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ACS Synthetic Biology

Yuchang Luo, Qinqin Zhao, Qian Liu, Yan Feng
2-Amino-1,3-propanediol (2-APD) is a chemical building block for the production of various value-added pharmaceuticals. However, the current manufacture of 2-APD predominantly relies on chemical processes by utilizing fossil fuel-derived and highly explosive raw materials. Herein, we established an artificial biosynthetic pathway for converting glucose to 2-APD in a metabolically engineered Escherichia coli. This artificial pathway employs an engineered heterogeneous aminotransferase RtxA for diverting dihydroxyacetone phosphate to generate 2-APD phosphate and an endogenous phosphatase for converting it into the target product 2-APD...
February 19, 2019: ACS Synthetic Biology
Duo Liu, Hong Liu, Hao Qi, Xue-Jiao Guo, Bin Jia, Jin-Lai Zhang, Ying-Jin Yuan
Synthetic chimeric biological system offers opportunities to illuminate principles of designing life and a primary step is constructing synthetic chimeric pathways. Here, we constructed yeast chimeric pathways by transferring the genes from Saccharomyces cerevisiae pathways into another budding yeast Yarrowia lipolytica for in vivo assembly. We efficiently diversified gene option, combination, localization order and copy number as expected. Convergence of two yeast pathways, especially mevalonic acid (MVA) pathways, remarkably enhanced synthesis of a lipophilic terpene, lycopene...
February 19, 2019: ACS Synthetic Biology
Xenia Meshik, Patrick O'Neill, Narasimhan Gautam
Cells experience physical deformations to the plasma membrane that can modulate cell behaviors like migration. Understanding the molecular basis for how physical cues affect dynamic cellular responses requires new approaches that can physically perturb the plasma membrane with rapid, reversible, subcellular control. Here we present an optogenetic approach based on light-inducible dimerization that alters plasma membrane properties by recruiting cytosolic proteins at high concentrations to a target site. Surprisingly, this polarized accumulation of proteins in a cell induces directional amoeboid migration in the opposite direction...
February 14, 2019: ACS Synthetic Biology
Yoshiki Moriizumi, Kazuhito V Tabata, Daisuke Miyoshi, Hiroyuki Noji
Molecular crowding is receiving great attention in cell-free synthetic biology because molecular crowding is a critical feature of natural cell discrimination from artificial cells. Further, it has significant and generic influences on biomolecular functions. Although there are reports on how the macromolecular crowder reagents affect cell-free systems such as transcription and translation, the second class of molecular crowder reagents with low molecular weight, osmolyte, was much less studied in cell-free systems...
February 14, 2019: ACS Synthetic Biology
Yucheng Guo, Chen Bao, Dacheng Ma, Yubing Cao, Yanda Li, Zhen Xie, Shao Li
Tumorigenesis is a complex process that is driven by a combination of the networks of genes and environmental factors; however, efficient approaches for identifying functional networks that are perturbed by the process of tumorigenesis is poorly understood. Then, the identification of functional synergistic modules and pathways is often limited by computational method and experiment measures. Here, we propose an integrated network-based approach for the systematic discovery of functional synergistic modules that are causal determinants of inflammation-induced tumorigenesis, by prioritizing candidate genes of integrating clinical-based and network-based genome-wide gene prediction methods and identify functional synergistic modules based on combinatorial CRISPR-Cas9 screens...
February 14, 2019: ACS Synthetic Biology
Emily E Wrenbeck, Matthew A Bedewitz, Justin R Klesmith, Syeda Noshin, Cornelius S Barry, Timothy A Whitehead
Enzymes are the ultimate entities responsible for chemical transformations in natural and engineered biosynthetic pathways. However, many natural enzymes suffer from suboptimal functional expression due to poor intrinsic protein stability. Further, stability enhancing mutations often come at the cost of impaired function. Here we demonstrate an automated protein engineering strategy for stabilizing enzymes while retaining catalytic function using deep mutational scanning coupled to multiple-filter based screening and combinatorial mutagenesis...
February 8, 2019: ACS Synthetic Biology
Byungjin Hwang, Sunghoon Heo, Namjin Cho, Hanna Seo, Duhee Bang
A typical molecular cloning procedure requires Sanger sequencing for sequence validation, which is cost-prohibitive and labor-intensive for large-scale clone analysis in genotype-phenotype studies. Here we present the cost-effective clone analysis platform TnClone, which uses next-generation sequencing based on Tn5 tagmentation to rapidly analyze a large number of clones from cell lysates. This method bypasses the extensive plasmid purification step. We also developed a user-friendly graphical user interface and provided general guidelines for conducting validation experiments...
February 6, 2019: ACS Synthetic Biology
Soo-Jung Kim, Matthew Leong, Matthew B Amrofell, Young Je Lee, Tae Seok Moon
The toehold switch consists of a cis-repressing switch RNA hairpin and a trans-acting trigger RNA. The binding of the trigger RNA to an unpaired toehold sequence of the switch hairpin allows for a branch migration process, exposing the start codon and ribosome binding site for translation initiation. In this work, we demonstrate that responses of toehold switches can be modulated by introducing an inhibitory hairpin that shortens the unpaired toehold region length. First, we investigated the effect of the toehold region length on output gene expression, showing that the second trigger RNA, which binds to the inhibitory hairpin, is necessary for output gene activation when the hairpin-to-hairpin spacing is short...
February 5, 2019: ACS Synthetic Biology
Stefan Hoffmann, Nan Hao, Keith E Shearwin, Katja M Arndt
Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. Controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modelled...
February 5, 2019: ACS Synthetic Biology
Tian Tian, Jing Wei Kang, Aram Kang, Taek Soon Lee
CRISPR interference (CRISPRi) via target guide RNA (gRNA) arrays and a deactivated Cas9 (dCas9) protein has been shown to simultaneously repress expression of multiple genomic DNA loci. By knocking down endogenous genes in competing pathways, CRISPRi technology can be utilized to redirect metabolic flux toward target metabolite. In this study, we constructed a CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway...
February 5, 2019: ACS Synthetic Biology
Maike Kortmann, Christina Mack, Meike Baumgart, Michael Bott
Pyruvate carboxylase is an anaplerotic carbon dioxide-fixing enzyme replenishing the tricarboxylic acid cycle with oxaloacetate during growth on sugars. In this study, we applied a lysine biosensor to identify pyruvate carboxylase variants in Corynebacterium glutamicum that enabled improved L-lysine production from glucose. A suitable reporter strain was transformed with a pyc gene library created by error-prone PCR and screened by fluorescence-activated cell sorting (FACS) for cells with increased fluorescence triggered by an elevated cytoplasmic lysine concentration...
February 1, 2019: ACS Synthetic Biology
Peng Geng, Sean P Leonard, Dennis M Mishler, Jeffrey E Barrick
Mobile genetic elements drive evolution by disrupting genes and rearranging genomes. Eukaryotes have evolved epigenetic mechanisms, including DNA methylation and RNA interference, that silence mobile elements and thereby preserve the integrity of their genomes. We created an artificial reprogrammable epigenetic system based on CRISPR interference to give engineered bacteria a similar line of defense against transposons and other selfish elements in their genomes. We demonstrate that this CRISPR interference with mobile elements (CRISPRi-ME) approach can be used to simultaneously repress two different transposon families in Escherichia coli, thereby increasing the evolutionary stability of costly protein expression...
January 31, 2019: ACS Synthetic Biology
Miaomiao Wang, Huimin Yu, Zhongyao Shen
The antisense RNA (asRNA) strategy is commonly used to block protein expression and downregulate the contents of metabolites in several microorganisms. Here, we show that the asRNA strategy can also be used to block gfp expression in Bacillus subtilis TS1726, which could further be utilized in the identification of new genes and functions. Via application of this strategy, biotin carboxylase II encoded by yngH (GeneID 939474) was identified to play a more significant role in maintaining acetyl-CoA carboxylase (ACCase) activity and enhancing surfactin synthesis compared to those of other ACCase subunits...
January 31, 2019: ACS Synthetic Biology
Yu Ping, Xiaodong Li, Baofu Xu, Wei Wei, Wenping Wei, Guoyin Kai, Zhihua Zhou, Youli Xiao
N-Methylpyrrolinium-derived alkaloids like tropane alkaloids, nicotine, and calystegines are valuable plant source specialized metabolites bearing pharmaceutical or biological activity. Microbial synthesis of the critical common intermediate N-methylpyrrolinium would allow for sustainable production of N-methylpyrrolinium-derived alkaloids. Here, we achieve the production of N-methylpyrrolinium both in Escherichia coli and in Saccharomyces cerevisiae by employing the biosynthetic genes derived from three different plants...
January 31, 2019: ACS Synthetic Biology
Masoud Norouzi, Andrew R Pickford, Louise E Butt, Helen A Vincent, Anastasia J Callaghan
The development of programmable regulators that precisely and predictably control gene expression is a major goal of synthetic biology. Consequently, rapid high-throughput biochemical methods capable of quantitatively analyzing all components of gene expression would be of value in the characterization and optimization of regulator performance. In this study we demonstrate a novel application of RNA arrays, involving the production of reporter-protein arrays, to gene expression analysis. This method enables simultaneous quantification of both the transcription and post-transcription/translation components of gene expression, and it also allows the assessment of the orthogonality of multiple regulators...
January 30, 2019: ACS Synthetic Biology
Yongkun Lv, Harley Edwards, Jingwen Zhou, Peng Xu
Conventional plasmid-based gene expression tends to introduce genetic instability and gene copy number variations that lead to degenerated production. The limited number of auxotrophic markers in Yarrowia lipolytica also restricts our ability to perform iterative genetic modifications and manipulate long gene clusters. To overcome these limitations, we combined the high recombination efficiency of the Cre-loxP system and the high integration rate of 26s rDNA, and developed a versatile framework to iteratively integrate multi-copy metabolic pathways in Y...
January 29, 2019: ACS Synthetic Biology
Jens Hausner, Michael Jordan, Christian Otten, Sylvestre Marillonnet, Daniela Buettner
Type III secretion (T3S) systems are essential pathogenicity factors of most Gram-negative bacteria and translocate effector proteins into plant or animal cells. T3S systems can, therefore, be used as tools for protein delivery into eukaryotic cells, for instance after transfer of the T3S gene cluster into non-pathogenic recipient strains. Here, we report the modular cloning of the T3S gene cluster from the plant-pathogenic bacterium Xanthomonas euvesicatoria. The resulting multi-gene construct encoded a functional T3S system and delivered effector proteins into plant cells...
January 29, 2019: ACS Synthetic Biology
Ling Li, Maiko Furubayashi, Takuya Hosoi, Takahiro Seki, Yusuke Otani, Shigeko Kawai-Noma, Kyoichi Saito, Daisuke Umeno
Longer-chain carotenoids have interesting physiological and electronic/photonic properties due to their long polyene structures. Establishing nonnatural biosynthetic pathways for longer-chain carotenoids in engineerable microorganisms will provide a platform to diversify and explore the potential of these molecules. We have previously reported the biosynthesis of nonnatural C50 carotenoids by engineering a C30-carotenoid backbone synthase (CrtM) from Staphylococcus aureus. In the present work, we conducted a series of experiments to engineer C60 carotenoid pathways...
January 28, 2019: ACS Synthetic Biology
Balwina Koopal, Aleksander J Kruis, Nico J Claassens, Franklin L Nobrega, John van der Oost
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 promoters that regulate the expression of the dcas9 gene. However, as dCas9-mediated gene silencing is very efficient, even slightly leaky dcas9 expression leads to significant background levels of repression of the target gene...
January 28, 2019: ACS Synthetic Biology
Mohammed Dwidar, Yohei Yokobayashi
Riboswitches are cis-acting RNA devices in mRNAs that control gene expression in response to chemical inputs. As RNA aptamers that recognize diverse classes of molecules can be isolated by in vitro selection, synthetic riboswitches hold promise for various applications in synthetic biology. One of the major drawbacks of riboswitches, however, is their limited dynamic range. High level of gene expression in the OFF state (leakage) is also a common problem. To address these challenges, we designed and constructed a dual-riboswitch plasmid in which two genes are controlled by theophylline-activated riboswitches...
January 25, 2019: ACS Synthetic Biology
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