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A genotoxic carcinogen, N-nitrosodimethylamine (NDMA), was detected as a synthesis impurity in some valsartan drugs in 2018, and other N-nitrosamines, such as N-nitrosodiethylamine (NDEA), were later detected in other sartan products. N-nitrosamines are pro-mutagens that can react with DNA following metabolism to produce DNA adducts, such as O6 -alkyl-guanine. The adducts can result in DNA replication miscoding errors leading to GC>AT mutations and increased risk of genomic instability and carcinogenesis. Both NDMA and NDEA are known rodent carcinogens in male and female rats. The DNA repair enzyme, methylguanine DNA-methyltransferase can restore DNA integrity via the removal of alkyl groups from guanine in an error-free fashion and this can result in nonlinear dose responses and a point of departure or "practical threshold" for mutation at low doses of exposure. Following International recommendations (ICHM7; ICHQ3C and ICHQ3D), we calculated permissible daily exposures (PDE) for NDMA and NDEA using published rodent cancer bioassay and in vivo mutagenicity data to determine benchmark dose values and define points of departure and adjusted with appropriate uncertainty factors (UFs). PDEs for NDMA were 6.2 and 0.6 μg/person/day for cancer and mutation, respectively, and for NDEA, 2.2 and 0.04 μg/person/day. Both PDEs are higher than the acceptable daily intake values (96 ng for NDMA and 26.5 ng for NDEA) calculated by regulatory authorities using simple linear extrapolation from carcinogenicity data. These PDE calculations using a bench-mark approach provide a more robust assessment of exposure limits compared with simple linear extrapolations and can better inform risk to patients exposed to the contaminated sartans.

The increased reliance on natural gas for heating worldwide makes the search for carbon-free alternatives imperative, especially if international decarbonisation targets are to be met. Hydrogen does not release carbon dioxide (CO2) at the point of use which makes it an appealing candidate to decarbonise domestic heating. Hydrogen can be produced from either 1) the electrolysis of water with no associated carbon emissions, or 2) from methane reformation (using steam) which produces CO2, but which is easily captured and storable during production. Hydrogen could be transported to the end-user via gas distribution networks similar to, and adapted from, those in use today. This would reduce both installation costs and end-user disruption. However, before hydrogen can provide domestic heat, it is necessary to assess the ‘risk’ associated with its distribution in direct comparison to natural gas. Here we develop a comprehensive and multi-faceted quantitative risk assessment tool to assess the difference in ‘risk’ between current natural gas distribution networks, and the potential conversion to a hydrogen based system. The approach uses novel experimental and modelling work, scientific literature, and findings from historic large scale testing programmes. As a case study, the risk assessment tool is applied to the newly proposed H100 demonstration (100% hydrogen network) project. The assessment includes the comparative risk of gas releases both upstream and downstream of the domestic gas meter. This research finds that the risk associated with the proposed H100 network (based on its current design) is lower than that of the existing natural gas network by a factor 0.88.

The kcat/KM ratio holds substantial significance as a comparative figure of merit in the evaluation of enzymes with uni-molecular mechanisms. However, its applicability in the evaluation of multi-reactant systems is frequently not justified. Here, we derive figures of merit for multi-reactant enzyme mechanisms based on the stabilization of the transition states and the kinetic Haldane equilibrium relationships. For most bi-molecular reactions, the ratio of kcat/KiaKb (where Kia is the dissociation constant of A and Kb is the Michaelis constant of B) is best suited for comparing enzyme performance, especially at non-saturating reactant concentrations. The value of this parameter for assessing the performance of a series of oxidoreductase mutants is demonstrated. Figures of merit for other common enzymatic mechanisms are derived, which enable a theory-based approach for comparing steady-state catalytic performances of enzymes with multiple reactants.

The enzymatic microenvironment can impact biocatalytic activity; however, these effects can be difficult to investigate as mutations and fusions can introduce multiple variables and overlapping effects. The fusion of a supercharged protein is a potentially facile means to alter the enzymatic microenvironment. We have investigated complexes made between a thermostable alcohol dehydrogenase (AdhD) and superfolding green fluorescent protein (sfGFP) mutants with extreme surface charges. Three charged sfGFP variants, -30, 0, and +36 were covalently attached to AdhD through the SpyCatcher/SpyTag system. Specific rates for the NAD+ -dependent oxidation of butane-2,3-diol were significantly increased in the -30 sfGFP complex, a mixed effect was seen for the 0 sfGFP complexes, and the rates were unaffected by +36 sfGFP complexation. Reactions performed at various pH values (7.8-9.8) and salt concentrations (7.75-500 mm) showed that there was a complex interplay between these effects that was consistent with fusion proteins affecting the local ionic strength, as opposed to the local pH. Steady-state kinetic analyses were performed with the -30 and 0 AdhD-sfGFP complexes. The overall catalytic efficiency was dependent on the charge of the fused sfGFP variant; the -30 sfGFP fusions exhibited the largest beneficial effects at pH 8.8. The impact of the fusions on the apparent ionic strength provides further insight into the effects of charged patches observed on metabolon-forming enzyme complexes.

Decarbonising the residential heating and cooking sector is essential to meet national and international carbon emission reduction targets. Hydrogen has been identified by the scientific community, industry, and policy makers as part of the solution to this challenge. Hydrogen has been used for decades in many industries, formerly making up approximately 50% of the Town Gas used for heating and cooking in UK homes in the mid 20th century. It is now crucial to ensure safety regulations are met, and public acceptance gained, before hydrogen can start being used for residential heating. Demonstration projects require hydrogen distribution networks to be odorised. This study examines the use of sulphur-based odorants, which are currently in use in the UK and Europe to odorise Natural Gas, to be used in a 100% hydrogen gas demonstration network in the UK. We undertook a comparative testing programme to evaluate the escape detection properties of odorised hydrogen against odorised methane and natural gas. This comparative approach will help address the question asked by UK and EU regulators: is hydrogen ‘as safe as’ natural gas? The results show that untrained participants can identify an escaping gas odorised with Odorant New Blend and standby odorant 2, in hydrogen, natural gas or methane, at the regulatory threshold of 1% gas in air. These results contribute to the safety-case of H100 led by SGN.

Development of the bioeconomy is driven by our ability to access the energy-rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value-added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two-stage bioprocessing system pairing the anaerobic fungus Piromyces indianae with the yeast Kluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose. P. indianae efficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, which K. marxianus consumed while producing 2.4 g/L ethyl acetate. An engineered strain of K. marxianus was also able to produce 550 mg/L 2-phenylethanol and 150 mg/L isoamyl alcohol from P. indianae hydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.

With the discovery of Western blotting as first described by Towbin et al. in 1979, the transfer and visualization of electrophoretically separated proteins on membranes has become the de facto method for the qualitative and quantitative detection of proteins of interest. In this method, proteins are resolved by electrophoresis on a polyacrylamide gel, followed by a transfer of the separated proteins onto a nitrocellulose or polyvinyl difluoride (PVDF) membrane. Once immobilized on these membranes, the protein of interest can be detected and visualized by exploiting antigen-antibody interactions. However, not all proteins are amenable to easy detection by Western blotting. Integral membrane proteins are a class of proteins that are attached to a biological membrane through a series of transmembrane segments that span the width of the membrane. Due to the inherent hydrophobicity of these proteins and their tendency to aggregate, the characterization and detection of these proteins can be challenging. In this methods chapter, we present a protocol for the easy detection and quantification of these proteins in the industrially important oleaginous yeast Yarrowia lipolytica. The first protocol describes a Western blotting procedure to quantify soluble cytosolic proteins of interest in Yarrowia lipolytica from its total cell lysate. The second protocol describes modifications to the first that are done to enhance detection and quantification of membrane-bound proteins in Yarrowia lipolytica from its total cell lysate, without the need for isolating the membrane-bound proteins, for use in Western blotting. The immunoblotting strategies described here should serve as an efficient and simple guide to quantify both cytosolic and the intractable membrane-bound proteins in Yarrowia lipolytica.

GaAs compound-based electronics attracted significant interest due to unique properties of GaAs like high electron mobility, high saturated electron velocity and low sensitivity to heat. However, GaAs compound-based electronics demand a significant decrease in their manufacturing costs to be a good competitor in the commercial markets. In this context, copper-based nanoparticle (NP) inks represent one of the most cost-effective metal inks as a proper candidate to be deposited as contact grids on GaAs. In addition, Inkjet-printing, as a low-cost back-end of the line process, is a flexible manufacturing method to deposit copper NP ink on GaAs. These printed copper NP structures need to be uncapped and fused via a sintering method in order to become conductive and form an ohmic contact with low contact resistivity. The main challenge for uncapping a copper-based NP ink is its rapid oxidation potential. Laser sintering, as a fast uncapping method for NPs, reduces the oxidation of uncapped copper. The critical point to combine these two well-known industrial methods of inkjet printing and laser sintering is to adjust the printing features and laser sintering power in a way that as much copper as possible is uncapped resulting in minimum contact resistivity and high conductivity. In this research, copper ink contact grids were deposited on n-doped GaAs by inkjet-printing. The printed copper ink was converted to a copper grid via applying the optimized settings of a picosecond laser. As a result, an ohmic copper on GaAs contact with a low contact resistivity (8 mΩ cm2) was realized successfully.

Background 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 33-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

Kluyveromyces marxianus is an emerging host for metabolic engineering. This thermotolerant yeast is the fastest growing eukaryote, has high flux through the TCA cycle, and can metabolize a broad range of C5, C6, and C12 carbon sources. In comparison to the common host Saccharomyces cerevisiae, this non-conventional yeast suffers from a lack of metabolic engineering tools to control gene expression over a wide transcriptional range. To address this issue, we designed a library of 25 native-derived promoters from K. marxanius CBS6556 that spans 87-fold transcriptional strength under glucose metabolism. Six promoters from the library were further characterized in both glucose and xylose as well as across various temperatures from 30 to 45 ​°C. The temperature study revealed that in most cases EGFP expression decreased with elevating temperature; however, two promoters, PSSA3 and PADH1, increased expression above 40 ​°C in both xylose and glucose. The six-promoter set was also validated in xylose for triacetic acid lactone (TAL) production. By controlling the expression level of heterologous 2-pyrone synthase (2-PS), the specific TAL titer increased over 8-fold at 37 ​°C. Cultures at 41 ​°C exhibited a similar TAL biosynthesis capability, while at 30 ​°C TAL levels were lower. Taken together, these results advance the metabolic engineering tool set in K. marxianus and further develop this new host for chemical biosynthesis., Highlights • Library of 25 K. marxianus promoters spans nearly 2 order magnitude of transcriptional strength under glucose metabolism. • Promoters show similar strength trends with both glucose and xylose metabolism. • KmNC1 promoter shows highest strength among reported K. marxianus native promoters. • KmNC1 promoter increased specific triacetic acid lactone (TAL) titer by 80% compared with previous studies.

Vascular plants, unlike bryophytes, have a strong root-shoot dichotomy in which the tissue systems are mutually interdependent; roots are completely dependent on shoots for photosynthetic sugars, and shoots are completely dependent on roots for water and mineral nutrients. Long-distance communication between shoot and root is therefore critical for the growth, development and survival of vascular plants, especially with regard to variable environmental conditions. However, this long-distance signalling does not appear an ancestral feature of land plants, and has likely arisen in vascular plants to service the radical alterations in body-plan seen in this taxon. In this review, we examine the defined hormonal root-to-shoot and shoot-to-root signalling pathways that coordinate the growth of vascular plants, with a particular view to understanding how these pathways may have evolved. We highlight the completely divergent roles of isopentenyl-adenine and trans-zeatin cytokinin species in long-distance signalling, and ask whether cytokinin can really be considered as a single class of hormones in the light of recent research. We also discuss the puzzlingly sparse evidence for auxin as a shoot-to-root signal, the evolutionary re-purposing of strigolactones and gibberellins as hormonal signals, and speculate on the possible role of sugars as long-distance signals. We conclude by discussing the 'design principles' of long-distance signalling in vascular plants.

The emergence of CRISPR-Cas9 for targeted genome editing and regulation has enabled the manipulation of desired traits and enhanced strain development of nonmodel microorganisms. The natural capacity of the yeast Kluyveromyces marxianus to produce volatile esters at high rate and at elevated temperatures make it a potentially valuable production platform for industrial biotechnology. Here, we identify the native localization of ethyl acetate biosynthesis in K. marxianus and use this information to develop a multiplexed CRISPRi system for redirecting carbon flux along central metabolic pathways, increasing ethyl acetate productivity. First, we identified the primary pathways of precursor and acetate ester biosynthesis. A genetic knockout screen revealed that the alcohol acetyltransferase Eat1 is the critical enzyme for ethyl, isoamyl, and phenylethyl acetate production. Truncation studies revealed that high ester biosynthesis is contingent on Eat1 mitochondrial localization. As ethyl acetate is formed from the condensation of ethanol and acetyl-CoA, we modulated expression of the TCA cycle and electron transport chain genes using a highly multiplexed CRISPRi approach. The simultaneous knockdown of ACO2b, SDH2, RIP1, and MSS51 resulted in a 3.8-fold increase in ethyl acetate productivity over the already high natural capacity. This work demonstrates that multiplexed CRISPRi regulation of central carbon flux, supported by a fundamental understanding of pathway biochemistry, is a potent strategy for metabolic engineering in nonconventional microorganisms.

Protein engineering provides a means to alter protein structure leading to new functions. Much work has focused on the engineering of enzyme active sites to enhance catalytic activity, however there is an increasing trend towards engineering other aspects of biocatalysts as these efforts can also lead to useful improvements. This tutorial discusses recent advances in engineering an enzyme's local chemical and physical environment, with the goal of enhancing enzyme reaction kinetics, substrate selectivity, and activity in harsh conditions (e.g., low or high pH). By introducing stimuli-responsiveness to these enzyme modifications, dynamic control of activity also becomes possible. These new biomolecular and protein engineering techniques are separate and independent from traditional active site engineering and can therefore be applied synergistically to create new biocatalyst technologies with novel functions.

This work combines the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus and the organic electrocatalyst TEMPO to create a bifunctional catalyst that selectively oxidizes primary and secondary alcohols. The active sites function independently, can be switched on by changing reaction conditions, and can selectively oxidize a mixture of 1- and 2-butanol. The NAD+-dependent enzyme catalyses the secondary alcohol oxidation at a rate 3-fold faster than the primary alcohol, while the covalently attached 4-glycidyl-TEMPO oxidizes 1-butanol and has negligible activity toward 2-butanol. This hybrid catalytic approach has potential value for selective alcohol oxidations as well as other electrochemical and enzymatic multistep processes in energy conversion and chemical synthesis.

The past decade has seen significant government and private investment in fundamental research and process development for the production of biofuels and chemicals from lignocellulosic biomass-derived sugars. This investment has helped create new metabolic engineering and synthetic biology approaches, novel homogeneous and heterogeneous catalysts, and chemical and biological routes that convert sugars, lignin, and waste products such as glycerol into hydrocarbon fuels and valuable chemicals. With the exception of ethanol, economical biofuels processes have yet to be realized. A potentially viable way forward is the integration of biological and chemical catalysis into processes that exploit the inherent advantages of each technology while circumventing their disadvantages. Microbial fermentation excels at converting sugars from low-cost raw materials streams into simple alcohols, acids, and other reactive intermediates that can be condensed into highly reduced, long and branched chain hydrocarbons and other industrially useful compounds. Chemical catalysis most often requires clean feed streams to avoid catalyst deactivation, but the chemical and petroleum industries have developed large scale processes for C-C coupling, hydrogenation, and deoxygenation that are driven by low grade heat and low-cost feeds such as hydrogen derived from natural gas. In this context, we suggest that there is a reasonably clear route to the high yield synthesis of biofuels from biomass- or otherwise derived-fermentable sugars: the microbial production of reactive intermediates that can be extracted or separated into clean feed stream for upgrading by chemical catalysis. When coupled with new metabolic engineering strategies that maximize carbon and energy yields during fermentation, biomass-to-fuels processes may yet be realized.

Eukaryotic biochemistry is organized throughout the cell in and on membrane-bound organelles. When engineering metabolic pathways this organization is often lost, resulting in flux imbalance and a loss of kinetic advantages from enzyme colocalization and substrate channeling. Here, we develop a protein-based scaffold for colocalizing multienzyme pathways on the membranes of intracellular lipid droplets. Scaffolds based on the plant lipid droplet protein oleosin and cohesin-dockerin interaction pairs recruited upstream enzymes in yeast ester biosynthesis to the native localization of the terminal reaction step, alcohol-O-acetyltransferase (Atf1). The native localization is necessary for high activity and pathway assembly in close proximity to Atf1 increased pathway flux. Screening a library of scaffold variants further showed that pathway structure can alter catalysis and revealed an optimized scaffold and pathway expression levels that produced ethyl acetate at a rate nearly 2-fold greater than unstructured pathways. This strategy should prove useful in spatially organizing other metabolic pathways with key lipid droplet-localized and membrane-bound reaction steps.

Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in KM and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased KM by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.

The heat protective performance of thermal protective fabrics is related to the multiscale structure of the fibrous material and the composite design of a multilayered fabric system. In this study, a molecular interpretation is provided to explain the structural changes within fibers to correlate with the mechanical properties of the outermost layer fabric made up of m-aramid blended with (5–10) % p-aramid upon various radiant heat exposures. An increase in crystallinity due to limited polymer chain movement but no change in lamellar spacing was observed when fabrics were exposed below the glass transition temperature which resulted in no change in mechanical properties. A noticeable increase in lamellar spacing and crystallinity was obtained when the temperature during the heat exposures is in the proximity of 375 °C of the fabric layer due to the fibrillar-to-lamellar transformation. Even though no visible changes were observed by scanning electron microscopy (SEM) in the fabric exposed to the aforementioned thermal conditions, we found that their mechanical properties are compromised due to the structural changes within fibers of the fabrics. Therefore, further use of fabrics could be potentially dangerous for the user. Furthermore, for expoheat exposures where the temperature of the fabrics reaches above the degradation point, thermal decomposition occurs which is noticed by its hard and brittle behavior. The inner layers of both the fabric systems mostly remain intact until the thermal degradation of the outermost layers. This study provides an in-depth understanding of molecular mechanisms of structural changes that are in line with changes in the mechanical properties. The understanding of the structure-mechanical property relationship could serve as basic knowledge for the design and fabrication of high-performance fabrics for various fire environments.

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems.

Millions of years of evolution have produced biological systems capable of efficient one-pot multi-step catalysis. The underlying mechanisms that facilitate these reaction processes are increasingly providing inspiration in synthetic chemistry. Substrate channelling, where intermediates between enzymatic steps are not in equilibrium with the bulk solution, enables increased efficiencies and yields in reaction and diffusion processes. Here, we review different mechanisms of substrate channelling found in nature and provide an overview of the analytical methods used to quantify these effects. The incorporation of substrate channelling into synthetic cascades is a rapidly developing concept, and recent examples of the fabrication of cascades with controlled diffusion and flux of intermediates are presented.

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl-CoA with an alcohol by alcohol-O-acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short- and medium-chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf-S.l). Atf1-S.l exhibited broad specificity towards acyl-CoAs with chain length from C4 to C10 and was specific towards 1-pentanol. The AATase screen also revealed new acyl-CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf-C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester-based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.

Self-assembled hydrogels based on the industrially-relevant 1,3:2,4-dibenzylidene sorbitol framework functionalised with reactive acyl hydrazide (DBS-CONHNH2) peripheral groups react with aldehydes without disrupting the nanoscale gel network, adapting gel performance, and dynamically selecting specific aldehyde components from complex mixtures.

Background With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver’s recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. Results The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. Conclusion In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

The development of biomaterials with embedded enzymatic activities has been driven by a range of applications including tissue engineering, biosensors, and bioenergy applications. Advances in the design and production of peptide-based biomaterials have inspired protein engineers to begin creating enzymes with self-assembling, biomaterial forming capabilities. Outfitting enzymes with cross-link forming domains allows biomaterials to be created with a range of benefits including simple low-cost production, homogenous dispersion of activity in the hydrogels, and the ability to colocalize enzymes to create multistep cascades in the hydrogels. Just as natural hydrogels have evolved to exhibit important material and catalytic properties, designed bifunctional proteins that enable colocalization of activity within biomaterials are poised to further advance a range of biocatalytic, biomedical, and biotechnological applications.

Enzyme–DNA nanostructures were designed to introduce new substrate–enzyme interactions into their reactions, which altered enzyme kinetics in a predictable manner. The designed enzymes demonstrate a new strategy of enzyme engineering based on the rational design of intermolecular interactions outside of the active site that enhance and control enzyme kinetics. Binding interactions between tetramethylbenzidine and DNA attached to horseradish peroxidase (HRP) resulted in a reduced Michaelis constant (KM) for the substrate. The enhancement increased with stronger interactions in the micromolar range, resulting in a 2.6 fold increase in kcat/KM. The inhibition effect of 4-nitrobenzoic hydrazide on HRP was also significantly enhanced by tuning the binding to HRP–DNA. Lastly, binding of a nicotinamide adenine dinucleotide (NAD(H)) cofactor mimic, nicotinamide mononucleotide (NMN(H)), to an aldo-keto reductase (AdhD) was enhanced by introducing NMN(H)–DNA interactions.

The yeast Yarrowia lipolytica has been widely studied for its ability to synthesize and accumulate intracellular lipids to high levels. Recent studies have identified native genes that enable growth on biomass-derived sugars, but these genes are not sufficiently expressed to facilitate robust metabolism. In this work, a CRISPR-dCas9 activation (CRISPRa) system in Y. lipolytica is developed and is used it to activate native β-glucosidase expression to support growth on cellobiose. A series of different transcriptional activators are compared for their effectiveness in Y. lipolytica, with the synthetic tripartite activator VPR yielding the highest activation. A VPR-dCas9 fusion is then targeted to various locations in a synthetic promoter driving hrGFP expression, and activation is achieved. Subsequently, the CRISPRa system is used to activate transcription of two different native β-glucosidase genes, facilitating enhanced growth on cellobiose as the sole carbon source. This work expands the synthetic biology toolbox for metabolic engineering in Y. lipolytica and demonstrates how the programmability of the CRISPR-Cas9 system can enable facile investigation of transcriptionally silent regions of the genome.

Carotenoids are a class of molecules with commercial value as food and feed additives with nutraceutical properties. Shifting carotenoid synthesis from petrochemical-based precursors to bioproduction from sugars and other biorenewable carbon sources promises to improve process sustainability and economics. In this work, we engineered the oleaginous yeast Yarrowia lipolytica to produce the carotenoid lycopene. To enhance lycopene production, we tested a series of strategies to modify host cell physiology and metabolism, the most successful of which were mevalonate pathway overexpression and alleviating auxotrophies previously engineered into the PO1f strain of Y. lipolytica. The beneficial engineering strategies were combined into a single strain, which was then cultured in a 1-L bioreactor to produce 21.1 mg/g DCW. The optimized strain overexpressed a total of eight genes including two copies of HMG1, two copies of CrtI, and single copies of MVD1, EGR8, CrtB, and CrtE. Recovering leucine and uracil biosynthetic capacity also produced significant enhancement in lycopene titer. The successful engineering strategies characterized in this work represent a significant increase in understanding carotenoid biosynthesis in Y. lipolytica, not only increasing lycopene titer but also informing future studies on carotenoid biosynthesis.

Background The thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the biochemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C5, C6, and C12 sugars into the volatile short-chain ester ethyl acetate. Strain engineering, however, has been limited due to a lack of advanced genome-editing tools and an incomplete understanding of ester and ethanol biosynthesis. Results Enabled by the design of hybrid RNA polymerase III promoters, this work adapts the CRISPR–Cas9 system from Streptococcus pyogenes for use in K. marxianus. The system was used to rapidly create functional disruptions to alcohol dehydrogenase (ADH) and alcohol-O-acetyltransferase (ATF) genes with putative function in ethyl acetate and ethanol biosynthesis. Screening of the KmATF disrupted strain revealed that Atf activity contributes to ethyl acetate biosynthesis, but the knockout reduced ethyl acetate titers by only ~15%. Overexpression experiments revealed that KmAdh7 can catalyze the oxidation of hemiacetal to ethyl acetate. Finally, analysis of the KmADH2 disrupted strain showed that the knockout almost completely eliminated ethanol production and resulted in the accumulation of acetaldehyde. Conclusions Newly designed RNA polymerase III promoters for sgRNA expression in K. marxianus enable a CRISPR–Cas9 genome-editing system for the thermotolerant yeast. This system was used to disrupt genes involved in ethyl acetate biosynthesis, specifically KmADH1–7 and KmATF. KmAdh2 was found to be critical for aerobic and anaerobic ethanol production. Aerobically produced ethanol supplies the biosynthesis of ethyl acetate catalyzed by KmAtf. KmAdh7 was found to exhibit activity toward the oxidation of hemiacetal, a possible alternative route for the synthesis of ethyl acetate. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0854-5) contains supplementary material, which is available to authorized users.

The engineering of Yarrowia lipolytica to accumulate lipids with high titers and productivities has been enabled with a handful of constitutive promoters for pathway engineering. However, the development of promoters that are both strong and lipid responsive could greatly benefit the bioproduction efficiency of lipid-derived oleochemicals in oleaginous yeast. In this study, a fatty acid regulated hybrid promoter for use in Y. lipolytica is engineered. A 200 bp upstream regulatory sequence in the peroxisomal acyl CoA oxidase 2 (POX2) promoter is identified. Further analysis of the promoter sequence reveal a regulatory sequence, that when used in tandem repeats, lead to a 48-fold induction of gene expression relative to glucose and fourfold higher than the native POX2 promoter. To date, this is the strongest inducible promoter reported in Y. lipolytica. Taken together, the results show that it is possible to engineer strong promoters that retain strong inducibility. These types of promoters will be useful in controlling metabolism and as fatty acid sensors.

New developments in nucleic acid nanotechnology and protein scaffold designs have enabled unparalleled control over the spatial organization of synthetic multienzyme cascade reactions. One of the goals of these new technologies is to create nanostructured enzyme cascade reactions that promote substrate channeling along the cascade and, in doing so, enhance cascade catalysis. The concept of substrate channeling has a long and rich history in biochemistry and has established methods of evaluation and quantification. In this Perspective, we review the most common of these methods and discuss them in the context of engineered multienzyme systems and natural bifunctional enzymes with known mechanisms of substrate channeling. In addition, we use experimental data and the results of simulations of coupled-enzyme reactions to develop a set of preliminary design rules for engineering multienzyme nanostructures. The design rules address the limitations on interenzyme distance and active site orientation in substrat...

Windows are a major source of energy inefficiency in buildings. In addition, heating by thermal radiation reduces the efficiency of photovoltaic panels. To help reduce heating by solar absorption in both of these cases, we developed a thin, transparent, bio-inspired, convective cooling layer for building windows and solar panels that contains microvasculature with millimeter-scale, fluid-filled channels. The thin cooling layer is composed of optically clear silicone rubber with microchannels fabricated using microfluidic engineering principles. Infrared imaging was used to measure cooling rates as a function of flow rate and water temperature. In these experiments, flowing room temperature water at 2 mL/min reduced the average temperature of a model 10×10 cm 2 window by approximately 7–9 °C. An analytic steady-state heat transfer model was developed to augment the experiments and make more general estimates as functions of window size, channel geometry, flow rate, and water temperature. Thin cooling layers may be added to one or more panes in multi-pane windows or as thin film non-structural central layers. Lastly, the color, optical transparency and aesthetics of the windows could be modulated by flowing different fluids that differ in their scattering or absorption properties.

The simulation of tunnel junctions is performed by using nonlocal band-to-band and trap assisted tunneling models that are capable of reproducing the experimental current-voltage characteristics of p++AlGaAs/ n++AlGaAs and p++AlGaAs/ n++GaAs based devices. These simulated characteristics are then implemented within a lattice matched InGaP/(In)GaAs/Ge multijunction solar cell (MJSC) to assess the performance as a function of tunnel junction layer doping in the regime where the TJ limits the performance of the MJSC. At 500 suns, a 4.6% absolute drop in simulated efficiency is observed for an AlGaAs/GaAs bottom TJ corresponding to a degenerately p-doped layer of 2.5 × 1019 cm-3 compared to a TJ with a doping of 4×1020 cm-3. A minimum p++ doping level of 3.3 × 10 19 cm-3 is required in order to avoid bottom TJ limitation up to 1000 suns concentration for an n++ doping of 2 × 1019 cm-3 based on the calibrated models. Furthermore, the effects of the peak and valley current densities are shown to have a strong influence on the efficiency over concentration within the TJ limiting regime.

A novel approach to the design of multijunction solar cells on silicon substrates for 1-sun applications is described. Models for device simulation, including porous silicon layers, are presented. A silicon bottom subcell is formed by diffusion of dopants into a silicon wafer. The top of the wafer is porosified to create a compliant layer, and a III-V buffer layer is then grown epitaxially, followed by middle and top subcells. Because of the resistivity of the porous material, these designs are best suited to high-efficiency 1-sun applications. Numerical simulations of a multijunction solar cell that incorporates a porous silicon-compliant membrane indicate an efficiency of 30.7% under AM1.5G, 1-sun for low-threading dislocation density, decreasing to 23.7% for a TDD of 107 cm-2.

The key characteristics of quantum dot (QD)-enhanced multijunction solar cells (MJSC) are explored theoretically by focusing on the generation and recombination rates throughout the QD layers in the middle subcell. The quantum dots are modeled using an effective medium to describe light absorption, confinement, and recombination properties. We report an 8% increase in the short-circuit current density accompanied by a 3% drop in an open-circuit voltage for a QD- enhanced MJSC at 1 sun illumination (1 kW/m2) compared with a control MJSC without QD. The drop in an open-circuit voltage is due in part to the increased recombination rates in the depletion region, decreased carrier lifetimes in the QDs, and the increased recombination rates resulting from carrier escape and capture. Overall, these contribute to an absolute increase in efficiency of over 1% for the studied QD-enhanced MJSC design for a QD density of 125 QD/μm2.

Understanding the molecular interactions between small molecules and double-stranded DNA has important implications on the design and development of DNA and DNA-protein nanomaterials. Such materials can be assembled into a vast array of 1-, 2-, and 3D structures that contain a range of chemical and physical features where small molecules can bind via intercalation, groove binding, and electrostatics. In this work, we use a series of simulation-guided binding assays and spectroscopy techniques to investigate the binding of selected organophosphtates, methyl parathion, paraoxon, their common enzyme hydrolysis product p-nitrophenol, and double-stranded DNA fragments and DNA DX tiles, a basic building block of DNA-based materials. Docking simulations suggested that the binding strength of each compound was DNA sequence-dependent, with dissociation constants in the micromolar range. Microscale thermophoresis and fluorescence binding assays confirmed sequence-dependent binding and that paraoxon bound to DNA with Kd's between ∼10 and 300 μM, while methyl parathion bound with Kd's between ∼10 and 100 μM. p-Nitrophenol also bound to DNA but with affinities up to 650 μM. Changes in biding affinity were due to changes in binding mode as revealed by circular dichroism spectroscopy. Based on these results, two DNA DX tiles were constructed and analyzed, revealing tighter binding to the studied compounds. Taken together, the results presented here add to our fundamental understanding of the molecular interactions of these compounds with biological materials and opens new possibilities in DNA-based sensors, DNA-based matrices for organophosphate extraction, and enzyme-DNA technologies for organophosphate hydrolysis.

Advances in DNA bionanotechnology have led to the ability to create structures with well-defined chemical and physical features at the nanoscale. Such nanostructures can be used to create spatially organized enzymatic cascades that promote substrate channeling and result in enhanced cascade kinetics. Here, we investigate the effects of substrate–scaffold interactions on the catalytic activity of an enzyme–DNA complex using horseradish peroxidase (HRP) and a nanoscale DNA scaffold with three addressable sites. Kinetic assays with a library of HRP substrates revealed that DNA scaffolding enhances HRP activity in a manner that is analogous to the Sabatier Principle. In this case, the binding of the substrate is to the scaffold and not to the catalyst, but the Sabatier trend holds: weak and strong binding substrates showed no enhancement in kinetics, whereas intermediately bound substrates result in >300% increase in enzyme activity.

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co-germinant. The aim of this study was to determine whether any other amino acids act as co-germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re-germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co-germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l-1. Conclusions: Histidine was found to be a co-germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.

In metabolic engineering, most available pathway engineering strategies aim to control enzyme expression by making changes at the transcriptional level with an underlying assumption that translation and functional expression follow suit. In this work, we engineer expression of a key reaction step in medium chain ester biosynthesis that does not follow this common assumption. The native Saccharomyces cerevisiae alcohol acyltransferses Eeb1 and Eht1 condense acyl-CoAs with ethanol to produce the corresponding ester, a reaction that is rate limiting in engineering ester biosynthesis pathways. By changing the N- and C-termini of Eeb1 to those of Eht1, Eeb1 localization is changed from the mitochondria to lipid droplets. The change has no significant effect on transcription, but increases protein expression by 23-fold thus enabling a 3-fold increase in enzyme activity. This system demonstrates one example of the impact of protein trafficking on functional pathway expression, and will guide future metabolic engineering of ester biosynthesis and, potentially, other pathways with critical membrane-bound enzymes., Highlights • Intracellular localization can control membrane-associated enzyme expression. • Terminal domain swapping of Eht1 and Eeb1 alters intracellular localization. • Eeb1 expression and activity are increased with lipid droplet targeting.

Four tunnel junction (TJ) designs for multijunction (MJ) solar cells under high concentration are studied to determine the peak tunnelling current and resistance change as a function of the doping concentration. These four TJ designs are: AlGaAs/AlGaAs, GaAs/GaAs, AlGaAs/InGaP and AlGaAs/GaAs. Time-dependent and time-average methods are used to experimentally characterize the entire current–voltage profile of TJ mesa structures. Experimentally calibrated numerical models are used to determine the minimum doping concentration required for each TJ design to operate within a MJ solar cell up to 2000-suns concentration. The AlGaAs/GaAs TJ design is found to require the least doping concentration to reach a resistance of

Organophosphate (OP) neurotoxins have contaminated the environment, contributed to millions of poisoning annually, and have been used as chemical weapons. Biomaterials incorporating the native activity of the OP hydrolase (OPH) enzyme are of interest for applications including OP sensing, environmental bioremediation and prophylactic decontamination. We have engineered and characterized four novel hydrogel-forming OPH variants by genetically fusing the OPH enzyme with alpha-helical leucine zipper domains (H), unstructured soluble linker domains (S) and polyhistidine purification tags. The appended H domains form physical cross-links between the enzymes and enable self-assembly of the enzymes into hydrogels. The addition of the H and S fusions significantly increased the expression levels of soluble protein. OPH constructs with biterminal H domains form hydrogels at lower protein weight percents and exhibit higher enzymatic activity than those variants modified with a single H domain fusion. Polyhistidine tags were not useful for purification but they were not benign, as the addition of the 6His tags increased the hydrogel-forming abilities of the proteins with a concomitant reduction in both the k(cat) and K(M) values. Active enzymatic hydrogels could be made from concentrated unpurified crude protein lysates, significantly simplifying the processing and utilization of the biomaterials. And, a simple proteinaceous bioactive surface coating exhibiting OPH activity is demonstrated. The hydrogels were stable over long-term storage, as activity was retained after cold storage in buffer after 5 months. These new protein constructs further show the use of rational protein design to create novel, bifunctional, self-assembling units for the formation of catalytic biomaterials.

3-[(3aR,4R,5S,7aS)-5-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy}-4-(4-fluorophenyl)octahydro-2H-isoindol-2-yl]cyclopent-2-en-1-one (17) is a high affinity, brain-penetrant, hydroisoindoline-based neurokinin-1 (NK(1)) receptor antagonist with a long central duration of action in preclinical species and a minimal drug-drug interaction profile. Positron emission tomography (PET) studies in rhesus showed that this compound provides 90% NK(1) receptor blockade in rhesus brain at a plasma level of 67 nM, which is about 10-fold more potent than aprepitant, an NK(1) antagonist marketed for the prevention of chemotherapy-induced and postoperative nausea and vomiting (CINV and PONV). The synthesis of this enantiomerically pure compound containing five stereocenters includes a Diels-Alder condensation, one chiral separation of the cyclohexanol intermediate, an ether formation using a trichloroacetimidate intermediate, and bis-alkylation to form the cyclic amine.

AIMS: To investigate the influence of chemical and physical factors on the rate and extent of germination of Clostridium difficile spores. METHODS AND RESULTS: Germination of C. difficile spores following exposure to chemical and physical germinants was measured by loss of either heat or ethanol resistance. Sodium taurocholate and chenodeoxycholate initiated germination together with thioglycollate medium at concentrations of 0.1-100 mmol l(-1) and 10-100 mmol l(-1) respectively. Glycine (0.2% w/v) was a co-factor required for germination with sodium taurocholate. There was no significant difference in the rate of germination of C. difficile spores in aerobic and anaerobic conditions (P > 0.05) however, the initial rate of germination was significantly increased at 37 degrees C compared to 20 degrees C (P < 0.05). The optimum pH range for germination was 6.5-7.5, with a decreased rate and extent of germination occurring at pH 5.5 and 8.5. CONCLUSIONS: This study demonstrates that sodium taurocholate and chenodeoxycholate initiate germination of C. difficile spores and is concentration dependant. Temperature and pH influence the rate and extent of germination. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript enhances the knowledge of the factors influencing the germination of C. difficile spores. This may be applied to the development of potential novel strategies for the prevention of C. difficile infection.

Previous work on human NK(1) antagonists in which the core of the structure is a substituted pyrrolidine has been disclosed. These compounds showed good binding affinity and functional IP activity, however, many did not exhibit the necessary brain penetration for good in vivo activity. The discovery and preparation of a novel 5,5-fused pyrrolidine core is presented in this paper. This scaffold maintains the excellent binding affinity and functional IP activity of the previously reported compounds, but also exhibits excellent brain penetration as observed in a gerbil foot-tapping assay. The determination of the core structural stereochemistry, which eventually led to the final synthesis of a single active diastereomer, is described.

The sporicidal activity of an odour-free peracetic acid-based disinfectant (Wofasteril®) and a widely-used dichloroisocyanurate preparation (Chlor-clean®) was assessed against spores of the hypervirulent strain of Clostridium difficile (ribotype 027), in the presence and absence of organic matter. In environmentally clean conditions, dichloroisocyanurate achieved a >3 log10 reduction in 3 minutes, but a minimum contact time of 9 minutes was required to reduce the viable spore load to below detection levels. Peracetic acid achieved a >3 log10 reduction in 30 minutes and was overall significantly less effective (P

The bacterially-expressed laccase, small laccase (SLAC) of Streptomyces coelicolor , was incorporated into electrodes of both direct electron transfer (DET) and mediated electron transfer (MET) designs for application in biofuel cells. Using the DET design, enzyme redox kinetics were directly observable using cyclic voltammetry, and a redox potential of 0.43 V (SHE) was observed. When mediated by an osmium redox polymer, the oxygen-reducing cathode retained maximum activity at pH 7, producing 1.5 mA/cm 2 in a planar configuration at 900 rpm and 40 °C, thus outperforming enzyme electrodes produced using laccase from fungal Trametes versicolor (0.2 mA/cm 2 ) under similar conditions. This improvement is directly attributable to differences in the kinetics of SLAC and fungal laccases. Maximum stability of the mediated SLAC electrode was observed at pH above the enzyme's relatively high isoelectric point, where the anionic enzyme molecules could form an electrostatic adduct with the cationic mediator. Porous composite SLAC electrodes with increased surface area produced a current density of 6.25 mA/cm 2 at 0.3 V (SHE) under the above conditions.

A set of closely related furylidene thiosemicarbazones was prepared and screened against various clinically important Gram-positive bacteria. One compound containing an ethylene spacer and a 5-nitrofuryl group was found to have promising activity against Clostridium difficile.