in our architecture include ad...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.6b00144

    authors: Song T,Garg S,Mokhtar R,Bui H,Reif J

    更新日期:2016-08-19 00:00:00

  • SBOLExplorer: Data Infrastructure and Data Mining for Genetic Design Repositories.

    abstract::This paper describes SBOLExplorer, a system that is used to provide intuitive searching within the SynBioHub genetic design repository. SynBioHub stores genetic constructs encoded in the SBOL data format. These constructs can represent genetic parts, circuits, and sequences. These constructs are often numerous, exist ...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00089

    authors: Zhang M,Zundel Z,Myers CJ

    更新日期:2019-10-18 00:00:00

  • Light-Inducible Recombinases for Bacterial Optogenetics.

    abstract::Optogenetic tools can provide direct and programmable control of gene expression. Light-inducible recombinases, in particular, offer a powerful method for achieving precise spatiotemporal control of DNA modification. However, to-date this technology has been largely limited to eukaryotic systems. Here, we develop opto...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00395

    authors: Sheets MB,Wong WW,Dunlop MJ

    更新日期:2020-02-21 00:00:00

  • Combinatory Biosynthesis of Prenylated 4-Hydroxybenzoate Derivatives by Overexpression of the Substrate-Promiscuous Prenyltransferase XimB in Engineered E. coli.

    abstract::Prenylated aromatic compounds are important intermediates in the biosynthesis of bioactive molecules such as 3-chromanols from plants, ubiquinones from prokaryotes and meroterpenoids from sponges. Biosynthesis of prenylated aromatic compounds using prokaryotic microorganisms has attracted increasing attention in the f...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.8b00070

    authors: He BB,Bu XL,Zhou T,Li SM,Xu MJ,Xu J

    更新日期:2018-09-21 00:00:00

  • Heterochiral DNA Strand-Displacement Based on Chimeric d/l-Oligonucleotides.

    abstract::Heterochiral DNA strand-displacement reactions enable sequence-specific interfacing of oligonucleotide enantiomers, making it possible to interface native d-nucleic acids with molecular circuits built using nuclease-resistant l-DNA. To date, all heterochiral reactions have relied on peptide nucleic acid (PNA), which p...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.9b00335

    authors: Young BE,Sczepanski JT

    更新日期:2019-12-20 00:00:00

  • Engineered platform for bioethylene production by a cyanobacterium expressing a chimeric complex of plant enzymes.

    abstract::Ethylene is an industrially important compound, but more sustainable production methods are desirable. Since cellulosomes increase the ability of cellulolytic enzymes by physically linking the relevant enzymes via dockerin-cohesin interactions, in this study, we genetically engineered a chimeric cellulosome-like compl...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/sb400197f

    authors: Jindou S,Ito Y,Mito N,Uematsu K,Hosoda A,Tamura H

    更新日期:2014-07-18 00:00:00

  • Systematic Identification of a Panel of Strong Constitutive Promoters from Streptomyces albus.

    abstract::Actinomycetes are important organisms for the biosynthesis of valuable natural products. However, only a limited number of well-characterized native constitutive promoters from actinomycetes are available for the construction and engineering of large biochemical pathways. Here, we report the discovery and characteriza...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.5b00016

    authors: Luo Y,Zhang L,Barton KW,Zhao H

    更新日期:2015-09-18 00:00:00

  • Exploiting Single Domain Antibodies as Regulatory Parts to Modulate Monoterpenoid Production in E. coli.

    abstract::Synthetic biology and metabolic engineering offer potentially green and attractive routes to the production of high value compounds. The provision of high-quality parts and pathways is crucial in enabling the biosynthesis of chemicals using synthetic biology. While a number of regulatory parts that provide control at ...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.0c00375

    authors: Wilkes J,Scott-Tucker A,Wright M,Crabbe T,Scrutton NS

    更新日期:2020-10-16 00:00:00

  • An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning of an Antimicrobial Resistance Protein.

    abstract::Understanding the molecular mechanisms underlying antibiotic resistance requires concerted efforts in enzymology and medicinal chemistry. Here we describe a new synthetic biology approach to antibiotic development, where the presence of tetracycline antibiotics is linked to a life-death selection in Saccharomyces cere...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.8b00121

    authors: Scott LH,Mathews JC,Flematti GR,Filipovska A,Rackham O

    更新日期:2018-08-17 00:00:00

  • Engineering Escherichia coli into a protein delivery system for mammalian cells.

    abstract::Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogen...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.5b00002

    authors: Reeves AZ,Spears WE,Du J,Tan KY,Wagers AJ,Lesser CF

    更新日期:2015-05-15 00:00:00

  • Biophysical, mutational, and functional investigation of the chromophore-binding pocket of light-oxygen-voltage photoreceptors.

    abstract::As light-regulated actuators, sensory photoreceptors underpin optogenetics and numerous applications in synthetic biology. Protein engineering has been applied to fine-tune the properties of photoreceptors and to generate novel actuators. For the blue-light-sensitive light-oxygen-voltage (LOV) photoreceptors, mutation...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/sb400205x

    authors: Diensthuber RP,Engelhard C,Lemke N,Gleichmann T,Ohlendorf R,Bittl R,Möglich A

    更新日期:2014-11-21 00:00:00

  • Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli.

    abstract::Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segrega...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.0c00008

    authors: Mukai T,Yoneji T,Yamada K,Fujita H,Nara S,Su'etsugu M

    更新日期:2020-06-19 00:00:00

  • Optogenetic Inhibition of Gαq Protein Signaling Reduces Calcium Oscillation Stochasticity.

    abstract::As fast terminators of G-protein coupled receptor (GPCR) signaling, regulators of G-protein signaling (RGS) serve critical roles in fine-tuning second messenger levels and, consequently, cellular responses to external stimuli. Here, we report the creation of an optogenetic RGS2 (opto-RGS2) that suppresses agonist-evok...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.8b00065

    authors: Hannanta-Anan P,Chow BY

    更新日期:2018-06-15 00:00:00

  • A System for Gene Expression Noise Control in Yeast.

    abstract::Gene expression noise arises from stochastic variation in the synthesis and degradation of mRNA and protein molecules and creates differences in protein numbers across populations of genetically identical cells. Such variability can lead to imprecision and reduced performance of both native and synthetic networks. In ...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.8b00279

    authors: Mundt M,Anders A,Murray SM,Sourjik V

    更新日期:2018-11-16 00:00:00

  • CRISPR-Cas9 Based Engineering of Actinomycetal Genomes.

    abstract::Bacteria of the order Actinomycetales are one of the most important sources of pharmacologically active and industrially relevant secondary metabolites. Unfortunately, many of them are still recalcitrant to genetic manipulation, which is a bottleneck for systematic metabolic engineering. To facilitate the genetic mani...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.5b00038

    authors: Tong Y,Charusanti P,Zhang L,Weber T,Lee SY

    更新日期:2015-09-18 00:00:00

  • Exploiting Spermidine N-Hydroxycinnamoyltransferase Diversity and Substrate Promiscuity to Produce Various Trihydroxycinnamoyl Spermidines and Analogues in Engineered Yeast.

    abstract::Trihydroxycinnamoyl spermidines (THCSpd) are plant specialized metabolites with promising pharmacological activities as antifungals, antibacterial, antiviral, and antidepressant drugs. However, their characterization and potential pharmaceutical exploitation are greatly impaired by the sourcing of these compounds, res...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.0c00391

    authors: Perrin J,Kulagina N,Unlubayir M,Munsch T,Carqueijeiro I,Dugé de Bernonville T,De Craene JO,Clastre M,St-Pierre B,Giglioli-Guivarc'h N,Gagneul D,Lanoue A,Courdavault V,Besseau S

    更新日期:2021-01-15 00:00:00

  • Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli.

    abstract::We have previously engineered green/red and red/far red photoreversible E. coli phytochrome and cyanobacteriochrome (CBCR) two-component systems (TCSs) and utilized them to program tailor-made gene expression signals for gene circuit characterization. Here, we transport the UV-violet/green photoreversible CBCR TCS Uir...

    journal_title:ACS synthetic biology

    pub_type: 杂志文章

    doi:10.1021/acssynbio.6b00068

    authors: Ramakrishnan P,Tabor JJ

    更新日期:2016-07-15 00:00:00