Direct fermentation route for the production of acrylic acid.

There have been growing concerns regarding the limited fossil resources and global climate changes resulting from modern civilization. Currently, finding renewable alternatives to conventional petrochemical processes has become one of the major focus areas of the global chemical industry sector. Since over 4.2 million tons of acrylic acid (AA) is annually employed for the manufacture of various products via petrochemical processes, this chemical has been the target of efforts to replace the petrochemical route by ecofriendly processes. However, there has been limited success in developing an approach combining the biological production of 3-hydroxypropionic acid (3-HP) and its chemical conversion to AA. Here, we report the first direct fermentative route for producing 0.12 g/L of AA from glucose via 3-HP, 3-HP-CoA, and Acryloyl-CoA, leading to a strain of Escherichia coli capable of directly producing acrylic acid. This route was developed through extensive screening of key enzymes and designing a novel metabolic pathway for AA.

Metabolic engineering of 3-hydroxypropionic acid biosynthesis in Escherichia coli.

3-Hydroxypropionic acid (3-HP) can be produced in microorganisms as a versatile platform chemical. However, owing to the toxicity of the intermediate product 3-hydroxypropionaldehyde (3-HPA), the minimization of 3-HPA accumulation is critical for enhancing the productivity of 3-HP. In this study, we identified a novel aldehyde dehydrogenase, GabD4 from Cupriavidus necator, and found that it possessed the highest enzyme activity toward 3-HPA reported to date. To augment the activity of GabD4, several variants were obtained by site-directed and saturation mutagenesis based on homology modeling. Escherichia coli transformed with the mutant GabD4_E209Q/E269Q showed the highest enzyme activity, which was 1.4-fold higher than that of wild type GabD4, and produced up to 71.9 g L(-1) of 3-HP with a productivity of 1.8 g L(-1) h(-1) . To the best of our knowledge, these are the highest 3-HP titer and productivity values among those reported in the literature. Additionally, our study demonstrates that GabD4 can be a key enzyme for the development of industrial 3-HP-producing microbial strains, and provides further insight into the mechanism of aldehyde dehydrogenase activity.

Roughening of Polyimide Surface for Inkjet Printing by Plasma Etching Using the Polyimide Masked with Polystyrene Nanosphere Array.

Two key conditions are required for the application of fine-line inkjet printing onto a flexible substrate such as polyimide (PI): linewidth control during the inkjetting process, and a strong adhesion of the polyimide surface to the ink after the ink solidifies. In this study, the properties of a polyimide surface that was roughened through etching in a He/SF6 plasma, using a polystyrene nanosphere array as the etch mask, were investigated. The near-atmospheric-pressure plasma system of the He/SF6 plasma that was used exhibits two notable properties in this context: similar to an atmospheric-pressure plasma system, it can easily handle inline substrate processing; and, similar to a vacuum system, it can control the process gas environment. Through the use of plasma etching, the polyimide surface masked the 120-nm-diameter polystyrene nanospheres, thereby forming a roughened nanoscale polyimide surface. This surface exhibited not only a greater hydrophobicity--with a contact angle of about 150° for water and about 30° for silver ink, indicating better silver linewidth control during the silver inkjetting process--but also a stronger adhesion to the silver ink sprayed onto it when compared with the flat polyimide surface.

Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes.

Understanding molecular recognition is of fundamental importance in applications such as therapeutics, chemical catalysis and sensor design. The most common recognition motifs involve biological macromolecules such as antibodies and aptamers. The key to biorecognition consists of a unique three-dimensional structure formed by a folded and constrained bioheteropolymer that creates a binding pocket, or an interface, able to recognize a specific molecule. Here, we show that synthetic heteropolymers, once constrained onto a single-walled carbon nanotube by chemical adsorption, also form a new corona phase that exhibits highly selective recognition for specific molecules. To prove the generality of this phenomenon, we report three examples of heteropolymer-nanotube recognition complexes for riboflavin, L-thyroxine and oestradiol. In each case, the recognition was predicted using a two-dimensional thermodynamic model of surface interactions in which the dissociation constants can be tuned by perturbing the chemical structure of the heteropolymer. Moreover, these complexes can be used as new types of spatiotemporal sensors based on modulation of the carbon nanotube photoemission in the near-infrared, as we show by tracking riboflavin diffusion in murine macrophages.

Structure and function of glucose binding protein-single walled carbon nanotube complexes.

Understanding the structure and function of glucose binding proteins (GBP) complexed with single walled carbon nanotubes (SWNTs) is important for the development of applications including fluorescent sensors and nanostructure particle tracking. Herein, circular dichroism (CD), thermal denaturation, photo-absorption spectroscopy and atomic force microscopy are used to study these nanostructures. The protein retains its glucose-binding activity after complexation and is thermally stable below 36 °C. However, the SWNT lowers the midpoint denaturation temperature (Tm) by 5 °C and 4 °C in the absence and presence of 10 mM glucose, respectively. This data highlights that using techniques such as CD and thermal denaturation may be necessary to fully characterize such protein-nanomaterial nanostructures.

Boronic acid library for selective, reversible near-infrared fluorescence quenching of surfactant suspended single-walled carbon nanotubes in response to glucose.

We describe the high-throughput screening of a library of 30 boronic acid derivatives to form complexes with sodium cholate suspended single-walled carbon nanotubes (SWNTs) to screen for their ability to reversibly report glucose binding via a change in SWNT fluorescence. The screening identifies 4-cyanophenylboronic acid which uniquely causes a reversible wavelength red shift in SWNT emission. The results also identify 4-chlorophenylboronic acid which demonstrates a turn-on fluorescence response when complexed with SWNTs upon glucose binding in the physiological range of glucose concentration. The mechanism of fluorescence modulation in both of these cases is revealed to be a photoinduced excited-state electron transfer that can be disrupted by boronate ion formation upon glucose binding. The results allow for the elucidation of design rules for such sensors, as we find that glucose recognition and transduction is enabled by para-substituted, electron-withdrawing phenyl boronic acids that are sufficiently hydrophobic to adsorb to the nanotube surface.

NoRSE: noise reduction and state evaluator for high-frequency single event traces.

UNLABELLED: NoRSE was developed to analyze high-frequency datasets collected from multistate, dynamic experiments, such as molecular adsorption and desorption onto carbon nanotubes. As technology improves sampling frequency, these stochastic datasets become increasingly large with faster dynamic events. More efficient algorithms are needed to accurately locate the unique states in each time trace. NoRSE adapts and optimizes a previously published noise reduction algorithm and uses a custom peak flagging routine to rapidly identify unique event states. The algorithm is explained using experimental data from our lab and its fitting accuracy and efficiency are then shown with a generalized model of stochastic datasets. The algorithm is compared to another recently published state finding algorithm and is found to be 27 times faster and more accurate over 55% of the generalized experimental space. NoRSE is written as an M-file for Matlab. AVAILABILITY: http://web.mit.edu/stranogroup/NoRSE.txt.

Expression screening of fusion partners from an E. coli genome for soluble expression of recombinant proteins in a cell-free protein synthesis system.

While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes.

Design, assembly, and activity of antisense DNA nanostructures.

Discrete DNA nanostructures allow simultaneous features not possible with traditional DNA forms: encapsulation of cargo, display of multiple ligands, and resistance to enzymatic digestion. These properties suggested using DNA nanostructures as a delivery platform. Here, DNA pyramids displaying antisense motifs are shown to be able to specifically degrade mRNA and inhibit protein expression in vitro, and they show improved cell uptake and gene silencing when compared to linear DNA. Furthermore, the activity of these pyramids can be regulated by the introduction of an appropriate complementary strand. These results highlight the versatility of DNA nanostructures as functional devices.

Transduction of glycan-lectin binding using near-infrared fluorescent single-walled carbon nanotubes for glycan profiling.

There is significant interest in developing new detection platforms for characterizing glycosylated proteins, despite the lack of easily synthesized model glycans or high affinity receptors for this analytical problem. In this work, we demonstrate a sensor array employing recombinant lectins as glycan recognition sites tethered via Histidine tags to Ni(2+) complexes that act as fluorescent quenchers for semiconducting single-walled carbon nanotubes (SWNTs) embedded in a chitosan hydrogel spot to measure binding kinetics of model glycans. We examine, as model glycans, both free and streptavidin-tethered biotinylated monosaccharides. Two higher-affined glycan-lectin pairs are explored: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. The dissociation constants (K(D)) for these pairs as free glycans (106 and 19 µM, respectively) and streptavidin-tethered (142 and 50 µM respectively) were found. The absolute detection limit for the current platform was found to be 2 µg of glycosylated protein or 100 ng of free glycan to 20 µg of lectin. Glycan detection (GlcNAc-streptavidin at 10 µM) is demonstrated at the single nanotube level as well by monitoring the fluorescence from individual SWNT sensors tethered to GafD lectin. Over a population of 1000 nanotubes, 289 of the SWNT sensors had signals strong enough to yield kinetic information (K(D) of 250 ± 10 µM). We are also able to identify the locations of "strong transducers" on the basis of dissociation constant (four sensors with K(D) < 10 µM) or overall signal modulation (eight sensors with >5% quench response). We report the key finding that the brightest SWNTs are not the best transducers of glycan binding. SWNTs ranging in intensity between 50 and 75% of the maximum show the greatest response. The ability to pinpoint strong-binding, single sensors is promising to build a nanoarray of glycan-lectin transducers as a high throughput method to profile glycans without protein labeling or glycan liberation pretreatment steps.

Single-molecule detection of H2O2 mediating angiogenic redox signaling on fluorescent single-walled carbon nanotube array.

Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), activate signal transduction pathways during angiogenesis and therefore play an important role in physiological development as well as various pathophysiologies. Herein, we utilize a near-infrared fluorescent single-walled carbon nanotube (SWNT) sensor array to measure the single-molecule efflux of H(2)O(2) from human umbilical vein endothelial cells (HUVEC) in response to angiogenic stimulation. Two angiogenic agents were investigated: the pro-angiogenic cytokine, vascular endothelial growth factor A (VEGF-A) and the recently identified inorganic pro-angiogenic factor, europium(III) hydroxide in nanorod form. The nanosensor array consists ofa SWNT embedded within a collagen matrix that exhibits high selectivity and sensitivity to single molecules of H(2)O(2). A calibration from 12.5 to 400 nM quantifies the production of H(2)O(2) at nanomolar concentration in HUVEC with 1 s temporal and 300 nm spatial resolutions. We find that the production of H(2)O(2) following VEGF stimulation is elevated outside of HUVEC, but not for stimulation via nanorods, while increased generation is observed in the cytoplasm for both cases, suggesting two distinct signaling pathways.

Near-infrared fluorescent sensors based on single-walled carbon nanotubes for life sciences applications.

Many properties of single-walled carbon nanotubes (SWCNTs) make them ideal candidates for sensors, particularly for biological systems. Both their fluorescence in the near-infrared range of 820-1600 nm, where absorption by biological tissues is often minimal, and their inherent photostability are desirable attributes for the design of in vitro and in vivo sensors. The mechanisms by which a target molecule can selectively alter the fluorescent emission include primarily changes in emission wavelength (i.e., solvatochromism) and intensity, including effects such as charge-transfer transition bleaching and exciton quenching. The central challenge lies in engineering the nanotube interface to be selective for the analyte of interest. In this work, we review the recent development in this area over the past few years, and describe the design rules that we have developed for detecting various analytes, ranging from stable small molecules and reactive oxygen species (ROS) or reactive nitrogen species (RNS) to macromolecules. Applications to in vivo sensor measurements using these sensors are also described. In addition, the emerging field of SWCNT-based single-molecule detection using band gap fluorescence and the recent efforts to accurately quantify and utilize this unique class of stochastic sensors are also described in this article.

Label-free, single protein detection on a near-infrared fluorescent single-walled carbon nanotube/protein microarray fabricated by cell-free synthesis.

Excessive sample volumes continue to be a major limitation in the analysis of protein-protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni(2+) chelated by Nα,Nα-bis(carboxymethyl)-L-lysine grafted to chitosan surrounding the SWNT. The Ni(2+) acts as a proximity quencher with the Ni(2+)/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein-protein interactions in the staurosporine-induced apoptosis of SH-SY5Y cells, confirming literature predictions.

Modulation of single-walled carbon nanotube photoluminescence by hydrogel swelling.

We demonstrate the use of hydrogel swelling as a mechanism to reversibly induce solvatochromic shifting in single-walled carbon nanotube (SWNT) near-infrared emission within a biocompatible hydrogel. The optical sensor reports the degree of the swelled state and glucose concentration when apo-glucose oxidase is used to cross-link the hydrogel. Photoluminescence emission maxima from dispersed nanotubes in a poly(vinyl alcohol) hydrogel shift as cross-linking is increased, with a maximum of -48 meV for the (6,5) nanotube. The Raman tangential mode also red shifts up to 17 cm(-1), indicative of nanotube lattice strain equivalent to an effective hydrostatic pressure of 3 GPa. While the electronic band gaps of SWNTs are known to either increase or decrease with uniaxial strain or lattice deformation depending on chiral vector, we show that the mechanism of detection is counterintuitively non-strain-dependent. Instead, the data are well-described by a model that accounts for changes in dielectric screening of the 1-D exciton, as the osmotic pressure forces conformational distortions in the PVA by rotating more polar groups to the nanotube surface. The model describes observed changes with hydration state and cross-linking density variation from 0 to 14%. Cross-linking with apo-glucose oxidase renders the hydrogel glucose responsive, and we demonstrate rapid and reversible detection of glucose from these systems after repeated cycling of 10 mM glucose. We also demonstrate detection and imaging in the near-infrared of implanted hydrogel sensors in a mouse tissue model, showing excellent signal-to-noise of 8.6 and contrast with integration times of 60 s.

Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system.

Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell-free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN-mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti-antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti-antisense ODNs enabled the stop-and-go expression of protein molecules. Through the on-demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components.

Tuning the expression level of recombinant proteins by modulating mRNA stability in a cell-free protein synthesis system.

In this work, it was discovered that the stability of mRNA in a cell-free extract could be controlled by using engineered T7 terminator sequences. Specifically, it was found that mRNA stability gradually decreased as the length of the stem structure of the T7 terminator was reduced sequentially. As a result of the controlled abundance of mRNA species, it was possible to manipulate the relative expression level of target proteins by employing the T7 terminator of adjusted stem lengths.

High-throughput, combinatorial engineering of initial codons for tunable expression of recombinant proteins.

We describe a high-throughput strategy for tuning the expression of recombinant proteins through engineering their early nucleotide sequences. After randomizing the +2 and +3 codons of the target genes, each of the variant genes was isolated in vivo and subsequently expressed using in vitro protein synthesis techniques. When several hundreds of clones were examined in parallel, it was found that expression levels of target genes varied as much as 70-fold depending on the identity of the codons in the randomized region. This broad and continuous distribution of expression levels enabled the selection of specific codon arrangements for the expression of target genes at a desired level. Furthermore, codon-dependent variations in protein expression were reproduced when the same genes were expressed in vivo. Thus, we expect that the methodology reported here could be utilized as a versatile platform for rapid expression of protein molecules at modulated levels either in vitro or in vivo.

Real-time monitoring of cell-free protein synthesis on a surface plasmon resonance chip.

Taking advantage of the "open" nature of cell-free protein synthesis, this study investigated the direct analysis of protein expression using a surface plasmon resonance sensor. During the on-chip incubation of the reaction mixture for cell-free protein synthesis, the expressed protein molecules were immobilized onto the surface of the chip, giving rise to a sensorgram signal, which enabled on-line monitoring of protein expression. In addition, we found that the expression of the aggregation-prone proteins could be effectively monitored. The ability to monitor these proteins was most likely through the instant isolation of the expressed protein molecules onto the solid surface of the chip.