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ACS Synthetic Biology

Ewa Maria Musiol-Kroll, Florian Zubeil, Thomas Schafhauser, Thomas Härtner, Andreas Kulik, John B McArthur, Irina Koryakina, Wolfgang Wohlleben, Stephanie Grond, Gavin J Williams, Sang Yup Lee, Tilmann Weber
During polyketide biosynthesis, acyltransferases (ATs) are the essential gatekeepers which provide the assembly lines with precursors and thus contribute greatly to structural diversity. Previously, we demonstrated that the discrete AT KirCII from the kirromycin antibiotic pathway accesses non-malonate extender units. Here, we exploit the promiscuity of KirCII to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by 'click' chemistry...
February 16, 2017: ACS Synthetic Biology
Aravind Natarajan, Charles H Haitjema, Robert Lee, Jason T Boock, Matthew P DeLisa
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...
February 16, 2017: ACS Synthetic Biology
Akihiro Furuya, Fuun Kawano, Takahiro Nakajima, Yoshibumi Ueda, Moritoshi Sato
We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein-protein interactions and genome editing, although the system could be improved further...
February 14, 2017: ACS Synthetic Biology
Yang Wang, Ralf Heermann, Kirsten Jung
Natural and synthetic scaffolds support enzyme organization in complexes, and they regulate their function and activity. Here we report that CipA and CipB, two small proteins that form protein crystalline inclusions (PCIs) in the cytoplasm of Photorhabdus luminescens, can be utilized as scaffolds to efficiently incorporate exogenous proteins into PCIs. We demonstrate that Cip-tagged GFP is assembled into fluorescent PCIs in P. luminescens and that in Escherichia coli Cip scaffolds can organize GFP or/and LacZ into bioactive PCIs, which could easily be isolated for in vitro catalysis...
February 10, 2017: ACS Synthetic Biology
Raj Chari, Nan Cher Yeo, Alejandro Chavez, George M Church
It has been possible to create tools to predict single guide RNA (sgRNA) activity in the CRISPR/Cas9 system derived from Streptococcus pyogenes due to the large amount of data that has been generated in sgRNA library screens. However, with the discovery of additional CRISPR systems from different bacteria, which show potent activity in eukaryotic cells, the approach of generating large data sets for each of these systems to predict their activity is not tractable. Here, we present a new guide RNA tool that can predict sgRNA activity across multiple CRISPR systems...
February 10, 2017: ACS Synthetic Biology
Patrick Basitta, Lucia Westrich, Manuela Rösch, Andreas Kulik, Bertolt Gust, Alexander Kristian Apel
The generation of novel secondary metabolites by reengineering or refactoring biochemical pathways is a rewarding but also challenging goal of synthetic biology. For this, the development of tools for the reconstruction of secondary metabolite gene clusters as well as the challenge of understanding the obstacles in this process is of great interest. The plug-and-play method AGOS (Artificial Gene Operon assembly System) was developed as a tool to consecutively assemble artificial gene operons into a destination vector and subsequently express them under the control of a de-repressed promoter in a Streptomyces host strain...
February 9, 2017: ACS Synthetic Biology
Alberto Sánchez-Pascuala, Víctor de Lorenzo, Pablo I Nikel
The Embden-Meyerhof-Parnas (EMP) pathway is generally considered to be the biochemical standard for glucose catabolism. Alas, its native genomic organization and the control of gene expression in Escherichia coli are both very intricate, which limits the portability of the EMP pathway to other biotechnologically important bacterial hosts that lack the route. In this work, the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (GlucoBrick) that enables their grouping in the form of functional modules at the user's will...
February 9, 2017: ACS Synthetic Biology
Xiaoyu Wang, Shuichi Hoshika, Raymond J Peterson, Myong-Jung Kim, Steven A Benner, Jason D Kahn
Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form additional nucleotide pairs that stabilize duplex DNA independent of the standard A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amount of molecular biology has been developed...
February 9, 2017: ACS Synthetic Biology
Yan Liang, Sarah Richardson, Jingwei Yan, Veronica T Benites, Clarabelle Cheng-Yue, Thu Tran, Jenny Mortimer, Aindrila Mukhopadhyay, Jay D Keasling, Henrik V Scheller, Dominique Loqué
Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. Meeting these challenges will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression-repression system to tightly control synthesis of transgene products...
February 8, 2017: ACS Synthetic Biology
Robert Sidney Cox Iii, James Alastair McLaughlin, Raik Gruenberg, Jake Beal, Anil Wipat, Herbert Sauro
As protein engineering becomes more sophisticated, practitioners increasingly need to share diagrams for communicating protein designs. To this end, we present a draft visual language, Protein Language, that describes the high-level architecture of an engineered protein with a few easy-to-draw glyphs, intended to be compatible with other biological diagram languages such as SBOL and SBGN. Protein Language con- sists of glyphs for representing important features (e.g., globular domains, recognition and localization sequences, sites of covalent modification, cleavage and catalysis), rules for composing these glyphs to represent complex architectures, and rules constraining the scaling and styling of diagrams...
February 7, 2017: ACS Synthetic Biology
Fabien Rideau, Chloé Le Roy, Elodie C T Descamps, Hélène Renaudin, Carole Lartigue, Cécile Bébéar
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 cloning and subsequent engineering of other mycoplasma genomes in yeast opens new possibilities for studies of the genomes of genetically intractable organisms. Here, we report the successful one-step cloning of the M...
February 7, 2017: ACS Synthetic Biology
Behnam Enghiad, Huimin Zhao
Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research. However, they have limited sequence specificity and availability. Here we report a Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by reannealing of the cleaved single stranded DNAs...
February 6, 2017: ACS Synthetic Biology
Ran Chao, Jing Liang, Ipek Tasan, Tong Si, Linyang Ju, Huimin Zhao
Transcription activator-like effector nuclease (TALEN) is a programmable genome editing tool with wide applications. Since TALENs perform cleavage of DNA as heterodimers, a pair of TALENs must be synthesized for each target genome locus. Conventionally, TALEN pairs are either expressed on separate vectors or synthesized separately and then subcloned to the same vector. Neither approach allows high-throughput construction of TALEN libraries for large-scale applications. Here we present a single-step assembly scheme to synthesize and express a pair of TALENs in a single-transcript format with the help of a P2A self-cleavage sequence...
February 2, 2017: ACS Synthetic Biology
Fabio Chizzolini, Michele Forlin, Noël Yeh Martín, Giuliano Berloffa, Dario Cecchi, Sheref S Mansy
Although RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, we investigated a series of ribosome binding sites (RBSs). Although RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including the GC content of the coding sequence...
February 2, 2017: ACS Synthetic Biology
Jing Wang, Le Yang, Xun Cui, Zhe Zhang, Lichun Dong, Ningzi Guan
We describe here a novel approach to enhance the transcription of target gene in cell-free systems by symmetrically introducing duplex aptamers upstream to T7 promoter in both the sense and antisense strands of double-stranded plasmids, which leads to the formation of a DNA bubble due to the none-complementary state of the ssDNA region harboring the aptamer sequences. With the presence of thrombins, the DNA bubble would be enlarged due to the binding of aptamers with thrombins. Consequently, the recognition region of the promoter contained in the DNA bubble can be more easily recognized and bound by RNA polymerases, and the separation efficiency of the unwinding region can also be significantly improved, leading to the enhanced expression of the target gene at the transcriptional level...
February 1, 2017: ACS Synthetic Biology
Ioannis Mougiakos, Elleke Fenna Bosma, Koen Weenink, Eric Michael Vossen, Kirsten Goijvaerts, John Van Der Oost, Richard van Kranenburg
Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR-Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e. Bacillus smithii, including a gene deletion, gene knock-out via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42°C and we employ the wide temperature growth range of B...
February 1, 2017: ACS Synthetic Biology
Hasan Baig, Jan Madsen
Constructing genetic logic circuits is an application of synthetic biology in which parts of the DNA of a living cell are engineered to perform a dedicated Boolean function triggered by an appropriate concentration of certain proteins or by different genetic components. These logic circuits work in a manner similar to electronic logic circuits, but they are much more stochastic and hence much harder to characterize. In this article, we introduce an approach to analyze the threshold value and timing of genetic logic circuits...
February 1, 2017: ACS Synthetic Biology
Xiaoxu Chen, Xiaoyu Yang, Yu Shen, Jin Hou, Xiaoming Bao
Malonyl-CoA is a precursor of a variety of compounds such as polyketides and flavonoids. In Saccharomyces cerevisiae, malonyl-CoA concentration is tightly regulated and therefore maintained at a very low level, limiting the production of malonyl-CoA-derived chemicals. Here we manipulated the phospholipid synthesis transcriptional regulators to control the malonyl-CoA levels and increase the downstream product. Through manipulating different regulators including Ino2p, Ino4p, Opi1p, and a series of synthetic Ino2p variants, combining with studying the inositol and choline effect, the engineered strain achieved a 9-fold increase of the titer of malonyl-CoA-derived product 3-hydroxypropionic acid, which is among the highest improvement relative to previously reported strategies...
January 29, 2017: ACS Synthetic Biology
Jie Wu, Aihua Deng, Qinyun Sun, Hua Bai, Zhaopeng Sun, Xiuling Shang, Yun Zhang, Qian Liu, Yong Liang, Shuwen Liu, Yongsheng Che, Tingyi Wen
Manipulating the bacterial genomes in an efficient manner is essential to biological and biotechnological research. Here, we reprogrammed the bacterial TA systems as the toxin counter-selectable cassette regulated by an antitoxin switch (TCCRAS) for genetic modifications in the extensively studied and utilized Gram-positive bacteria, B. subtilis and Corynebacterium glutamicum. In the five characterized type II TA systems, the RelBE complex can specifically and efficiently regulate cell growth and death by the conditionally controlled antitoxin RelB switch, thereby serving as a novel counter-selectable cassette to establish the TCCRAS system...
January 26, 2017: ACS Synthetic Biology
Svetlana V Harbaugh, Michael S Goodson, Kateri Dillon, Sarah Zabarnick, Nancy Kelley-Loughnane
Riboswitches are RNA-based 'sensors' that utilize chemically-induced structural changes in the 5'-untranslated region of mRNA to regulate expression of downstream genes. Coupling a specific riboswitch with a reporter gene system translates chemical detection by the cell into a quantifiable reporter protein signal. For the majority of reporter gene systems the readout signal is only expressed in the presence of the target analyte. This makes it difficult to determine the viability and localization of the uninduced biosensor when it is used for 'real-word' applications...
January 25, 2017: ACS Synthetic Biology
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