Synergistic substrate cofeeding stimulates reductive metabolism

Advanced bioproduct synthesis via reductive metabolism requires coordinating carbons, ATP and reducing agents, which are generated with varying efficiencies depending on metabolic pathways. Substrate mixtures with direct access to multiple pathways may optimally satisfy these biosynthetic requirements. However, native regulation favouring preferential use precludes cells from co-metabolizing multiple substrates. Here we explore mixed substrate metabolism and tailor pathway usage to synergistically stimulate carbon reduction. By controlled cofeeding of superior ATP and NADPH generators as ‘dopant’ substrates to cells primarily using inferior substrates, we circumvent catabolite repression and drive synergy in two divergent organisms. Glucose doping in Moorella thermoacetica stimulates CO2 reduction (2.3 g gCDW−1 h−1) into acetate by augmenting ATP synthesis via pyruvate kinase. Gluconate doping in Yarrowia lipolytica accelerates acetate-driven lipogenesis (0.046 g gCDW−1 h−1) by obligatory NADPH synthesis through the pentose cycle. Together, synergistic cofeeding produces CO2-derived lipids with 38% energy yield and demonstrates the potential to convert CO2 into advanced bioproducts. This work advances the systems-level control of metabolic networks and CO2 use, the most pressing and difficult reduction challenge

Comparative Avian Cognition : Physical and Social Problem Solving in Corvids and Parrots

Within the last few decades, investigations of problem solving in avian species such as corvids have challenged the few that complex cognitive abilities are unique to the primate lineage, and provide a compelling case for the convergent evolution of cognition in a range of large-brained, socially complex species. Despite these advances, there is still much that is unknown about how corvids acquire and use information to solve problems in both their social and physical environments, and comparably little research has focused on other large-brained avian taxa such as parrots. The research presented in this thesis investigates both physical and social cognition in two parrot and two corvid species by examining how individuals interact with and acquire information about their physical world, and whether birds will use physical information to benefit conspecifics. Observational data provide new evidence for a novel form of tool use among a highly explorative species, the greater vasa parrot (Coracopsis vasa), and empirical data show that exploration may provide these birds with information about how novel objects (including potential tools) behave. An additional experiment with kea (Nestor notabilis) and New Caledonian crows (Corvus moneduloides) suggests that information acquired during exploration may aid in problem solving, although individuals do not change their exploratory behaviour in order to acquire functional information about objects that is relevant to a specific task. An investigation of social cognition in ravens (Corvus corax) shows that subjects can attend to multiple dynamic stimuli in order to obtain a food reward, but do not use this ability to provide food to an affiliate or non-affiliate partner. Taken together, the results of these studies suggest that both parrots and corvids are adept at attending to and learning about different types of physical information which can aid problem solving, but do not intentionally seek this information or use it to benefit others

Designing a New Entry Point into Isoprenoid Metabolism by Exploiting Fructose-6-Phosphate Aldolase Side Reactivity of <i>Escherichia coli</i>

The 2C-methyl-d-erythritol-4-phosphate (MEP) pathway in <i>Escherichia coli</i> has been highlighted for its potential to provide access to myriad isoprenoid chemicals of industrial and therapeutic relevance and discover antibiotic targets to treat microbial human pathogens. Here, we describe a metabolic engineering strategy for the <i>de novo</i> construction of a biosynthetic pathway that produces 1-dexoxy-d-xylulose-5-phosphate (DXP), the precursor metabolite of the MEP pathway, from the simple and renewable starting materials d-arabinose and hydroxyacetone. Unlike most metabolic engineering efforts in which cell metabolism is reprogrammed with enzymes that are highly specific to their desired reaction, we highlight the promiscuous activity of the native <i>E. coli</i> fructose-6-phosphate aldolase as central to the metabolic rerouting of carbon to DXP. We use mass spectrometric isotopomer analysis of intracellular metabolites to show that the engineered pathway is able to support <i>in vivo</i> DXP biosynthesis in <i>E. coli</i>. The engineered DXP synthesis is further able to rescue cells that were chemically inhibited in their ability to produce DXP and to increase terpene titers in strains harboring the non-native lycopene pathway. In addition to providing an alternative metabolic pathway to produce isoprenoids, the results here highlight the potential role of pathway evolution to circumvent metabolic inhibitors in the development of microbial antibiotic resistance

The effects of Fe-S operons on the amorphadiene production.

<p>Different concentrations of IPTG were represented by bars with different colors. The experiment was repeated four times and the standard errors of four replicates were presented as error bars. The two tailed p-values of student’s t-test were carried out to compare certain conditions and presented as P in the figure.</p

The cross-lapping in vitro assembly (CLIVA) method.

<p>(A) Illustration of the design at one junction between two modules (blue and red). The cross-lapping primer consists of gene specific sequence (GSS) and tag sequence complementary to adjacent primer’s GSS. The phosphorothioate modifications were indicated as cycles. An “Ox/y” designation was used to define the primers, where O denoted overlap; x was the length of overlap which had one modification at each y base pairs of the sequence. (B) Illustration of assembling of multiple DNA modules into one plasmid. </p

Effects of UPLC gradient on chromatography separation of the DXP pathway intermediates.

<p>Different concentrations (0.8, 0.4, 2.6, 2.6, 0.5 and 2 µM respectively) of DXP, MEP, CDP-ME, CDP-MEP, MEC, and HMBPP were prepared in 1 mL acidic extraction solution and purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#s4" target="_blank">METHODS AND MATERIALS</a>. Quantification m/z ratio for each compound was extracted from total ion chromatography as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t001" target="_blank">Table 1</a> and traces were overlaid. (A) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a> except 40% aqueous solution was used in step 3 and 4; (B) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a> except 50% aqueous solution was used in step 3 and 4; (C) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a>; (D) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a> except 70% aqueous solution was used in step 3 and 4; (E) UPLC gradient described was employed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047513#pone-0047513-t002" target="_blank">Table 2</a> except 80% aqueous solution was used in step 3 and 4.</p

The effects of fosmidomycin inhibition of dxr on DXP metabolism.

<p>Twenty five microgram per milliliter fosmidomycin was used to inhibit MEC biosynthesis at 4 h after induction, and the metabolites of BL21 Gold (DE3) harboring pET-SIDF and pAC-LYC were analyzed before and 2 h after inhibition; Presented data were average of triplicates and standard errors were drawn on the plot.</p

The performance of different combinations of DXP pathway genes in <i>E</i>. <i>coli</i>.

<p>(A) 48h amorphadiene yield. Different concentrations of IPTG were represented by bars with different colors. The experiment was repeated four times and the standard errors were shown. (B) The correlation of pathway modules with amorphadiene yield at optimal IPTG inductions. (C) Early response of intracellular metabolites at 3h after induction. The gray areas indicated the overexpressed section of DXP pathway. The experiment was repeated twice and the averages were shown. </p

Method for increased gene targeting.

<p>Cells are grown in the presence of hydroxyurea to induce cell cycle arrest in S-phase with high HR activity (a). <i>Y</i>. <i>lipolytica</i> YB-392 cells untreated or arrested at the large-budded stage are shown. HU-arrested cells are transformed with an antibiotic resistance cassette bearing the marker flanked by short regions of homology to the promoter and terminator of the target gene (b). Homologous recombination between the cassette and genomic DNA leads to replacement of the target gene with the marker (c). Antibiotic-resistant colonies are screened by PCR to distinguish between random and targeted integration using primer sets specific to each integration outcome (d).</p

Mobile phase gradient used for the separation of DXP intermediates in the UPLC method.

*<p>Aqueous solution: 15 mM acetic acid and 10 mM tributylamine.</p