Abstract：
:The extracellular expression of recombinant proteins using laboratory strains of Escherichia coli is now routinely achieved using naturally secreted substrates, such as YebF or the osmotically inducible protein Y (OsmY), as carrier molecules. However, secretion efficiency through these pathways needs to be improved for most synthetic biology and metabolic engineering applications. To address this challenge, we developed a generalizable survival-based selection strategy that effectively couples extracellular protein secretion to antibiotic resistance and enables facile isolation of rare mutants from very large populations (i.e., 1010-12 clones) based simply on cell growth. Using this strategy in the context of the YebF pathway, a comprehensive library of E. coli single-gene knockout mutants was screened and several gain-of-function mutations were isolated that increased the efficiency of extracellular expression without compromising the integrity of the outer membrane. We anticipate that this user-friendly strategy could be leveraged to better understand the YebF pathway and other secretory mechanisms-enabling the exploration of protein secretion in pathogenesis as well as the creation of designer E. coli strains with greatly expanded secretomes-all without the need for expensive exogenous reagents, assay instruments, or robotic automation.
journal_name
ACS Synth Bioljournal_title
ACS synthetic biologyauthors
Natarajan A,Haitjema CH,Lee R,Boock JT,DeLisa MPdoi
10.1021/acssynbio.6b00366subject
Has Abstractpub_date
2017-05-19 00:00:00pages
875-883issue
5issn
2161-5063journal_volume
6pub_type
杂志文章abstract：:Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on prope...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00248
更新日期：2017-02-17 00:00:00
abstract：:Metabolic engineering of Escherichia coli was performed to construct a 100% rationally engineered strain capable of overproducing L-isoleucine, an important branched-chain amino acid. The thrABC (encoding L-threonine biosynthetic enzymes), ilvA (encoding feedback-resistant threonine dehydratase), ilvIH (encoding feedb...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb300071a
更新日期：2012-11-16 00:00:00
abstract：:One limitation of gene-directed enzyme prodrug therapy (GDEPT) is the difficulty in selectively and efficiently transducing cancer cells with the gene encoding a prodrug-converting enzyme. To circumvent this issue, we sought to move the selectivity from the gene delivery level to the protein level. We developed fusion...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00036
更新日期：2019-05-17 00:00:00
abstract：:Cell-free systems, as part of the synthetic biology field, have become a critical platform in biological studies. However, there is a lack of research into developing a switch for a dynamical control of the transcriptional and translational process. The optogenetic tool has been widely proven as an ideal control switc...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00211
更新日期：2020-08-21 00:00:00
abstract：:In many biotechnological applications, it is useful for gene expression to be regulated by multiple signals, as this allows the programming of complex behavior. Here we implement, in Escherichia coli, a system that compares the concentration of two signal molecules, and tunes GFP expression proportionally to their rel...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00109
更新日期：2017-10-20 00:00:00
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-gu...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00299
更新日期：2020-09-18 00:00:00
abstract：:Nanostructures formed by self-assembled peptides have been increasingly exploited as functional materials for a wide variety of applications, from biotechnology to energy. However, it is sometimes challenging to assemble free short peptides into functional supramolecular structures, since not all peptides have the abi...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00415
更新日期：2020-12-18 00:00:00
abstract：:Cell-free systems provide a versatile platform for the development of low-cost, easy-to-use sensors for diverse analytes. However, sensor affinity dictates response sensitivity, and improving binding affinity can be challenging. Here, we describe efforts to address this problem while developing a biosensor for vitamin...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00429
更新日期：2020-02-21 00:00:00
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
更新日期：2019-10-18 00:00:00
abstract：:Mycoplasma hominis is a minimal human pathogen that is responsible for genital and neonatal infections. Despite many attempts, there is no efficient genetic tool to manipulate this bacterium, limiting most investigations of its pathogenicity and its uncommon energy metabolism that relies on arginine. The recent clonin...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00379
更新日期：2017-05-19 00:00:00
abstract：:The availability of different host chassis will greatly expand the range of applications in synthetic biology. Members of the Acetobacteraceae family of Gram-negative bacteria form an attractive class of nonmodel microorganisms that can be exploited to produce industrial chemicals, food and beverage, and biomaterials....
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00168
更新日期：2019-04-19 00:00:00
abstract：:Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We id...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb500363y
更新日期：2015-07-17 00:00:00
abstract：:A gene-directed chemical communication pathway between synthetic protocell signaling transmitters (lipid vesicles) and receivers (proteinosomes) was designed, built and tested using a bottom-up modular approach comprising small molecule transcriptional control, cell-free gene expression, porin-directed efflux, substra...
journal_title：ACS synthetic biology
pub_type： 信件
doi：10.1021/acssynbio.7b00306
更新日期：2018-02-16 00:00:00
abstract：:For infectious diseases, rapid and accurate identification of the pathogen is critical for effective management and treatment, but diagnosis remains challenging, particularly in resource-limited areas. Methods that accurately detect pathogen nucleic acids can provide robust, accurate, rapid, and ultrasensitive technol...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00507
更新日期：2020-06-19 00:00:00
abstract：:Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00166
更新日期：2017-02-17 00:00:00
abstract：:Multidrug resistance (MDR) is a globally relevant problem that requires novel approaches. Two-component systems are a promising, yet untapped target for novel antibacterials. They are prevalent in bacteria and absent in mammals, and their activity can be modulated upon perception of various stimuli. Screening pre-exis...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00288
更新日期：2017-07-21 00:00:00
abstract：:Living organisms employ endogenous negative feedback loops to maintain homeostasis despite environmental fluctuations. A pressing open challenge in Synthetic Biology is to design and implement synthetic circuits to control host cells' behavior, in order to regulate and maintain desired conditions. To cope with the hig...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00220
更新日期：2017-03-17 00:00:00
abstract：:The ATLAS of Biochemistry is a repository of both known and novel predicted biochemical reactions between biological compounds listed in the Kyoto Encyclopedia of Genes and Genomes (KEGG). ATLAS was originally compiled based on KEGG 2015, though the number of KEGG reactions has increased by almost 20 percent since the...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00052
更新日期：2020-06-19 00:00:00
abstract：:Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each conde...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00176
更新日期：2017-01-20 00:00:00
abstract：:Recent advances in the design and construction of synthetic multicelled systems in E. coli and S. cerevisiae suggest that it may be possible to implement sophisticated distributed algorithms with these relatively simple organisms. However, existing design frameworks for synthetic biology do not account for the unique ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb300034m
更新日期：2012-08-17 00:00:00
abstract：:RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA),...
journal_title：ACS synthetic biology
pub_type： 信件
doi：10.1021/acssynbio.7b00237
更新日期：2018-03-16 00:00:00
abstract：:While inducing agents are often used to redirect resources from growth and proliferation toward product outputs, they can be prohibitively expensive on the industrial scale. Previously, we developed an autonomously guided protein production system based on the rewiring of E. coli's native quorum sensing (QS) signal tr...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00261
更新日期：2016-09-16 00:00:00
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
更新日期：2019-08-16 00:00:00
abstract：:Melatonin is a commercially attractive tryptophan-derived hormone. Here we describe a bioprocess for the production of melatonin using Escherichia coli to high titers. The first engineered strain produced 0.13 g/L of melatonin from tryptophan under fed-batch fermentation conditions. A 4-fold improvement on melatonin t...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00065
更新日期：2020-06-19 00:00:00
abstract：:Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00388
更新日期：2017-07-21 00:00:00
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
更新日期：2015-09-18 00:00:00
abstract：:Improved plants are necessary to meet human needs. Agrobacterium-mediated transformation is the most common method used to rewire plant capabilities. For plant gene delivery, DNA constructs are assembled into binary T-DNA vectors that rely on broad host range origins for bacterial replication. Here we present pLX vect...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00354
更新日期：2017-10-20 00:00:00
abstract：:The ability of RNA to sense, regulate, and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00041
更新日期：2018-07-20 00:00:00
abstract：:Bacterial biofilms are three-dimensional networks of cells entangled in a self-generated extracellular polymeric matrix composed of proteins, lipids, polysaccharides, and nucleic acids. Biofilms can establish themselves on virtually any accessible surface and lead to varying impacts ranging from infectious diseases to...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00192
更新日期：2019-07-19 00:00:00
abstract：:In this work, we develop methodologies for analyzing and cross comparing metabolic models. We investigate three important metabolic networks to discuss the complexity of biological organization of organisms, modeling, and system properties. In particular, we analyze these metabolic networks because of their biotechnol...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb300102k
更新日期：2013-05-17 00:00:00
