Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin

Adenosine Triphosphate and Carbon Efficient Route to Second Generation Biofuel Isopentanol.

Climate change necessitates the development of CO2 neutral or negative routes to chemicals currently produced from fossil carbon. In this paper we demonstrate a pathway from the renewable resource glucose to next generation biofuel isopentanol by pairing the isovaleryl-CoA biosynthesis pathway from Myxococcus xanthus and a butyryl-CoA reductase from Clostridium acetobutylicum. The best plasmid and Escherichia coli strain combination makes 80.50 ± 8.08 (SD) mg/L of isopentanol after 36 h under microaerobic conditions with an oleyl alcohol overlay. In addition, the system also shows a strong preference for isopentanol production over prenol in microaerobic conditions. Finally, the pathway requires zero adenosine triphosphate and can be paired theoretically with nonoxidative glycolysis, the combination being redox balanced from glucose thus avoiding unnecessary carbon loss as CO2. These pathway properties make the isovaleryl-CoA pathway an attractive isopentanol production route for further optimization

Microbial production of advanced biofuels

Concerns over climate change have necessitated a rethinking of our transportation infrastructure. One possible alternative to carbon-polluting fossil fuels are biofuels produced from a renewable carbon source using engineered microorganisms. Two biofuels, ethanol and biodiesel, have been made inroads to displacing petroleum-based fuels, but their penetration has been limited by the amounts that can be used in conventional engines and by cost. Advanced biofuels that mimic petroleum-based fuels are not limited by the amounts that can be used in existing transportation infrastructure, but have had limited penetration due to costs. In this review, we will discuss the advances in engineering microbial metabolism to produce advanced biofuels and prospects for reducing their costs

DeviceEditor visual biological CAD canvas

<p>Abstract</p> <p>Background</p> <p>Biological Computer Aided Design (bioCAD) assists the <it>de novo </it>design and selection of existing genetic components to achieve a desired biological activity, as part of an integrated design-build-test cycle. To meet the emerging needs of Synthetic Biology, bioCAD tools must address the increasing prevalence of combinatorial library design, design rule specification, and scar-less multi-part DNA assembly.</p> <p>Results</p> <p>We report the development and deployment of web-based bioCAD software, DeviceEditor, which provides a graphical design environment that mimics the intuitive visual whiteboard design process practiced in biological laboratories. The key innovations of DeviceEditor include visual combinatorial library design, direct integration with scar-less multi-part DNA assembly design automation, and a graphical user interface for the creation and modification of design specification rules. We demonstrate how biological designs are rendered on the DeviceEditor canvas, and we present effective visualizations of genetic component ordering and combinatorial variations within complex designs.</p> <p>Conclusions</p> <p>DeviceEditor liberates researchers from DNA base-pair manipulation, and enables users to create successful prototypes using standardized, functional, and visual abstractions. Open and documented software interfaces support further integration of DeviceEditor with other bioCAD tools and software platforms. DeviceEditor saves researcher time and institutional resources through correct-by-construction design, the automation of tedious tasks, design reuse, and the minimization of DNA assembly costs.</p

Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

<p>Abstract</p> <p>Background</p> <p>Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol.</p> <p>Results and conclusion</p> <p><it>Saccharomyces cerevisiae </it>was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (<it>S. cerevisiae</it>, <it>Escherichia coli</it>, <it>Clostridium beijerinckii</it>, and <it>Ralstonia eutropha</it>) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the <it>C. beijerinckii </it>3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the <it>R. eutropha </it>isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from <it>S. cerevisiae </it>or <it>E. coli </it>rather than that from <it>R. eutropha</it>. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from <it>C. beijerinckii </it>(<it>bcd </it>and <it>etfAB</it>) did not improve butanol production significantly as previously reported in <it>E. coli</it>. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.</p