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ACS Synthetic Biology

Karolina Z Kalbarczyk, Emily J Mazeau, Kent M Rapp, Nicholas Marchand, Mattheos A G Koffas, Cynthia H Collins
Recent environmental concerns have intensified the need to develop systems to degrade waste biomass for use as an inexpensive carbon source for microbial chemical production. Current approaches to biomass utilization rely on pretreatment processes that include expensive enzymatic purification steps for the requisite cellulases. We aimed to engineer a synthetic microbial community to synergistically degrade cellulose by compartmentalizing the system with multiple specialized Bacillus megaterium strains. EGI1, an endoglucanase, and Cel9AT, a multimodular cellulase, were targeted for secretion from B...
September 18, 2018: ACS Synthetic Biology
Ankanahalli N Nanjaraj Urs, Yiling Hu, Pengwei Li, Zhiguang Yuchi, Yihua Chen, Yan Zhang
Rose has been entwined with human culture and history. "Blue rose" in English signifies unattainable hope or an impossible mission as it does not exist naturally and is not breedable regardless of centuries of effort by gardeners. With the knowledge of genes and enzymes involved in flower pigmentation and modern genetic technologies, synthetic biologists have undertaken the challenge of producing blue rose by engineering the complicated vacuolar flavonoid pigmentation pathway and resulted in a mauve-colored rose...
September 18, 2018: ACS Synthetic Biology
Teresa Cordero, Arantxa Rosado, Eszter Majer, Alfonso Jaramillo, Guillermo Rodrigo, José-Antonio Daròs
Due to autotrophic growing capacity and extremely rich secondary metabolism, plants should be preferred targets of synthetic biology. However, developments in plants usually run below those in other taxonomic groups. In this work we engineered genetic circuits capable of logic YES, OR and AND Boolean computation in plant tissues with a visual output signal. The circuits, which are deployed by means of Agrobacterium tumefaciens, perform with the conditional activity of the MYB transcription factor Rosea1 from Antirrhinum majus inducing the accumulation of anthocyanins, plant endogenous pigments that are directly visible to the naked eye or accurately quantifiable by spectrophotometric analysis...
September 18, 2018: ACS Synthetic Biology
Emanuel M Avrahami, Shahar Levi, Eyal Zajfman, Clil Regev, Oshrit Ben-David, Eyal Arbely
Lysine deacetylases (KDACs) are enzymes that catalyze the hydrolysis of acyl groups from acyl-lysine residues. The recent identification of thousands of putative acylation sites, including specific acetylation sites, created an urgent need for biochemical methodologies aimed at better characterizing KDAC-substrate specificity and evaluating KDACs activity. To address this need, we utilized genetic code expansion technology to coexpress site-specifically acylated substrates with mammalian KDACs, and study substrate recognition and deacylase activity in live Escherichia coli...
September 18, 2018: ACS Synthetic Biology
Alice Checcucci, George diCenzo, Veronica Ghini, Marco Bazzicalupo, Anke Becker, Francesca Decorosi, Johannes Döhlemann, Camilla Fagorzi, Turlough M Finan, Marco Fondi, Claudio Luchinat, Paola Turano, Tiziano Vignolini, Carlo Viti, Alessio Mengoni
Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbour a multipartite genome composed of more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacterium Sinorhizobium meliloti...
September 17, 2018: ACS Synthetic Biology
Aziz M Al'Khafaji, Daniel Deatherage, Amy Brock
Lineage tracking delivers essential quantitative insight into dynamic, probabilistic cellular processes, such as somatic tumor evolution and differentiation. Methods for high diversity lineage quantitation rely on sequencing a population of DNA barcodes. However, manipulation of specific individual lineages is not possible with this approach. To address this challenge, we developed a functionalized lineage tracing tool, Control of Lineages by Barcode Enabled Recombinant Transcription (COLBERT), that enables high diversity lineage tracing and lineage-specific manipulation of gene expression...
September 17, 2018: ACS Synthetic Biology
Yuan Yu, Pengcheng Chang, Huan Yu, Huiyong Ren, Danning Hong, Zeyan Li, Ying Wang, Hao Song, Yi-Xin Huo, Chun Li
α-amyrin is a plant-derived pentacyclic triterpenoid, with a lot of important physiological and pharmacological activities. The formation of α-amyrin from (3S)-2,3-oxidosqualene is catalyzed by α-amyrin synthase (α-AS), a member of the oxidosqualene cyclase (OSC) protein family. However, α-amyrin is not yet commercially developed due to its extremely low productivity in plant. The engineered Saccharomyces cerevisiae with efficient α-amyrin production pathway could be used as an alternative and sustainable solution to produce α-amyrin from renewable raw materials...
September 14, 2018: ACS Synthetic Biology
Oliver Purcell, Jicong Cao, Isaak E Müller, Ying-Chou Chen, Timothy K Lu
RNA interference (RNAi) is widely used as a research tool for studying biological systems and implementing artificial genetic circuits that function by modulating RNA concentrations. Here we engineered Saccharomyces cerevisiae containing a heterologous Saccharomyces castelli RNAi system as a test-bed for RNAi-based circuits. Unlike prior approaches, we describe a strategy that leverages repeat-structured siRNA precursors with incrementally sized stems formed from 23 bp-repeats to achieve modular RNAi-based gene regulation...
September 14, 2018: ACS Synthetic Biology
Daniel J Wiegand, Henry H Lee, Nili Ostrov, George M Church
The fast growing bacterium Vibrio natriegens is an emerging microbial host for biotechnology. Harnessing its productive cellular components may offer a compelling platform for rapid protein production and prototyping of metabolic pathways or genetic circuits. Here, we report the development of a V. natriegens cell-free expression system. We devised a simplified crude extract preparation protocol and achieved >260 μg/mL of superfolder GFP in a small-scale batch reaction after 3 h. Culturing conditions, including growth media and cell density, significantly affect translation kinetics and protein yield of extracts...
September 12, 2018: ACS Synthetic Biology
David Öling, Lina Lawenius, William Shaw, Sonya Clark, Ross Kettleborough, Tom Ellis, Niklas Larsson, Mark Wigglesworth
Site saturation mutagenesis (SSM) is a powerful mutagenesis strategy for protein engineering and directed evolution experiments. However, limiting factors using this method are either biased representation of variants, or limiting library size. To overcome these hurdles, we generated large scale targeted synthetic SSM libraries using massively parallel oligonucleotide synthesis and benchmarked this against an error-prone (epPCR) library. The yeast glucose activated GPCR-Gpr1 was chosen as a prototype to evolve novel glucose sensors...
September 12, 2018: ACS Synthetic Biology
Benjamin Jester, Christine E Tinberg, Matthew S Rich, David Baker, Stanley Fields
Biosensors are important components of many synthetic biology and metabolic engineering applications. Here, we report a second generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization. The biosensors, comprising one ligand-binding domain monomer fused to a DNA-binding domain and another fused to a transcriptional activation domain, activate reporter gene expression in response to steroid binding and receptor dimerization...
September 11, 2018: ACS Synthetic Biology
Kei Motomura, Kosuke Sano, Satoru Watanabe, Akihiro Kanbara, Abdel-Hady Gamal Nasser, Takeshi Ikeda, Takenori Ishida, Hisakage Funabashi, Akio Kuroda, Ryuichi Hirota
Recent progress in genetic engineering and synthetic biology have greatly expanded the production capabilities of cyanobacteria, but concerns regarding biosafety issues and the risk of contamination of cultures in outdoor culture conditions remain to be resolved. With this dual goal in mind, we applied the recently established biological containment strategy based on phosphite (H3 PO3 , Pt) dependency to the model cyanobacterium Synechococcus elongatus PCC 7942 ( Syn 7942). Pt assimilation capability was conferred on Syn 7942 by the introduction of Pt dehydrogenase (PtxD) and hypophosphite transporter (HtxBCDE) genes that allow the uptake of Pt, but not phosphate (H3 PO4 , Pi)...
September 11, 2018: ACS Synthetic Biology
Jakeb M Reis, Xiuling Xu, Sherin McDonald, Ryan Woloschuk, Anna Jaikaran, Frederick Vizeacoumar, G Andrew Woolley, Maruti Uppalapati
Nature provides an array of proteins that change conformation in response to light. The discovery of a complementary array of proteins that bind only the light-state or dark-state conformation of their photoactive partner proteins would allow each light-switchable protein to be used as an optogenetic tool to control protein-protein interactions. However, as many photoactive proteins have no known binding partner, the advantages of optogenetic control - precise spatial and temporal resolution - are currently restricted to a few well-defined natural systems...
September 11, 2018: ACS Synthetic Biology
Joseph T Decker, Matthew S Hall, Beatriz Peñalver-Bernabé, Rachel B Blaisdell, Lauren N Liebman, Jacqueline S Jeruss, Lonnie D Shea
Dynamic systems biology aims to identify the molecular mechanisms governing cell fate decisions through the analysis of living cells. Large scale molecular information from living cells can be obtained from reporter constructs that provide activities for either individual transcription factors or multiple factors binding to the full promoter following CRISPR/Cas9 directed insertion of luciferase. In this report, we investigated the design criteria to obtain reporters that are specific and responsive to transcription factor (TF) binding and the integration of TF binding activity with genetic reporter activity...
September 10, 2018: ACS Synthetic Biology
Benjamin J Des Soye, Samuel R Davidson, Matthew T Weinstock, Daniel G Gibson, Michael C Jewett
A new wave of interest in cell-free protein synthesis (CFPS) systems has shown their utility for producing proteins at high titers, establishing genetic regulatory element libraries ( e.g., promoters, ribosome binding sites) in nonmodel organisms, optimizing biosynthetic pathways before implementation in cells, and sensing biomarkers for diagnostic applications. Unfortunately, most previous efforts have focused on a select few model systems, such as Escherichia coli. Broadening the spectrum of organisms used for CFPS promises to better mimic host cell processes in prototyping applications and open up new areas of research...
September 6, 2018: ACS Synthetic Biology
Pierre Crozet, Francisco J Navarro, Felix Willmund, Payam Mehrshahi, Kamil Bakowski, Kyle J Lauersen, Maria-Esther Pérez-Pérez, Pascaline Auroy, Aleix Gorchs Rovira, Susana Sauret-Gueto, Justus Niemeyer, Benjamin Spaniol, Jasmine Theis, Raphael Trösch, Lisa-Desiree Westrich, Konstantinos Vavitsas, Thomas Baier, Wolfgang Hübner, Felix de Carpentier, Mathieu Cassarini, Antoine Danon, Julien Henri, Christophe H Marchand, Marcello de Mia, Kevin Sarkissian, David C Baulcombe, Gilles Peltier, José-Luis Crespo, Olaf Kruse, Poul-Erik Jensen, Michael Schroda, Alison G Smith, Stéphane D Lemaire
Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii...
September 5, 2018: ACS Synthetic Biology
Sun-Young Kang, Kyung Taek Heo, Young-Soo Hong
One of the optimization strategies of an artificial biosynthetic metabolic flux with a multienzyme pathway is when the enzyme concentrations are present at the appropriate ratios rather than at their maximum expression. Thus, many recent research efforts have focused on the development of tools that fine-tune the enzyme expression, and these research efforts have facilitated the search for the optimum balance between pathway expression and cell viability. However, the rational approach has some limitations in finding the most optimized expression ratio in in vivo systems...
September 4, 2018: ACS Synthetic Biology
Xiaopei Xu, Zhaoxia Du, Renmei Liu, Ting Li, Yuzheng Zhao, Xianjun Chen, Yi Yang
Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In this study, we developed a single-component light-switchable gene expression system for yeast cells, termed yLightOn system. The yLightOn system is independent of exogenous cofactors, and exhibits more than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics, and high spatial resolution. We demonstrated the usefulness of the yLightOn system in regulating cell growth and cell cycle by stringently controlling the expression of His3 and ΔN Sic1 genes, respectively...
September 4, 2018: ACS Synthetic Biology
Mohammad ElGamacy, Murray Coles, Patrick Ernst, Hongbo Zhu, Marcus D Hartmann, Andreas Plückthun, Andrei N Lupas
Designing proteins with novel folds remains a major challenge, as the biophysical properties of the target fold are not known a priori and no sequence profile exists to describe its features. Therefore, most computational design efforts so far have been directed toward creating proteins that recapitulate existing folds. Here we present a strategy centered upon the design of novel intramolecular interfaces that enables the construction of a target fold from a set of starting fragments. This strategy effectively reduces the amount of computational sampling necessary to achieve an optimal sequence, without compromising the level of topological control...
September 4, 2018: ACS Synthetic Biology
Martina Garni, Tomaz Einfalt, Roland Goers, Cornelia G Palivan, Wolfgang Meier
Compartmentalization of functional biological units, cells, and organelles serves as an inspiration for the development of biomimetic materials with unprecedented properties and applications in biosensing and medicine. Because of the complexity of cells, the design of ideal functional materials remains a challenge. An elegant strategy to obtain cell-like compartments as novel materials with biofunctionality is the combination of synthetic micrometer-sized giant unilamellar vesicles (GUVs) with biomolecules because it enables studying the behavior of biomolecules and processes within confined cavities...
September 4, 2018: ACS Synthetic Biology
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