Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

Tetrahydrofolate levels influence 2-aminoacrylate stress in Salmonella enterica.

In Salmonella enterica, the absence of the RidA deaminase results in the accumulation of the reactive enamine 2-aminoacrylate (2AA). The resulting 2AA stress impacts metabolism and prevents growth in some conditions by inactivating a specific target pyridoxal 5'-phosphate (PLP)-dependent enzyme(s). The detrimental effects of 2AA stress can be overcome by changing the sensitivity of a critical target enzyme or modifying flux in one or more nodes in the metabolic network. The catabolic L-alanine racemase DadX is a target of 2AA, which explains the inability of an alr ridA strain to use L-alanine as the sole nitrogen source. Spontaneous mutations that suppressed the growth defect of the alr ridA strain were identified as lesions in folE, which encodes GTP cyclohydrolase and catalyzes the first step of tetrahydrofolate (THF) synthesis. The data here show that THF limitation resulting from a folE lesion, or inhibition of dihydrofolate reductase (FolA) by trimethoprim, decreases the 2AA generated from endogenous serine. The data are consistent with an increased level of threonine, resulting from low folate levels, decreasing 2AA stress.IMPORTANCERidA is an enamine deaminase that has been characterized as preventing the 2-aminoacrylate (2AA) stress. In the absence of RidA, 2AA accumulates and damages various cellular enzymes. Much of the work describing the 2AA stress system has depended on the exogenous addition of serine to increase the production of the enamine stressor. The work herein focuses on understanding the effect of 2AA stress generated from endogenous serine pools. As such, this work describes the consequences of a subtle level of stress that nonetheless compromises growth in at least two conditions. Describing mechanisms that alter the physiological consequences of 2AA stress increases our understanding of endogenous metabolic stress and how the robustness of the metabolic network allows perturbations to be modulated.

Development and evaluation of a drug-in-adhesive transdermal delivery system for delivery of olanzapine.

OBJECTIVES: Olanzapine (OZP) is a safe and effective atypical antipsychotic drug used in treating schizophrenia and bipolar disorders. The dosage forms currently on the market for OZP are administered via oral or intramuscular routes. However, there are many problems associated with oral and intramuscular routes of drug administration. Thus, our aim was to develop a drug-in-adhesive transdermal delivery system (TDS) that can deliver OZP for 3 days. METHODS: We determined passive permeation, effect of oleic acid as chemical enhancer, and delivery of OZP across different skin types. Based on preliminary studies and saturation solubility of OZP in different pressure-sensitive adhesives (PSAs), we formulated and characterized solution-based TDS in acrylate PSA and suspension-based TDS in silicone and PIB PSA, with oleic acid as chemical enhancer. RESULTS: Acrylate solution-based TDS, silicone, and PIB suspension-based TDS delivered 58.97 ± 6.59 µg/sq.cm, 129.34 ± 16.59 µg/sq.cm, and 245.00 ± 2.51 µg/sq.cm, respectively, using in vitro permeation testing. PIB PSA suspension-based TDS met the 3 days desired target delivery. Skin irritation testing using In vitro EpiDermTM skin irritation test (EPI-200-SIT) kit found PIB TDS to be nonirritant. CONCLUSION: The PIB PSA suspension-based TDS could serve as a potentially effective transdermal delivery system for olanzapine.

Production of propionate using metabolically engineered strains of Clostridium saccharoperbutylacetonicum.

The carboxylic acid propionate is a valuable platform chemical with applications in various fields. The biological production of this acid has become of great interest as it can be considered a sustainable alternative to petrochemical synthesis. In this work, Clostridium saccharoperbutylacetonicum was metabolically engineered to produce propionate via the acrylate pathway. In total, the established synthetic pathway comprised eight genes encoding the enzymes catalyzing the conversion of pyruvate to propionate. These included the propionate CoA-transferase, the lactoyl-CoA dehydratase, and the acryloyl-CoA reductase from Anaerotignum neopropionicum as well as a D-lactate dehydrogenase from Leuconostoc mesenteroides subsp. mesenteroides. Due to difficulties in assembling all genes on one plasmid under the control of standard promoters, the PtcdB-tcdR promoter system from Clostridium difficile was integrated into a two-plasmid system carrying the acrylate pathway genes. Several promoters were analyzed for their activity in C. saccharoperbutylacetonicum using the fluorescence-activating and absorption-shifting tag (FAST) as a fluorescent reporter to identify suitable candidates to drive tcdR expression. After selecting the lactose-inducible PbgaL promoter, engineered C. saccharoperbutylacetonicum strains produced 0.7 mM propionate upon induction of gene expression. The low productivity was suspected to be a consequence of a metabolic imbalance leading to acryloyl-CoA accumulation in the cells. To even out the proposed imbalance, the propionate-synthesis operons were rearranged, thereby increasing the propionate concentration by almost four-fold. This study is the first one to report recombinant propionate production using a clostridial host strain that has opened a new path towards bio-based propionate to be improved further in subsequent work. KEY POINTS: • Determination of promoter activities in C. saccharoperbutylacetonicum using FAST. • Implementation of propionate production in C. saccharoperbutylacetonicum. • Elevation of propionate production by 375% to a concentration of 3 mM.

The Cysteine Desulfurase IscS Is a Significant Target of 2-Aminoacrylate Damage in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes eight members of the Rid protein superfamily. PA5339, a member of the RidA subfamily, is required for full growth and motility of P. aeruginosa. Our understanding of RidA integration into the metabolic network of P. aeruginosa is at an early stage, with analyses largely guided by the well-established RidA paradigm in Salmonella enterica. A P. aeruginosa strain lacking RidA has a growth and motility defect in a minimal glucose medium, both of which are exacerbated by exogenous serine. All described ridA mutant phenotypes are rescued by supplementation with isoleucine, indicating the primary generator of the reactive metabolite 2-aminoacrylate (2AA) in ridA mutants is a threonine/serine dehydratase. However, the critical (i.e., phenotype determining) targets of 2AA leading to growth and motility defects in P. aeruginosa remained undefined. This study was initiated to probe the effects of 2AA stress on the metabolic network of P. aeruginosa by defining the target(s) of 2AA that contribute to physiological defects of a ridA mutant. Suppressor mutations that restored growth to a P. aeruginosa ridA mutant were isolated, including an allele of iscS (encoding cysteine desulfurase). Damage to IscS was identified as a significant cause of growth defects of P. aeruginosa during enamine stress. A suppressing allele encoded an IscS variant that was less sensitive to damage by 2AA, resulting in a novel mechanism of phenotypic suppression of a ridA mutant. IMPORTANCE 2-aminoacrylate (2AA) is a reactive metabolite formed as an intermediate in various enzymatic reactions. In the absence of RidA, this metabolite can persist in vivo where it attacks and inactivates specific PLP-dependent enzymes, causing metabolic defects and organism-specific phenotypes. This work identifies the cysteine desulfurase IscS as the critical target of 2AA in Pseudomonas aeruginosa. A single substitution in IscS decreased sensitivity to 2AA and suppressed growth phenotypes of a ridA mutant. Here, we provide the first report of suppression of a ridA mutant phenotype by altering the sensitivity of a target enzyme to 2AA.

Genome-scale metabolic modelling enables deciphering ethanol metabolism via the acrylate pathway in the propionate-producer Anaerotignum neopropionicum.

BACKGROUND: Microbial production of propionate from diluted streams of ethanol (e.g., deriving from syngas fermentation) is a sustainable alternative to the petrochemical production route. Yet, few ethanol-fermenting propionigenic bacteria are known, and understanding of their metabolism is limited. Anaerotignum neopropionicum is a propionate-producing bacterium that uses the acrylate pathway to ferment ethanol and CO2 to propionate and acetate. In this work, we used computational and experimental methods to study the metabolism of A. neopropionicum and, in particular, the pathway for conversion of ethanol into propionate. RESULTS: Our work describes iANEO_SB607, the first genome-scale metabolic model (GEM) of A. neopropionicum. The model was built combining the use of automatic tools with an extensive manual curation process, and it was validated with experimental data from this and published studies. The model predicted growth of A. neopropionicum on ethanol, lactate, sugars and amino acids, matching observed phenotypes. In addition, the model was used to implement a dynamic flux balance analysis (dFBA) approach that accurately predicted the fermentation profile of A. neopropionicum during batch growth on ethanol. A systematic analysis of the metabolism of A. neopropionicum combined with model simulations shed light into the mechanism of ethanol fermentation via the acrylate pathway, and revealed the presence of the electron-transferring complexes NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn) and acryloyl-CoA reductase-EtfAB, identified for the first time in this bacterium. CONCLUSIONS: The realisation of the GEM iANEO_SB607 is a stepping stone towards the understanding of the metabolism of the propionate-producer A. neopropionicum. With it, we have gained insight into the functioning of the acrylate pathway and energetic aspects of the cell, with focus on the fermentation of ethanol. Overall, this study provides a basis to further exploit the potential of propionigenic bacteria as microbial cell factories.

2-Aminoacrylate stress damages diverse PLP-dependent enzymes in vivo.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for a class of enzymes that catalyze diverse reactions in central metabolism. The catalytic mechanism of some PLP-dependent enzymes involves the generation of reactive enamine intermediates like 2-aminoacrylate (2AA). 2AA can covalently modify PLP in the active site of some PLP-dependent enzymes and subsequently inactivate the enzyme through the formation of a PLP-pyruvate adduct. In the absence of the enamine/imine deaminase RidA, Salmonella enterica experiences 2AA-mediated metabolic stress. Surprisingly, PLP-dependent enzymes that generate endogenous 2AA appear to be immune to its attack, while other PLP-dependent enzymes accumulate damage in the presence of 2AA stress; however, structural determinants of 2AA sensitivity are unclear. In this study, we refined a molecular method to query proteins from diverse systems for their sensitivity to 2AA in vivo. This method was then used to examine active site residues of Alr, a 2AA-sensitive PLP-dependent enzyme, that affect its sensitivity to 2AA in vivo. Unexpectedly, our data also showed that a low level of 2AA stress can persist even in the presence of a functional RidA. In summary, this study expands our understanding of 2AA metabolism and takes an initial step toward characterizing the structural determinants influencing enzyme susceptibility to damage by free 2AA.

Effect of Fatty Acid Polyunsaturation on Synthesis and Properties of Emulsion Polymers Based on Plant Oil-Based Acrylic Monomers.

This study demonstrated that polymerization behavior of plant oil-based acrylic monomers (POBMs) synthesized in one-step transesterification reaction from naturally rich in oleic acid olive, canola, and high-oleic soybean oils is associated with a varying mass fraction of polyunsaturated fatty acid fragments (linoleic (C18:2) and linolenic (C18:3) acid esters) in plant oil. Using miniemulsion polymerization, a range of stable copolymer latexes was synthesized from 60 wt.% of each POBM and styrene to determine the impact of POBM chemical composition (polyunsaturation) on thermal and mechanical properties of the resulted polymeric materials. The unique composition of each plant oil serves as an experimental tool to determine the effect of polyunsaturated fatty acid fragments on POBM polymerization behavior and thermomechanical properties of crosslinked films made from POBM-based latexes. The obtained results show that increasing polyunsaturation in the copolymers results in an enhanced crosslink density of the latex polymer network which essentially impacts the mechanical properties of the films (both Young's modulus and toughness). Maximum toughness was observed for crosslinked latex films made from 50 wt.% of each POBM in the monomer feed.

Acrylate protects a marine bacterium from grazing by a ciliate predator.

Cleavage of dimethylsulfoniopropionate (DMSP) can deter herbivores in DMSP-producing eukaryotic algae; however, it is unclear whether a parallel defence mechanism operates in marine bacteria. Here we demonstrate that the marine bacterium Puniceibacterium antarcticum SM1211, which does not use DMSP as a carbon source, has a membrane-associated DMSP lyase, DddL. At high concentrations of DMSP, DddL causes an accumulation of acrylate around cells through the degradation of DMSP, which protects against predation by the marine ciliate Uronema marinum. The presence of acrylate can alter the grazing preference of U. marinum to other bacteria in the community, thereby influencing community structure.

Combining High-Throughput Synthesis and High-Throughput Protein Crystallography for Accelerated Hit Identification.

Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and -esters from diverse building blocks suitable for mmol scale synthesis on 96-well format and on a high-throughput nanoscale format in a highly automated fashion. High-throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID-19 causing agent, SARS-CoV-2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects.

Heterodimeric GW7604 Derivatives: Modification of the Pharmacological Profile by Additional Interactions at the Coactivator Binding Site.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of (Z)-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative 16 showed extraordinarily high affinity to the estrogen receptor (ER) ß (RBA = 110%), inhibited effectively the coactivator recruitment (IC50 = 20.88 nM (ERα) and 28.34 nM (ERß)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC50 = 18.5 nM (ERα) and 7.5 nM (ERß)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 µM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC50 = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC50 = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.

The Rid family member RutC of Escherichia coli is a 3-aminoacrylate deaminase.

The Rid protein family (PF14588, IPR006175) is divided into nine subfamilies, of which only the RidA subfamily has been characterized biochemically. RutC, the founding member of one subfamily, is encoded in the pyrimidine utilization (rut) operon that encodes a pathway that allows Escherichia coli to use uracil as a sole nitrogen source. Results reported herein demonstrate that RutC has 3-aminoacrylate deaminase activity and facilitates one of the reactions previously presumed to occur spontaneously in vivo. RutC was active with several enamine-imine substrates, showing similarities and differences in substrate specificity with the canonical member of the Rid superfamily, Salmonella enterica RidA. Under standard laboratory conditions, a Rut pathway lacking RutC generates sufficient nitrogen from uracil for growth of E. coli. These results support a revised model of the Rut pathway and provide evidence that Rid proteins may modulate metabolic fitness, rather than catalyzing essential functions.

A kinetic model of the central carbon metabolism for acrylic acid production in Escherichia coli.

Acrylic acid is a value-added chemical used in industry to produce diapers, coatings, paints, and adhesives, among many others. Due to its economic importance, there is currently a need for new and sustainable ways to synthesise it. Recently, the focus has been laid in the use of Escherichia coli to express the full bio-based pathway using 3-hydroxypropionate as an intermediary through three distinct pathways (glycerol, malonyl-CoA, and ß-alanine). Hence, the goals of this work were to use COPASI software to assess which of the three pathways has a higher potential for industrial-scale production, from either glucose or glycerol, and identify potential targets to improve the biosynthetic pathways yields. When compared to the available literature, the models developed during this work successfully predict the production of 3-hydroxypropionate, using glycerol as carbon source in the glycerol pathway, and using glucose as a carbon source in the malonyl-CoA and ß-alanine pathways. Finally, this work allowed to identify four potential over-expression targets (glycerol-3-phosphate dehydrogenase (G3pD), acetyl-CoA carboxylase (AccC), aspartate aminotransferase (AspAT), and aspartate carboxylase (AspC)) that should, theoretically, result in higher AA yields.

Benzophenone Accumulates over Time from the Degradation of Octocrylene in Commercial Sunscreen Products.

Benzophenone is a mutagen, carcinogen, and endocrine disruptor. Its presence in food products or food packaging is banned in the United States. Under California Proposition 65, there is no safe harbor for benzophenone in any personal care products, including sunscreens, anti-aging creams, and moisturizers. The purpose of this study was to determine (1) if benzophenone was present in a wide variety of commercial sun protection factor (SPF)/sunscreen products, (2) whether benzophenone concentration in the product increased over time, and (3) if the degradation of octocrylene was the likely source for benzophenone contamination. Benzophenone concentration was assayed in nine commercial sunscreen products from the European Union and eight from the United States (in triplicate), including two single ingredient sources of octocrylene. These same SPF items were subjected to the United States Food and Drug Administration (U.S. FDA)-accelerated stability aging protocol for 6 weeks. Benzophenone was measured in the accelerated-aged products. Sixteen octocrylene-containing product lines that were recently purchased had an average concentration of 39 mg/kg benzophenone, ranging from 6 mg/kg to 186 mg/kg. Benzophenone was not detectable in the product that did not contain octocrylene. After subjecting the 17 products to the U.S. FDA-accelerated stability method, the 16 octocrylene-containing products had an average concentration of 75 mg/kg, ranging from 9.8 mg/kg to 435 mg/kg. Benzophenone was not detectable in the product that did not contain octocrylene. Benzophenone was detected in the pure octocrylene manufactured ingredient. Octocrylene generates benzophenone through a retro-aldol condensation. In vivo, up to 70% of the benzophenone in these sunscreen products may be absorbed through the skin. U.S. FDA has established a zero tolerance for benzophenone as a food additive. In the United States, there were 2999 SPF products containing octocrylene in 2019. The safety of octocrylene as a benzophenone generator in SPF or any consumer products should be expeditiously reviewed by regulatory agencies.

In vivo interpretation of model predicted inhibition in acrylate pathway engineered Lactococcus lactis.

To maximize the productivity of engineered metabolic pathway, in silico model is an established means to provide features of enzyme reaction dynamics. In our previous study, Escherichia coli engineered with acrylate pathway yielded low propionic acid titer. To understand the bottleneck behind this low productivity, a kinetic model was developed that incorporates the enzymatic reactions of the acrylate pathway. The resulting model was capable of simulating the fluxes reported under in vitro studies with good agreement, suggesting repression of propionyl-CoA transferase (Pct) by carboxylate metabolites as the main limiting factor for propionate production. Furthermore, the predicted flux control coefficients of the pathway enzymes under steady state conditions revealed that the control of flux is shared between Pct and lactoyl-CoA dehydratase. Increase in lactate concentration showed gradual decrease in flux control coefficients of Pct that in turn confirmed the control exerted by the carboxylate substrate. To interpret these in silico predictions under in vivo system, an organized study was conducted with a lactic acid bacteria strain engineered with acrylate pathway. Analysis reported a decreased product formation rate on attainment of inhibitory titer by suspected metabolites and supported the model.

Two novel fish paralogs provide insights into the Rid family of imine deaminases active in pre-empting enamine/imine metabolic damage.

Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.

Investigation into the influence of an acrylic acid acceptor in organic D-π-A sensitizers against phototoxicity.

Photodynamic therapy (PDT) is a non-invasive, selective, and cost-effective cancer therapy. We previously reported that thiophene-based organic D-π-A sensitizers consist of an electron-donating (D) moiety, a π-conjugated bridge (π) moiety, and an electron-accepting (A) moiety, and are readily accessible and stable templates for photosensitizers that could be used in PDT. In addition, acrylic acid acceptor-containing photosensitizers exert a high level of phototoxicity. This study was an investigation into 1) the possibility of increasing phototoxicity by introducing another carboxyl group or by replacing a carboxyl group with a pyridinium group, and 2) the importance of an alkene in the acrylic acid acceptor for phototoxicity. A review of the design, synthesis, and evaluation of sensitizers revealed that neither dicarboxylic acid nor pyridinium photosensitizers enhance phototoxicity. An evaluation of a photosensitizer without an alkene in the acrylic acid moiety revealed that the alkene was not indispensable in the pursuit of phototoxicity. The obtained results provided new insight into the design of ideal D-π-A photosensitizers for PDT.

Contribution of calpain to protein degradation, variation in myowater properties and the water-holding capacity of pork during postmortem ageing.

To investigate calpain's effect on protein degradation, myowater properties, and the water-holding capacity (WHC), porcine longissimus muscles were incubated with control buffer, PD150,606 (calpain-specific inhibitor) and MG-262 (multiple-protease inhibitor) and assigned to an ageing period of 1, 4 or 7 d. Over 7 d of storage, no significant differences (P > 0.05) were observed in desmin or integrin expression between the MG-262 and PD150,606 groups, which indicated that calpain played a major role in protein proteolysis. Compared to those in the control group, muscle samples subjected to PD150,606 and MG-262 exhibited higher water mobility and a poorer WHC. Additionally, there were no significant differences in myowater properties or the WHC between the two groups at 1 d postmortem (P > 0.05). Calpain regulated the distribution and mobility of myowater, which contributed to a higher WHC in the early postmortem period (before 4 d), but other proteases tended to take over at a later stage.

Two new secondary metabolites, saccharochlorines A and B, from a marine bacterium Saccharomonospora sp. KCTC-19160.

Two new chlorinated secondary metabolites, saccharochlorines A and B (1 and 2), were isolated from the saline cultivation of a marine-derived bacterium Saccharomonospora sp. (KCTC-19160). The chemical structures of the saccharochlorines were elucidated by 2D NMR and MS spectroscopic data. Saccharochlorines A and B (1 and 2) exhibit weak inhibition of ß-secretase (BACE1) in biochemical inhibitory assay, but they induced the release of Aß (1-40) and Aß (1-42) in H4-APP neuroglial cells. This discrepancy might be derived from the differences between the cellular and sub-cellular environments or the epigenetic stimulation of BACE1 expression.

A Novel Biosynthetic Pathway for the Production of Acrylic Acid through ß-Alanine Route in Escherichia coli.

Acrylic acid (AA) is an important industrial chemical used for several applications including superabsorbent polymers and acrylate esters. Here, we report the development of a new biosynthetic pathway for the production of AA from glucose in metabolically engineered Escherichia coli through the ß-alanine (BA) route. The AA production pathway was partitioned into two modules: an AA forming downstream pathway and a BA forming upstream pathway. We first validated the operation of the downstream pathway in vitro and in vivo, and then constructed the downstream pathway by introducing efficient enzymes (Act, Acl2, and YciA) screened out of various microbial sources and optimizing the expression levels. For the direct fermentative production of AA from glucose, the downstream pathway was introduced into the BA producing E. coli strain. The resulting strain could successfully produce AA from glucose in flask cultivation. AA production was further enhanced by expressing the upstream genes (panD and aspA) under the constitutive BBa_J23100 promoter. Replacement of the native promoter of the acs gene with the BBa_J23100 promoter in the genome increased AA production to 55.7 mg/L in flask. Fed-batch fermentation of the final engineered strain allowed production of 237 mg/L of AA in 57.5 h, representing the highest AA titer reported to date.