Reshaping the 2-Pyrone Synthase Active Site for Chemoselective Biosynthesis of Polyketides.

Engineering enzymes with novel reactivity and applying them in metabolic pathways to produce valuable products are quite challenging due to the intrinsic complexity of metabolic networks and the need for high in vivo catalytic efficiency. Triacetic acid lactone (TAL), naturally generated by 2-pyrone synthase (2PS), is a platform molecule that can be produced via microbial fermentation and further converted into value-added products. However, these conversions require extra synthetic steps under harsh conditions. We herein report a biocatalytic system for direct generation of TAL derivatives under mild conditions with controlled chemoselectivity by rationally engineering the 2PS active site and then rewiring the biocatalytic pathway in the metabolic network of E. coli to produce high-value products, such as kavalactone precursors, with yields up to 17 mg/L culture. Computer modeling indicates sterics and hydrogen-bond interactions play key roles in tuning the selectivity, efficiency and yield.

Mechanisms of solid-state nanopore enlargement under electrical stress.

We present a thorough exploration of nanopore growth under electrical stress in electrolyte solution, and demonstrate that despite their superficial similarities, nanopore formation by controlled breakdown (CBD) and nanopore growth under moderate voltage stress are fundamentally different processes. In particular, we demonstrate that unlike the CBD process, nanopore growth is primarily driven by the level of ionic current passing through the nanopore, rather than the strength of the electric field generating the current, and that enlargement has a much weaker pH dependence than does CBD pore formation. In combination with other works in the field, our results suggest that despite clear current-dependence, Joule heating is unlikely to be the main driver of pore growth during electrical stress, pointing instead toward electrochemical dissolution of membrane material along the pore walls. While the chemistry underlying the growth process remains unclear, the dependence of growth rate on current allows decoupling of the pore enlargement mechanism from the possibility of forming additional nanopores during the growth process, providing a practical method by which to rapidly enlarge a nanopore without risking opening a second nanopore.

Solid-state nanopore fabrication by automated controlled breakdown.

Solid-state nanopores are now well established as single-biomolecule sensors that hold great promise as sensing elements in diagnostic and sequencing applications. However, until recently this promise has been limited by the expensive, labor-intensive, and low-yield methods used to fabricate low-noise and precisely sized pores. To address this problem, we pioneered a low-cost and scalable solid-state nanopore fabrication method, termed controlled breakdown (CBD), which is rapidly becoming the method of choice for fabricating solid-state nanopores. Since its initial development, nanopore research groups around the world have applied and adapted the CBD method in a variety of ways, with varying levels of success. In this work, we present our accumulated knowledge of nanopore fabrication by CBD, including a detailed description of the instrumentation, software, and procedures required to reliably fabricate low-noise and precisely sized solid-state nanopores with a yield of >85% in less than 1 h. The assembly instructions for the various custom instruments can be found in the Supplementary Manual, and take approximately a day to complete, depending on the unit that the user is building and their level of skill with mechanical and electrical assembly. Unlike traditional beam-based nanopore fabrication technologies, the methods presented here are accessible to non-experts, lowering the cost of, and technical barriers to, fabricating nanoscale pores in thin solid-state membranes.

Precise DNA Concentration Measurements with Nanopores by Controlled Counting.

Using a solid-state nanopore to measure the concentration of clinically relevant target analytes, such as proteins or specific DNA sequences, is a major goal of nanopore research. This is usually achieved by measuring the capture rate of the target analyte through the pore. However, progress is hindered by sources of systematic error that are beyond the level of control currently achievable with state-of-the-art nanofabrication techniques. In this work, we show that the capture rate process of solid-state nanopores is subject to significant sources of variability, both within individual nanopores over time and between different nanopores of nominally identical size, which are absent from theoretical electrophoretic capture models. We experimentally reveal that these fluctuations are inherent to the nanopore itself and make nanopore-based molecular concentration determination insufficiently precise to meet the standards of most applications. In this work, we present a simple method by which to reduce this variability, increasing the reliability, accuracy, and precision of single-molecule nanopore-based concentration measurements. We demonstrate controlled counting, a concentration measurement technique, which involves measuring the simultaneous capture rates of a mixture of both the target molecule and an internal calibrator of precisely known concentration. Using this method on linear DNA fragments, we show empirically that the requirements for precisely controlling the nanopore properties, including its size, height, geometry, and surface charge density or distribution, are removed while allowing for higher-precision measurements. The quantitative tools presented herein will greatly improve the utility of solid-state nanopores as sensors of target biomolecule concentration.

Interfacing solid-state nanopores with gel media to slow DNA translocations.

We demonstrate the ability to slow DNA translocations through solid-state nanopores by interfacing the trans side of the membrane with gel media. In this work, we focus on two reptation regimes: when the DNA molecule is flexible on the length scale of a gel pore, and when the DNA behaves as persistent segments in tight gel pores. The first regime is investigated using agarose gels, which produce a very wide distribution of translocation times for 5 kbp dsDNA fragments, spanning over three orders of magnitude. The second regime is attained with polyacrylamide gels, which can maintain a tight spread and produce a shift in the distribution of the translocation times by an order of magnitude for 100 bp dsDNA fragments, if intermolecular crowding on the trans side is avoided. While previous approaches have proven successful at slowing DNA passage, they have generally been detrimental to the S/N, capture rate, or experimental simplicity. These results establish that by controlling the regime of DNA movement exiting a nanopore interfaced with a gel medium, it is possible to address the issue of rapid biomolecule translocations through nanopores-presently one of the largest hurdles facing nanopore-based analysis-without affecting the signal quality or capture efficiency.

Kinetics of nanopore fabrication during controlled breakdown of dielectric membranes in solution.

Nanopore fabrication by controlled breakdown (CBD) overcomes many of the challenges of traditional nanofabrication techniques, by reliably forming solid-state nanopores sub-2 nm in size in a low-cost and scalable way for nucleic acid analysis applications. Herein, the breakdown kinetics of thin dielectric membranes immersed in a liquid environment are investigated in order to gain deeper insights into the mechanism of solid-state nanopore formation by high electric fields. For various fabrication conditions, we demonstrate that nanopore fabrication time is Weibull-distributed, in support of the hypothesis that the fabrication mechanism is a stochastic process governed by the probability of forming a connected path across the membrane (i.e. a weakest-link problem). Additionally, we explore the roles that various ions and solvents play in breakdown kinetics, revealing that asymmetric pH conditions across the membrane can significantly affect nanopore fabrication time for a given voltage polarity. These results, characterizing the stochasticity of the nanopore fabrication process and highlighting the parameters affecting it, should assist researchers interested in exploiting the potential of CBD for nanofluidic channel fabrication, while also offering guidance towards the conceivable manufacturing of solid-state nanopore-based technologies for DNA sequencing applications.

Solvent isotope effects on alkane formation by cyanobacterial aldehyde deformylating oxygenase and their mechanistic implications.

The reaction catalyzed by cyanobacterial aldehyde deformylating oxygenase is of interest both because of its potential application to the production of biofuels and because of the highly unusual nature of the deformylation reaction it catalyzes. Whereas the proton in the product alkane derives ultimately from the solvent, the identity of the proton donor in the active site remains unclear. To investigate the proton transfer step, solvent isotope effect (SIE) studies were undertaken. The rate of alkane formation was found to be maximal at pH 6.8 and to be the same in D2O or H2O within experimental error, implying that proton transfer is not a kinetically significant step. However, when the ratio of protium to deuterium in the product alkane was measured as a function of the mole fraction of D2O, a (D2O)SIEobs of 2.19 ± 0.02 was observed. The SIE was invariant with the mole fraction of D2O, indicating the involvement of a single protic site in the reaction. We interpret this SIE as most likely arising from a reactant state equilibrium isotope effect on a proton donor with an inverse fractionation factor, for which Φ = 0.45. These observations are consistent with an iron-bound water molecule being the proton donor to the alkane in the reaction.

Control of DNA capture by nanofluidic transistors.

We report the use of an array of electrically gated ~200 nm solid-state pores as nanofluidic transistors to manipulate the capture and passage of DNA. The devices are capable of reversibly altering the rate of DNA capture by over 3 orders of magnitude using sub-1 V biasing of a gate electrode. This efficient gating originates from the counter-balance of electrophoresis and electroosmosis, as revealed by quantitative numerical simulations. Such a reversible electronically tunable biomolecular switch may be used to manipulate nucleic acid delivery in a fluidic circuit, and its development is an important first step toward active control of DNA motion through solid-state nanopores for sensing applications.