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ACS Synthetic Biology

Peng-Fei Xia, Guo-Chang Zhang, Berkley Walker, Seung-Oh Seo, Suryang Kwak, Jingjing Liu, Heejin Kim, Donald Ort, Shu-Guang Wang, Yong-Su Jin
Global climate change caused by the emission of anthropogenic greenhouse gasses (GHGs) is a grand challenge to humanity. To alleviate the trend, the consumption of fossil fuels needs to be largely reduced and alternative energy technologies capable of controlling GHG emissions are anticipated. In this study, we introduced a synthetic reductive pentose phosphate pathway (rPPP) into a xylose-fermenting Saccharomyces cerevisiae strain SR8 to achieve simultaneous lignocellulosic bioethanol production and carbon dioxide recycling...
October 17, 2016: ACS Synthetic Biology
Bryan S Der, Emerson Glassey, Bryan A Bartley, Casper Enghuus, Daniel B Goodman, D Benjamin Gordon, Christopher A Voigt, Thomas E Gorochowski
DNAplotlib ( is a computational toolkit for the programmable visualization of highly customizable, standards-compliant genetic designs. Functions are provided to aid with both visualization tasks and to extract and overlay associated experimental data. High-quality output is produced in the form of vector-based PDFs, rasterized images, and animated movies. All aspects of the rendering process can be easily customized or extended by the user to cover new forms of genetic part or regulation...
October 17, 2016: ACS Synthetic Biology
Andrew Kd Younger, Neil Chandra Dalvie, Austin G Rottinghaus, Joshua N Leonard
Efforts to engineer microbial factories have benefitted from mining biological diversity and high throughput synthesis of novel enzymatic ensembles, yet screening and optimizing metabolic pathways remain rate-limiting steps. Metabolite-responsive biosensors may help to address these persistent challenges by enabling the monitoring of metabolite levels in individual cells and metabolite-responsive feedback control. We are currently limited to naturally-evolved biosensors, which are insufficient for monitoring many metabolites of interest...
October 17, 2016: ACS Synthetic Biology
Joshua Fern, Dominic Scalise, Angelo Cangialosi, Dylan Howie, Leo Potters, Rebecca Schulman
Chemical circuits can coordinate elaborate sequences of events in cells and tissues, from the self-assembly of biological complexes to the sequence of embryonic development. However, autonomously directing the timing of events in synthetic systems using chemical signals remains challenging. Here we demonstrate that a simple synthetic DNA strand-displacement circuit can release target sequences of DNA into solution at a constant rate after a tunable delay that can range from hours to days. The rates of DNA release can be tuned to the order of 1-100 nM per day...
October 17, 2016: ACS Synthetic Biology
Pieter Coussement, David Bauwens, Jo Maertens, Marjan De Mey
Combinatorial engineering approaches are becoming increasingly popular, yet they are hindered by the lack of specialized techniques for both efficient introduction of sequence variability and assembly of numerous DNA parts, required for the construction of lengthy multigene pathways. In this contribution, we introduce a new combinatorial multigene pathway assembly scheme based on Single Strand Assembly (SSA) methods and Golden Gate Assembly, exploiting the strengths of both assembly techniques. With a minimum of intermediary steps and an accompanying set of well-characterized and ready-to-use genetic parts, the developed workflow allows effective introduction of various libraries and efficient assembly of multigene pathways...
October 11, 2016: ACS Synthetic Biology
Yihao Zhang, Long Qian, Weijia Wei, Yu Wang, Beining Wang, Pingping Lin, Wenchao Liu, Luze Xu, Xiang Li, Dongming Liu, Sida Cheng, Jiaofeng Li, Yixuan Ye, Hang Li, Xiaohan Zhang, Yiming Dong, Xuejin Zhao, Cuihua Liu, Haoqian M Zhang, Qi Ouyang, Chunbo Lou
We developed an in vitro DNA detection system using a pair of dCas9 proteins linked to split halves of luciferase. Luminescence was induced upon co-localization of the reporter pair to a ~44 bp target sequence defined by sgRNAs. We used the system to detect Mycobacterium tuberculosis DNA with high specificity and sensitivity. The re-programmability of dCas9 was further leveraged in an array design that accesses sequence information across the entire genome.
October 10, 2016: ACS Synthetic Biology
Alvaro R Lara, Karim E Jaén, Juan Carlos Sigala, Martina Mühlmann, Lars Regestein, Jochen Büchs
Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (Pvgb) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker protein, was used as a reporter...
October 7, 2016: ACS Synthetic Biology
Melchior du Lac, Andrew H Scarpelli, Andrew K D Younger, Declan G Bates, Joshua N Leonard
For many applications in microbial synthetic biology, optimizing a desired function requires careful tuning of the degree to which various genes are expressed. One challenge for predicting such effects or interpreting typical characterization experiments is that in bacteria such as E. coli, genome copy number varies widely across different phases and rates of growth, which also impacts how and when genes are expressed from different loci. While such phenomena are relatively well-understood at a mechanistic level, our quantitative understanding of such processes is essentially limited to ideal exponential growth...
October 7, 2016: ACS Synthetic Biology
Christian B Winiger, Ryan W Shaw, Myong-Jung Kim, Jennifer D Moses, Mariko F Matsuura, Steven A Benner
2,4-Diaminopyrimidine (trivially K) and imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (trivially X) form a nucleobase pair with Watson-Crick geometry as part of an artificially expanded genetic information system (AEGIS). Neither K nor X can form a Watson-Crick pair with any natural nucleobase. Further, neither K nor X has an accessible tautomeric form or a protonated/deprotonated state that can form a Watson-Crick pair with any natural nucleobase. In vitro experiments show how DNA polymerase I from E. coli manages replication of DNA templates with one K:X pair, but fails with templates containing two adjacent K:X pairs...
October 7, 2016: ACS Synthetic Biology
Jackson K B Cahn, Caroline A Werlang, Armin Baumschlager, Sabine Brinkmann-Chen, Stephen L Mayo, Frances H Arnold
The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity...
October 5, 2016: ACS Synthetic Biology
Min-Ji Heo, Hwi-Min Jung, Jaeyong Um, Sang-Woo Lee, Min-Kyu Oh
Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively produce n-butanol in a defined medium under micro-aerobic condition. The butanol synthetic pathway genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in metabolically engineered E. coli, resulting in 0.82 g/L butanol production. To increase butanol production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA. For this purpose, the 5'-untranslated region sequence of gltA encoding citrate synthase was designed using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9 genome editing method to reduce its expression level...
October 4, 2016: ACS Synthetic Biology
Takuya Matsumoto, Kou Furuta, Tsutomu Tanaka, Akihiko Kondo
We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling. Here, sortase A expression was controlled by the lac promoter to trigger metabolic channeling by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). We tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A...
October 4, 2016: ACS Synthetic Biology
Naoki Wada, Yasuhiro Kazuki, Kanako Kazuki, Toshiaki Inoue, Kiichi Fukui, Mitsuo Oshimura
Replication, segregation, gene expression and inheritance are essential features of all eukaryotic chromosomes. To delineate the extent of conservation of chromosome functions between humans and plants during evolutionary history, we have generated the first human cell line containing an Arabidopsis chromosome. The Arabidopsis chromosome was mitotically stable in hybrid cells following cell division, and initially existed as a translocated chromosome. During culture, the translocated chromosomes then converted to two types of independent plant chromosomes without human DNA sequences, with the reproducibility...
October 4, 2016: ACS Synthetic Biology
Kulika Chomvong, Eric Lin, Michael Blaisse, Abigail E Gillespie, Jamie H D Cate
Cellobiose phosphorylase (CBP) cleaves cellobiose-abundant in plant biomass-to glucose and glucose 1-phosphate. However, the pentose sugar xylose, also abundant in plant biomass, acts as a mixed-inhibitor and a substrate for the reverse reaction, limiting the industrial potential of CBP. Preventing xylose, which lacks only a single hydroxymethyl group relative to glucose, from binding to the CBP active site poses a spatial challenge for protein engineering, since simple steric occlusion cannot be used to block xylose binding without also preventing glucose binding...
October 3, 2016: ACS Synthetic Biology
Patrick M Caveney, S Elizabeth Norred, Charles W Chin, Jonathan B Boreyko, Brandon S Razooky, Scott T Retterer, C Patrick Collier, Michael L Simpson
Episodic gene expression, with periods of high expression separated by periods of no expression, is a pervasive biological phenomenon. This bursty pattern of expression draws from a finite reservoir of expression machinery in a highly time variant way, i.e., requiring no resources most of the time but drawing heavily on them during short intense bursts, that intimately links expression bursting and resource sharing. Yet, most recent investigations have focused on specific molecular mechanisms intrinsic to the bursty behavior of individual genes, while little is known about the interplay between resource sharing and global expression bursting behavior...
September 26, 2016: ACS Synthetic Biology
Bryn L Adams
Escherichia coli (E. coli) has played a pivotal role in the development of genetics and molecular biology as scientific fields. It is therefore not surprising that synthetic biology (SB) was built upon E. coli and continues to dominate the field. However, scientific capabilities have advanced from simple gene mutations to the insertion of rationally designed, complex synthetic circuits and creation of entirely synthetic genomes. The point is rapidly approaching where E. coli is no longer an adequate host for the increasingly sophisticated genetic designs of SB...
September 26, 2016: ACS Synthetic Biology
Ryan M Phelan, Daniel Sachs, Shayne J Petkiewicz, Jesus F Barajas, Jacquelyn M Blake-Hedges, Mitchell G Thompson, Amanda Reider Apel, Blake J Rasor, Leonard Katz, Jay D Keasling
Streptomyces have a rich history as producers of important natural products and this genus of bacteria has recently garnered attention for its potential applications in the broader context of synthetic biology. However, the dearth of genetic tools available to control and monitor protein production precludes rapid and predictable metabolic engineering that is possible in hosts such as Escherichia coli or Saccharomyces cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor protein production in this host...
September 26, 2016: ACS Synthetic Biology
Jordan W Monk, Sean P Leonard, Colin W Brown, Michael J Hammerling, Catherine Mortensen, Alejandro E Gutierrez, Nathan Y Shin, Ella Watkins, Dennis M Mishler, Jeffrey E Barrick
By introducing engineered tRNA and aminoacyl-tRNA synthetase pairs into an organism, its genetic code can be expanded to incorporate nonstandard amino acids (nsAAs). The performance of these orthogonal translation systems (OTSs) varies greatly, however, with respect to the efficiency and accuracy of decoding a reassigned codon as the nsAA. To enable rapid and systematic comparisons of these critical parameters, we developed a toolkit for characterizing any Escherichia coli OTS that reassigns the amber stop codon (TAG)...
September 20, 2016: ACS Synthetic Biology
Masahiro Kanno, Shota Atsumi
Cyanobacteria have attracted much attention as a means to directly recycle carbon dioxide into valuable chemicals that are currently produced from petroleum. However, the titers and productivities achieved are still far below the level required in industry. To make a more industrially applicable production scheme, glycerol, a byproduct of biodiesel production, can be used as an additional carbon source for photomixotrophic chemical production. Glycerol is an ideal candidate due to its availability and low-cost...
September 19, 2016: ACS Synthetic Biology
Akiyoshi Higo, Atsuko Isu, Yuki Fukaya, Toru Hisabori
In recent years, studies on the development of gene regulation tools in cyanobacteria have been extensively conducted towards efficient production of valuable chemicals. However, there is considerable scope for improving the economic feasibility of production. To improve a recently reported gene induction system using anhydrotetracycline (aTc)-TetR and an endogenous gene repression system using small antisense RNA in the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena), we constructed a positive feedback loop, in which gfp and a small antisense RNA for tetR are controlled by an aTc-inducible promoter...
September 16, 2016: ACS Synthetic Biology
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