Laboratory-Evolved Mutants of an Exogenous Global Regulator, IrrE from Deinococcus radiodurans, Enhance Stress Tolerances of Escherichia coli

The tolerance of cells toward different stresses is very important for industrial strains of microbes, but difficult to improve by the manipulation of single genes. Traditional methods for enhancing cellular tolerances are inefficient and time-consuming. Recently, approaches employing global transcriptional or translational engineering methods have been increasingly explored. We found that an exogenous global regulator, irrE from an extremely radiation-resistant bacterium, Deinococcus radiodurans, has the potential to act as a global regulator in Escherichia coli, and that laboratory-evolution might be applied to alter this regulator to elicit different phenotypes for E. coli.To extend the methodology for strain improvement and to obtain higher tolerances toward different stresses, we here describe an approach of engineering irrE gene in E. coli. An irrE library was constructed by randomly mutating the gene, and this library was then selected for tolerance to ethanol, butanol and acetate stresses. Several mutants showing significant tolerances were obtained and characterized. The tolerances of E. coli cells containing these mutants were enhanced 2 to 50-fold, based on cell growth tests using different concentrations of alcohols or acetate, and enhanced 10 to 100-fold based on ethanol or butanol shock experiments. Intracellular reactive oxygen species (ROS) assays showed that intracellular ROS levels were sharply reduced for cells containing the irrE mutants. Sequence analysis of the mutants revealed that the mutations distribute cross all three domains of the protein.To our knowledge, this is the first time that an exogenous global regulator has been artificially evolved to suit its new host. The successes suggest the possibility of improving tolerances of industrial strains by introducing and engineering exogenous global regulators, such as those from extremophiles. This new approach can be applied alone or in combination with other global methods, such as global transcriptional machinery engineering (gTME) for strain improvements

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by <i>in vitro</i> transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2′-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2′-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis

An evolved xylose transporter from <it>Zymomonas mobilis </it>enhances sugar transport in <it>Escherichia coli</it>

Abstract Background Xylose is a second most abundant sugar component of lignocellulose besides glucose. Efficient fermentation of xylose is important for the economics of biomass-based biorefineries. However, sugar mixtures are sequentially consumed in xylose co-fermentation with glucose due to carbon catabolite repression (CCR) in microorganisms. As xylose transmembrance transport is one of the steps repressed by CCR, it is therefore of interest to develop a transporter that is less sensitive to the glucose inhibition or CCR. Results The glucose facilitator protein Glf transporter from Zymomonas mobilis, also an efficient transporter for xylose, was chosen as the target transporter for engineering to eliminate glucose inhibition on xylose uptake. The evolution of Glf transporter was carried out with a mixture of glucose and xylose in E. coli. Error-prone PCR and random deletion were employed respectively in two rounds of evolution. Aided by a high-throughput screening assay using xylose analog p-nitrophenyl-β-D-xylopyranoside (pNPX) in 96-well plates, a best mutant 2-RD5 was obtained that contains several mutations, and a deletion of 134 residues (about 28% of total residues), or three fewer transmembrane sections (TMSs). It showed a 10.8-fold improvement in terms of pNPX transport activity in the presence of glucose. The fermentation performance results showed that this mutant improved xylose consumption by 42% with M9 minimal medium containing 20 g L-1 xylose only, while with the mixture sugar of xylose and glucose, 28% more glucose was consumed, but no obvious co-utilization of xylose was observed. Further glucose fed-batch experiments suggested that the intracellular metabolism of xylose was repressed by glucose. Conclusions Through random mutagenesis and partial deletion coupled with high-throughput screening, a mutant of the Glf transporter (2-RD5) was obtained that relieved the inhibition of xylose transport by glucose. The fermentation tests revealed that 2-RD5 was advantageous in xylose and glucose uptakes, while no obvious advantage was seen for xylose co-consumption when co-fermented with glucose. Further efforts could focus on reducing CCR-mediated repression of intracellular metabolism of xylose. Glf should also serve as a useful model to further exploit the molecular mechanism of xylose transport and the CCR-mediated inhibition.</p

Investigation on Modeling and Formation Mechanism of Dynamic Rotational Error for Spindle-Rolling Bearing System

In the field of precision machining, the spindle-rolling bearing (SRB) system is widely used on the machine tool as one of the most fundamental and important components. The rotational error motions of the SRB system have significant effects on the machining accuracy (contour accuracy and surface roughness). Over the past decades, much work has been focused on the measurement of spindle balancing and rotational error motions, the vibrations response induced by the nonlinear stiffness and surface waviness of the bearing. However, the formative mechanism of the rotational error motions for the SRB system is not well understood. In this paper, the dynamic model of the SRB system considering the bearing nonlinearity is established. Seeking to reveal the effects of surface waviness of the bearing raceway, unbalance mass and disturbance force on the dynamic rotational error, the modeling method and formative mechanism of the dynamic rotational error for the SRB system is explored both theoretically and experimentally. Then, numerical simulation is performed to analyze the influence of the bearing raceway waviness, unbalance mass and disturbance force on the dynamic rotational error. An experimental setup is established based on a typical SRB system and a series of experiments are carried out. The experimental results are in good agreement with the theoretical and simulation results, which can demonstrate the feasibility and validity of the modeling method. Furthermore, this method can be effectively applied to the design and development phases of an SRB system to improve dynamic rotational accuracy

Recognition of an Unnatural Base Pair by Tool Enzymes from Bacteriophages and Its Application in the Enzymatic Preparation of DNA with an Expanded Genetic Alphabet

Unnatural base pairs (UBPs) have been developed to expand the genetic alphabet in vitro and in vivo. UBP dNaM-dTPT3 and its analogues have been successfully used to construct the first set of semi-synthetic organisms, which suggested the great potential of UBPs to be used for producing novel synthetic biological parts. Two prerequisites for doing so are the facile manipulation of DNA containing UBPs with common tool enzymes, including DNA polymerases and ligases, and the easy availability of UBP-containing DNA strands. Besides, for the application of UBPs in phage synthetic biology, the recognition of UBPs by phage enzymes is essential. Here, we first explore the recognition of dNaM–dTPT3 by a family B DNA polymerase from bacteriophage, T4 DNA polymerase D219A. Results from primer extension, steady-state kinetics, and gap-filling experiments suggest that T4 DNA polymerase D219A can efficiently and faithfully replicate dNaM–dTPT3, and efficiently fill a gap by inserting dTPT3TP or its analogues opposite dNaM. We then systematically explore the recognition of dNaM–dTPT3 and its analogues by different DNA ligases from bacteriophages and find that these DNA ligases are generally able to efficiently ligate the DNA nick next to dNaM–dTPT3 or its analogues, albeit with slightly different efficiencies. These results suggest more enzymatic tools for the manipulation of dNaM–dTPT3 and indicate the potential use of dNaM–dTPT3 for expanding the genetic alphabet in bacteriophages. Based on these results, we next develop and comprehensively optimize an upgraded method for enzymatic preparation of unnatural nucleobase (UB)-containing DNA oligonucleotides with good simplicity and universality

Selection of 2′-Fluoro-Modified Aptamers with Optimized Properties

RNA or single-stranded DNA aptamers with 2′-F pyrimidines have been pursued to increase resistance to nucleases, and while it seems likely that these and other modifications, including the modification of purines, could be used to optimize additional properties, this has been much less explored because such aptamers are challenging to discover. Using a thermostable DNA polymerase, SFM4-3, which was previously evolved to accept nucleotides with 2′-modifications, we now report the selection of 2′-F purine aptamers that bind human neutrophil elastase (HNE). Two aptamers were identified, 2fHNE-1 and 2fHNE-2, that bind HNE with reasonable affinity. Interestingly, the 2′-F substituents facilitate the selection of specific interactions with HNE and overcome nonspecific electrostatic interactions that can otherwise dominate. The data demonstrate that inclusion of only a few 2′-F substituents can optimize properties far beyond simple nuclease resistance and that SFM4-3 should prove valuable for the further exploration and production of aptamers with properties optimized for various applications