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11:22 EST 6th December 2019 | BioPortfolio

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Showing PubMed Articles 1–25 of 402 from ACS synthetic biology

mpCRISTAR: Multiple plasmid approach for CRISPR/Cas9 and TAR-mediated multiplexed refactoring of natural product biosynthetic gene clusters.

Multiplexed refactoring provides a tool for rapid transcriptional optimization of biosynthetic gene clusters (BGCs) through simultaneous replacement of multiple native promoters with synthetic counterparts. Here, we present the mpCRISTAR, a multiple plasmid-based CRISPR/Cas9 and TAR (transformation-associated recombination), that enables a rapid and highly efficient, multiplexed refactoring of natural product BGCs in yeast. A series of CRISPR plasmids with different auxotrophic markers that could be stably ...

Multiple-site diversification of regulatory sequences enables inter-species operability of genetic devices.

The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e. ho1 and pcyA genes for synthesis of phycocyanobilin, the ccaS/ccaR system from Synechocystis and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by [i] reassembling them together in a single broad host range, stan...

Enzymatic O-glycosylation of etoposide aglycone by exploration of the substrate promiscuity for glycosyltransferases.

The 4-O-β-D-glucopyranoside of DMEP ((-)-4-desmethylepipodophyllotoxin) (GDMEP), a natural product from Podophyllum hexandrum, is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic O-glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glyc...

Evolutionary Outcomes of Diversely-Functionalized Aptamers Isolated from In Vitro Evolution.

Expanding the chemical diversity of aptamers remains an important thrust in the field in order to increase their func-tional potential. Previously, our group developed LOOPER, which enables the incorporation of up to 16 unique modi-fication through-out a ssDNA sequence, and applied it to the in vitro evolution of thrombin binders. As LOOPER-derived highly-modified nucleic acids polymers are governed by two interrelated evolutionary variables, namely functional modifications and sequence, the evolution of th...

Codon selection affects recruitment of ribosome-associating factors during translation.

An intriguing aspect of protein synthesis is how co-translational events are managed inside the cell. In this study, we developed an in vivo bimolecular fluorescence complementation assay coupled to SecM stalling (BiFC-SecM) to study how codon usage influences the interactions of ribosome-associating factors that occur co-translationally. We profiled ribosomal associations of a number of proteins, and observed differential association of chaperone proteins TF, DnaK, GroEL and translocation factor Ffh as a r...

Double-check base editing (DBE) for efficient A to G conversions.

With the development of CRISPR/Cas9 technology, a new generation of editing methods that convert specific bases has enabled precise single-base mutations. To date, conversion of cytosine to thymidine and adenine to guanine has been achieved using the cytidine deaminase APOBEC1 and adenosine deaminase (TadA), respectively. However, the base editing efficiency can be unacceptably low in some cell types or at certain target loci. One reason might be the lack of a selective pressure against the survival of non-...

Orthogonal protein-responsive mRNA switches for mammalian synthetic biology.

The lack of available genetic modules is a fundamental issue in mammalian synthetic biology. Especially, the variety of genetic parts for translational control are limited. Here we report a new set of synthetic mRNA-based translational switches by engineering RNA-binding proteins (RBPs) and RBP-binding RNA motifs (aptamers) that perform strong translational repression. We redesigned the RNA motifs with RNA scaffolds and improved the efficiency of the repression to target RBPs. Using new and previously repor...

Unravelling the Biosynthetic Flexibility of UK-2A Enables Enzymatic Synthesis of its Structural Variants.

Emerging antimicrobial resistant fungal pathogens are a growing threat, and fungicides with novel modes of action are urgently needed to prevent critical failures in global food security. Fenpicoxamid, the prodrug of UK-2A, is a member of a new class of antifungal agents that displays no cross-resistance to other fungicides. Rational engineering of its structure using a biosynthetic approach is a promising avenue for developing more potent fungicides. Herein, through in vitro enzymatic reconstitution, we el...

High-Throughput, Lysis-free Screening for Sulfatase Activity Using Autodisplay in Microdroplets.

Directed evolution of enzymes toward improved catalytic performance has become a powerful tool in protein engineering. To be effective, a directed evolution campaign requires the use of high-throughput screening. In this study we describe the development of an ultra high-throughput lysis-free procedure to screen for improved sulfatase activity by combining microdroplet-based single-variant activity sorting with autodisplay. For the first step in a 4-step screening procedure we quantitatively screened >10 v...

Identification of Three Antimicrobials Activating Serotonin Receptor 4 in Colon Cells.

The serotonin receptor 4b (5-HTR) is expressed throughout the gastrointestinal tract, and its agonists are used in the treatment of irritable bowel syndrome with constipation (IBS-C). Today, there are no rapid assays for the identification of 5-HTR agonists. Here, we developed a luciferase-based 5-HTR assay capable of assessing one compound per second with a 38-fold dynamic range and nM limit of detection for serotonin. We used the assay to screen more than 1000 natural products and anti-infection agents an...

Engineering new branches of the kynurenine pathway to produce oxo-(2-aminophenyl) and quinoline scaffolds in yeast.

The kynurenine pathway, named after its non-proteinogenic amino acid precursor L-kynurenine, is responsible for the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+) in eukaryotes. Oxo-(2-aminophenyl) and quinoline molecules downstream from L-kynurenine also serve as antagonists of several receptors of the central nervous system in mammals. In this study, we engineered new biosynthetic routes in yeast Saccharomyces cerevisiae to produce a suite of L-kynurenine-derived natural products. Overex...

Detecting changes in the Caenorhabditis elegans intestinal environment using an engineered bacterial biosensor.

Caenorhabditis elegans has become a key model organism within biology. In particular, the transparent gut, rapid growing time and ability to create a defined gut microbiota make it an ideal candidate organism for understanding and engineering the host microbiota. Here we present the development of an experimental model which can be used to characterise whole-cell bacterial biosensors in vivo. A dual-plasmid sensor system responding to isopropyl β-D-1-thiogalactopyranoside was developed and fully characteri...

Exploring Chemical Biosynthetic Design Space with Transform-MinER.

Transform-MinER (Transforming Molecules in Enzyme Reactions) is a web application facilitating the exploration of chemical biosynthetic space, guiding the user towards promising start points for enzyme design projects or directed evolution experiments. Two types of search are possible: Molecule Search allows a user to submit a source substrate enabling Transform-MinER to search for enzyme reactions acting on similar substrates; whereas Path Search additionally allows a user to submit a target molecule enabl...

Optimizing oleaginous yeast cell factories for flavonoids and hydroxylated flavonoids biosynthesis.

Plants possess myriads of secondary metabolites with a broad spectrum of health-promoting benefits. Up to date, plant extraction is still the primary route to produce high-value natural products, which inherently suffers from economics and scalability issues. Heterologous expression of plant biosynthetic gene clusters in microbial host is considered as a feasible approach to overcoming these limitations. Oleaginous yeast produces large amount of lipid bodies, the abundant membrane structure and the lipophil...

Off-colony screening of biosynthetic libraries by rapid laser-enabled mass spectrometry.

Leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address t...

An aldolase-catalyzed new metabolic pathway for the assimilation of formaldehyde and methanol to synthesize 2-keto-4-hydroxybutyrate and 1,3-propanediol in Escherichia coli.

Formaldehyde (HCHO) is an important intermediate in the metabolism of one-carbon (C1) compounds such as methanol, formate and methane. The ribulose monophosphate (RuMP) pathway is the mostly studied HCHO assimilation route and the 3-hexulose-6-phosphate synthase (Hps) plays an important role for HCHO fixation. In this study, we proposed and selected a pyruvate-dependent aldolase to channel HCHO into 2-keto-4-hydroxybutyrate as an important intermediate for biosynthesis. By combining this reaction with three...

Repurposing protein degradation for optogenetic modulation of protein activities.

Non-neuronal optogenetic approaches empower precise regulation of protein dynamics in live cells but often require target-specific protein engineering. To address this challenge, we developed a generalizable light-modulated protein stabilization system (GLIMPSe) to control intracellular protein level independent of its functionality. We applied GLIMPSe to control two distinct classes of proteins: mitogen-activated protein kinase phosphatase 3 (MKP3), a negative regulator of the extracellu-lar signal-regulat...

Simultaneous visualization of multiple gene expression in single cells using an engineered multicolor reporter toolbox and approach of spectral crosstalk correction.

Synthetic biology aims to make biology easier to engineer and focuses on the design and construction of core components that can be modeled, understood, and tuned to meet specific performance criteria, and the assembly of these smaller parts and devices into larger integrated systems to solve specific problems. Here, we designed and engineered a multicolor fluorescent reporter toolbox to simultaneously monitor the activities of multiple genes in single cells. The toolbox contained standardized and well-char...

Model-driven engineering of N-linked glycosylation in Chinese Hamster Ovary cells.

Chinese Hamster Ovary (CHO) cells are used for industrial production of protein-based therapeutics (i.e. 'biologics'). Here we describe a method for combining systems-level kinetic models with a synthetic biology platform for multi-gene overexpression to rationally perturb N-linked glycosylation. Specifically, we sought to increase galactose incorporation on a secreted Immunoglobulin G (IgG) protein. We rationally design, build, and test a total of 23 transgenic cell pools that express single or three-gene ...

Design and control of extrachromosomal elements in AM1.

Genetic tools are a prerequisite to engineer cellular factories for synthetic biology and biotechnology. AM1 is an important platform organism of a future C1-bioeconomy. However, its application is currently limited by the availability of genetic tools. Here we systematically tested regions to maintain extrachromosal DNA in . We used three elements to construct mini-chromosomes that are stably inherited at single copy number and can be shuttled between and . These mini-chromosomes are compatible among ea...

Models for DNA design tools: The trouble with metaphors is they don't go away.

Synthetic biology relies heavily on DNA design tools to enable manipulating DNA in silico. Existing tools, however, are falling short of enabling aspirations for the field that emphasize efficient, automated design pipelines. We review existing DNA design tools, identifying underlying similarities in how they model correlations between DNA structure and function and suggest that iterating the existing model is unlikely to overcome limitations in matching software applications to design aspirations. The curr...

Development of a new biocontainment strategy in model cyanobacterium strains.

Recent synthetic biology efforts have raised biosafety concerns for possible release of engineered cyanobacteria into natural environments. To address the issues, we developed a controllable metal ion induced biocontainment system for two model cyanobacteria. First, six ion-inducible promoters were respectively evaluated in both PCC 7942 and the fast-growing cyanobacterium UTEX 2973, leading to the identification of an iron ion-repressed promoter P with low leakage and a reduction fold of 5.4 and 7.9, res...

Model-assisted fine-tuning of central carbon metabolism in yeast through dCas9-based regulation.

Engineering Saccharomyces cerevisiae for industrial-scale production of valuable chemicals involves extensive modulation of its metabolism. Here, we identified novel gene expression fine-tuning set-ups to enhance endogenous metabolic fluxes towards increasing levels of acetyl-CoA and malonyl-CoA. dCas9-based transcriptional regulation was combined together with a malonyl-CoA responsive intracellular biosensor to select for beneficial set-ups. The candidate genes for screening were predicted using a genome-s...

In vitro transcriptional regulation via nucleic-acid-based transcription factors.

Cells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in in vitro gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic-acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates displaying cis-regulatory ssDN...

Functional Expression of GFP-Fused Class I Lanthipeptides in .

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides, with several having antimicrobial activity. The biosynthetic machinery responsible for modification of the class I lanthipeptide nisin provides a means for modification of a diverse range of lanthipeptides. However, literature regarding expression of class I lanthipeptides in a malleable Gram-negative host such as is limited. Here, we coexpressed precursor class I lanthipeptides fused to green fluorescent protein (GFP) a...


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