GeMS: an advanced software package for designing synthetic genes

A user-friendly, advanced software package for gene design is described. The software comprises an integrated suite of programs—also provided as stand-alone tools—that automatically performs the following tasks in gene design: restriction site prediction, codon optimization for any expression host, restriction site inclusion and exclusion, separation of long sequences into synthesizable fragments, T(m) and stem–loop determinations, optimal oligonucleotide component design and design verification/error-checking. The output is a complete design report and a list of optimized oligonucleotides to be prepared for subsequent gene synthesis. The user interface accommodates both inexperienced and experienced users. For inexperienced users, explanatory notes are provided such that detailed instructions are not necessary; for experienced users, a streamlined interface is provided without such notes. The software has been extensively tested in the design and successful synthesis of over 400 kb of genes, many of which exceeded 5 kb in length

Rational Design and Assembly of Synthetic Trimodular Polyketide Synthases

SummaryType I polyketide synthases (PKSs) consist of modules that add two-carbon units in polyketide backbones. Rearranging modules from different sources can yield novel enzymes that produce unnatural products, but the rules that govern module-module communication are still not well known. The construction and assay of hybrid bimodular units with synthetic PKS genes were recently reported. Here, we describe the rational design of trimodular PKSs by combining bimodular units. A cloning-expression system was developed to assemble and test 54 unnatural trimodular PKSs flanked by the loading module and the thioesterase from the erythromycin synthase. Remarkably, 96% of them produced the expected polyketide. The obtained results represent an important milestone toward the ultimate goal of making new bioactive polyketides by rational design. Additionally, these results show a path for the production of customized tetraketides by fermentation, which can be an important source of advanced intermediates to facilitate the synthesis of complex products

Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase

AbstractBackground: Non-ribosomal peptide synthetases (NRPSs) found in bacteria abd fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4′-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4′-phosphopantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4′-phosphopantetheine.Results: A truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His6) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, < 1% was converted to the Phe thioester. In contrast, >80% of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP.Conclusions: Our finding indicates the presence of an almost equimolar amount of 4′-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase

Thermal stabilization of thymidylate synthase by engineering two disulfide bridges across the dimer interface

Thermal inactivation of oligomeric enzymes is most often irreversible and is frequently accompanied by precipitation. We have engineered two symmetry related disulfide bridges (155-188′ and 188-155′) across the subunit interface of Lactobacillus casei thymidylate synthase, at sites chosen on the basis of an algorithm for the introduction of stereochemically unstrained bridges into proteins. In this communication, we demonstrate a remarkable enhancement in the thermal stability of the covalently cross-linked double disulfide containing dimeric enzyme. The mutant enzyme remains soluble and retains secondary structure even at 90°C, in contrast to the wild-type enzyme which precipitates at 52°C. Furthermore, the mutant enzyme has a temperature optimum of 55°C and possesses appreciable enzymatic activity at 65°C. Cooling restores complete activity, in the mutant protein, demonstrating reversible thermal unfolding. The results suggest that inter-subunit crosslinks can impart appreciable thermal stability in multimeric enzymes

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5°C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 Å crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures

Increasing aridity will not offset CO2_{2} fertilization in fast-growing eucalypts with access to deep soil water

Rising atmospheric [CO$_{2}$] (C$_{a}$) generally enhances tree growth if nutrients are not limiting. However, reduced water availability and elevated evaporative demand may offset such fertilization. Trees with access to deep soil water may be able to mitigate such stresses and respond more positively to C$_{a}$. Here, we sought to evaluate how increased vapor pressure deficit and reduced precipitation are likely to modify the impact of elevated C$_{a}$ (eC$_{a}$) on tree productivity in an Australian Eucalyptus saligna Sm. plantation with access to deep soil water. We parameterized a forest growth simulation model (GOTILWA+) using data from two field experiments on E. saligna: a 2‐year whole‐tree chamber experiment with factorial C$_{a}$ (ambient =380, elevated =620 μmol mol$^{-1}$) and watering treatments, and a 10‐year stand‐scale irrigation experiment. Model evaluation showed that GOTILWA+ can capture the responses of canopy C uptake to (1) rising vapor pressure deficit (D) under both C$_{a}$ treatments; (2) alterations in tree water uptake from shallow and deep soil layers during soil dry‐down; and (3) the impact of irrigation on tree growth. Simulations suggest that increasing C$_{a}$ up to 700 μmol mol$^{-1}$ alone would result in a 33% increase in annual gross primary production (GPP) and a 62% increase in biomass over 10 years. However, a combined 48% increase in D and a 20% reduction in precipitation would halve these values. Our simulations identify high D conditions as a key limiting factor for GPP. They also suggest that rising Ca will compensate for increasing aridity limitations in E. saligna trees with access to deep soil water under non‐nutrient limiting conditions, thereby reducing the negative impacts of global warming upon this eucalypt species. Simulation models not accounting for water sources available to deep‐rooting trees are likely to overestimate aridity impacts on forest productivity and C stocks

Stellar Astrophysics and Exoplanet Science with the Maunakea Spectroscopic Explorer (MSE)

The Maunakea Spectroscopic Explorer (MSE) is a planned 11.25-m aperture facility with a 1.5 square degree field of view that will be fully dedicated to multi-object spectroscopy. A rebirth of the 3.6m Canada-France-Hawaii Telescope on Maunakea, MSE will use 4332 fibers operating at three different resolving powers (R ~ 2500, 6000, 40000) across a wavelength range of 0.36-1.8mum, with dynamical fiber positioning that allows fibers to match the exposure times of individual objects. MSE will enable spectroscopic surveys with unprecedented scale and sensitivity by collecting millions of spectra per year down to limiting magnitudes of g ~ 20-24 mag, with a nominal velocity precision of ~100 m/s in high-resolution mode. This white paper describes science cases for stellar astrophysics and exoplanet science using MSE, including the discovery and atmospheric characterization of exoplanets and substellar objects, stellar physics with star clusters, asteroseismology of solar-like oscillators and opacity-driven pulsators, studies of stellar rotation, activity, and multiplicity, as well as the chemical characterization of AGB and extremely metal-poor stars.Comment: 31 pages, 11 figures; To appear as a chapter for the Detailed Science Case of the Maunakea Spectroscopic Explore

Measurement of the ttbar Production Cross Section in ppbar Collisions at sqrt{s}=1.96 TeV using Lepton + Jets Events with Secondary Vertex b-tagging

We present a measurement of the ttbar production cross section using events with one charged lepton and jets from ppbar collisions at a center-of-mass energy of 1.96 TeV. In these events, heavy flavor quarks from top quark decay are identified with a secondary vertex tagging algorithm. From 162 pb-1 of data collected by the Collider Detector at Fermilab, a total of 48 candidate events are selected, where 13.5 +- 1.8 events are expected from background contributions. We measure a ttbar production cross section of 5.6^{+1.2}_{-1.1} (stat.) ^{+0.9}_{0.6} (syst.) pb.Comment: 28 pages, 20 figures. Published in Physical Review