Transcriptional regulation of living materials via extracellular electron transfer.

Engineered living materials combine the advantages of biological and synthetic systems by leveraging genetic and metabolic programming to control material-wide properties. Here, we demonstrate that extracellular electron transfer (EET), a microbial respiration process, can serve as a tunable bridge between live cell metabolism and synthetic material properties. In this system, EET flux from Shewanella oneidensis to a copper catalyst controls hydrogel cross-linking via two distinct chemistries to form living synthetic polymer networks. We first demonstrate that synthetic biology-inspired design rules derived from fluorescence parameterization can be applied toward EET-based regulation of polymer network mechanics. We then program transcriptional Boolean logic gates to govern EET gene expression, which enables design of computational polymer networks that mechanically respond to combinations of molecular inputs. Finally, we control fibroblast morphology using EET as a bridge for programmed material properties. Our results demonstrate how rational genetic circuit design can emulate physiological behavior in engineered living materials.

CsrA selectively modulates sRNA-mRNA regulator outcomes.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact directly with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcases CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

CsrA Shows Selective Regulation of sRNA-mRNA Networks.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcase CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

RNP-Based Control Systems for Genetic Circuits in Synthetic Biology Beyond CRISPR.

Ribonucleoproteins (RNPs) are RNA-protein complexes utilized natively in both prokaryotes and eukaryotes to regulate essential processes within the cell. Over the past few years, many of these native systems have been adapted to provide control over custom genetic targets. Engineered RNP-based control systems allow for fine-tune regulation of desired targets, by providing customizable nucleotide-nucleotide interactions. However, as there have been several engineered RNP systems developed recently, identifying an optimal system for various bioprocesses is challenging. Here, we review the most successful engineered RNP systems and their applications to survey the current state of the field. Additionally, we provide selection criteria to provide users a streamlined method for identifying an RNP control system most useful to their own work. Lastly, we discuss future applications of RNP control systems and how they can be utilized to address the current grand challenges of the synthetic biology community.

Production of 1-octanol in Escherichia coli by a high flux thioesterase route.

1-octanol is a valuable molecule in the chemical industry, where it is used as a plasticizer, as a precursor in the production of linear low-density polyethylene (LLDPE), and as a growth inhibitor of tobacco plant suckers. Due to the low availability of eight-carbon acyl chains in natural lipid feedstocks and the selectivity challenges in petrochemical routes to medium-chain fatty alcohols,1-octanol sells for the highest price among the fatty alcohol products. As an alternative, metabolic engineers have pursued sustainable 1-octanol production via engineered microbes. Here, we report demonstration of gram per liter titers in the model bacterium Escherichia coli via the development of a pathway composed of a thioesterase, an acyl-CoA synthetase, and an acyl-CoA reductase. In addition, the impact of deleting fermentative pathways was explored E. coli K12 MG1655 strain for production of octanoic acid, a key octanol precursor. In order to overcome metabolic flux barriers, bioprospecting experiments were performed to identify acyl-CoA synthetases with high activity towards octanoic acid and acyl-CoA reductases with high activity to produce 1-octanol from octanoyl-CoA. Titration of expression of key pathway enzymes was performed and a strain with the full pathway integrated on the chromosome was created. The final strain produced 1-octanol at 1.3 g/L titer and a >90% C8 specificity from glycerol. In addition to the metabolic engineering efforts, this work addressed some of the technical challenges that arise when quantifying 1-octanol produced from cultures grown under fully aerobic conditions where evaporation and stripping are prevalent.

Metabolic engineering of ß-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone.

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the ß-oxidation cycle at an advantageous reaction, and introducing active ß-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli ß-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding ß-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to ß-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

Highly Active C8-Acyl-ACP Thioesterase Variant Isolated by a Synthetic Selection Strategy.

Microbial metabolism is an attractive route for producing medium chain length fatty acids, e.g., octanoic acid, used in the oleochemical industry. One challenge to this strategy is the lack of enzymes that are both highly active in a microbial host and selective toward substrates with desired chain length. Of the many steps in fatty acid biosynthesis, the thioesterase is the most widely used enzyme for controlling chain length. Thioesterases hydrolyze the thioester bond between fatty acids and the acyl-carrier protein (ACP) or coenzyme A (CoA) cofactor. The functional role of thioesterases varies between organisms ( i.e., bacteria vs plant) and therefore so do the substrate specificities. As a result, microbial biocatalysts that utilize a heterologous thioesterase either produce high titers of fatty acids with mixed chain lengths or low titers of products with a narrow chain length distribution. To search for highly active enzymes that selectively hydrolyze octanoyl-ACP, we developed a genetic selection based on the lipoic acid requirement of Escherichia coli. We used the selection to identify variants in a randomly mutagenized library of the C8-specific Cuphea palustris FatB1 thioesterase. After optimizing expression of the thioesterase, E. coli cultures produced 1.7 g/L of octanoic acid with >90% specificity from a single chromosomal copy of this thioesterase. In vitro studies confirmed the mutant thioesterase possessed a 15-fold increase in kcat compared to its native sequence. The high level of specific activity allowed for low levels of expression while maintaining fatty acid titer. The low expression requirement will allow metabolic engineers to use more cellular resources to address other limitations in the pathway and maximize overall productivity.

Nickel-centred proton reduction catalysis in a model of [NiFe] hydrogenase.

Hydrogen production through water splitting is one of the most promising solutions for the storage of renewable energy. [NiFe] hydrogenases are organometallic enzymes containing nickel and iron centres that catalyse hydrogen evolution with performances that rival those of platinum. These enzymes provide inspiration for the design of new molecular catalysts that do not require precious metals. However, all heterodinuclear NiFe models reported so far do not reproduce the Ni-centred reactivity found at the active site of [NiFe] hydrogenases. Here, we report a structural and functional NiFe mimic that displays reactivity at the Ni site. This is shown by the detection of two catalytic intermediates that reproduce structural and electronic features of the Ni-L and Ni-R states of the enzyme during catalytic turnover. Under electrocatalytic conditions, this mimic displays high rates for H2 evolution (second-order rate constant of 2.5 × 104 M-1 s-1; turnover frequency of 250 s-1 at 10 mM H+ concentration) from mildly acidic solutions.

Novel B(Ar')2(Ar'') hetero-tri(aryl)boranes: a systematic study of Lewis acidity.

A series of homo- and hetero-tri(aryl)boranes incorporating pentafluorophenyl, 3,5-bis(trifluoromethyl)phenyl, and pentachlorophenyl groups, four of which are novel species, have been studied as the acidic component of frustrated Lewis pairs for the heterolytic cleavage of H2. Under mild conditions eight of these will cleave H2; the rate of cleavage depending on both the electrophilicity of the borane and the steric bulk around the boron atom. Electrochemical studies allow comparisons of the electrophilicity with spectroscopic measurements of Lewis acidity for different series of boranes. Discrepancies in the correlation between these two types of measurements, combined with structural characterisation of each borane, reveal that the twist of the aryl rings with respect to the boron-centred trigonal plane is significant from both a steric and electronic perspective, and is an important consideration in the design of tri(aryl)boranes as Lewis acids.

Facile Protocol for Water-Tolerant "Frustrated Lewis Pair"-Catalyzed Hydrogenation.

Despite rapid advances in the field of metal-free, "frustrated Lewis pair" (FLP)-catalyzed hydrogenation, the need for strictly anhydrous reaction conditions has hampered wide-scale uptake of this methodology. Herein, we report that, despite the generally perceived moisture sensitivity of FLPs, 1,4-dioxane solutions of B(C6F5)3 actually show appreciable moisture tolerance and can catalyze hydrogenation of a range of weakly basic substrates without the need for rigorously inert conditions. In particular, reactions can be performed directly in commercially available nonanhydrous solvents without subsequent drying or use of internal desiccants.

Spectroscopic Characterization of the Bridging Amine in the Active Site of [FeFe] Hydrogenase Using Isotopologues of the H-Cluster.

The active site of [FeFe] hydrogenase contains a catalytic binuclear iron subsite coordinated by CN(-) and CO ligands as well as a unique azadithiolate (adt(2-)) bridging ligand. It has been established that this binuclear cofactor is synthesized and assembled by three maturation proteins HydE, -F, and -G. By means of in vitro maturation in the presence of (15)N- and (13)C-labeled tyrosine it has been shown that the CN(-) and CO ligands originate from tyrosine. The source of the bridging adt(2-) ligand, however, remains unknown. In order to identify the nitrogen of the bridging amine using HYSCORE spectroscopy and distinguish its spectroscopic signature from that of the CN(-) nitrogens, we studied three isotope-labeled variants of the H-cluster ((15)N-adt(2-)/C(14)N(-), (15)N-adt(2-)/C(15)N(-), and (14)N-adt(2-)/C(15)N(-)) and extracted accurate values of the hyperfine and quadrupole couplings of both CN(-) and adt(2-) nitrogens. This will allow an evaluation of isotopologues of the H-cluster generated by in vitro bioassembly in the presence of various (15)N-labeled potential precursors as possible sources of the bridging ligand.

Artificially maturated [FeFe] hydrogenase from Chlamydomonas reinhardtii: a HYSCORE and ENDOR study of a non-natural H-cluster.

Hydrogenases are enzymes that catalyze the oxidation of H2 as well as the reduction of protons to form H2. The active site of [FeFe] hydrogenase is referred to as the "H-cluster" and consists of a "classical" [4Fe-4S] cluster connected via a bridging cysteine thiol group to a unique [2Fe]H sub-cluster, containing CN(-) and CO ligands as well as a bidentate azadithiolate ligand. It has been recently shown that the biomimetic [Fe2(adt)(CO)4(CN)2](2-) (adt(2-) = azadithiolate) complex resembling the diiron sub-cluster can be inserted in vitro into the apo-protein of [FeFe] hydrogenase, which contains only the [4Fe-4S] part of the H-cluster, resulting in a fully active enzyme. This synthetic tool allows convenient incorporation of a variety of diiron mimics, thus generating hydrogenases with artificial active sites. [FeFe] hydrogenase from Chlamydomonas reinhardtii maturated with the biomimetic complex [Fe2(pdt)(CO)4(CN)2](2-) (pdt(2-) = propanedithiolate), in which the bridging adt(2-) ligand is replaced by pdt(2-), can be stabilized in a state strongly resembling the active oxidized (Hox) state of the native protein. This state is EPR active and the signal originates from the mixed valence Fe(I)Fe(II) state of the diiron sub-cluster. Taking advantage of the variant with (15)N and (13)C isotope labeled CN(-) ligands we performed HYSCORE and ENDOR studies on this hybrid protein. The (13)C hyperfine couplings originating from both CN(-) ligands were determined and assigned. Only the (15)N coupling from the CN(-) ligand bound to the terminal iron was observed. Detailed orientation selective ENDOR and HYSCORE experiments at multiple field positions enabled the extraction of accurate data for the relative orientations of the nitrogen and carbon hyperfine tensors. These data are consistent with the crystal structure assuming a g-tensor orientation following the local symmetry of the binuclear sub-cluster.

Electronic control of the protonation rates of Fe-Fe bonds.

Protonation at metal-metal bonds is of fundamental interest in the context of the function of the active sites of hydrogenases and nitrogenases. In diiron dithiolate complexes bearing carbonyl and electron-donating ligands, the metal-metal bond is the highest occupied molecular orbital (HOMO) with a "bent" geometry. Here we show that the experimentally measured rates of protonation (kH) of this bond and the energy of the HOMO as measured by the oxidation potential of the complexes (E1/2(ox)) correlate in a linear free energy relationship: ln kH = ((F(c - ßE1/2(ox)))/(RT)), where c is a constant and ß is the dimensionless Brønsted coefficient. The value of ß of 0.68 is indicative of a strong dependence upon energy of the HOMO: measured rates of protonation vary over 6 orders of magnitude for a change in E1/2(ox) of ca. 0.55 V (ca. 11 orders of magnitude/V). This relationship allows prediction of protonation rates of systems that are either too fast to measure experimentally or that possess additional protonation sites. It is further suggested that the nature of the bridgehead in the dithiolate ligand can exert a stereoelectronic influence: bulky substituents destabilize the HOMO, thereby increasing the rate of protonation.

The mixed diol-dithiol 2,2-bis(sulfanylmethyl)propane-1,3-diol: characterization of key intermediates on a new synthetic pathway.

A new synthetic route to 2,2-bis(sulfanylmethyl)propane-1,3-diol, (II), is described starting from the commercially available 2,2-bis(hydroxymethyl)propane-1,3-diol. The structures of two intermediates on this route are described. 5,5-Dimethenyl-2,2-dimethyl-1,3-dioxane bis(thiocyanate) (systematic name: {[5-(cyanosulfanyl)-2,2-dimethyl-1,3-dioxan-5-yl]sulfanyl}formonitrile), C(10)H(14)N(2)O(2)S(2), (X), crystallizes in the space group P2(1)/c with no symmetry relationship between the two thiocyanate groups. There is a short intramolecular N...S contact for one thiocyanate group, while the second group is positioned such that this type of interaction is not possible. 1,3-(Hydroxymethyl)propane-1,3-diyl bis(thiocyanate), C(7)H(10)N(2)O(2)S(2), (XI), also features a single short N···S contact in the solid state. Hydrogen bonding between two molecules of compound (XI) results in the formation of dimers in the crystal, which are then linked together by a second hydrogen-bond interaction between the dimers. In addition, the structures of two intermediates from an unsuccessful alternative synthesis of (II) are reported. 2,2-Bis(chloromethyl)propane-1,3-diol, C(5)H(10)Cl(2)O(2), (VI), crystallized as an inversion twin with a minor twin fraction of 0.43 (6). It forms a zigzag structure as a result of intermolecular hydrogen bonding. The structure of 9,9-dimethyl-2,4,8,10-tetraoxa-3λ(4)-thiaspiro[5.5]undecan-3-one, C(8)H(14)O(5)S, (VII), shows evidence for a weak S···O contact with a distance of 3.2529 (11) Å.