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Raspberry ketone (RK), a natural product derived from raspberry fruit, is commonly utilized as a flavoring agent in foods and as an active component for weight loss. Metabolic engineering has enabled microorganisms to produce RK more efficiently and cost-effectively. However, the biosynthesis of RK is hindered by an unbalanced synthetic pathway and a deficiency of precursors, including tyrosine and malonyl-CoA. In this study, we constructed and optimized the RK synthetic pathway in Escherichia coli using a static metabolic engineering strategy to enhance the biosynthesis of tyrosine from glucose, thereby achieving the de novo production of RK. Additionally, the synthetic and consumption pathways of malonyl-CoA were dynamically regulated by p -coumaric acid-responsive biosensor to balance the metabolic flux distribution between cell growth and RK biosynthesis. Following pathway optimization, the medium components and fermentation conditions were further refined, resulting in a significant increase in the RK titer to 415.56 mg/L. The optimized strain demonstrated a 32.4-fold increase in the RK titer while maintaining a comparable final OD 600 to the initial strain. Overall, the implemented static and dynamic regulatory strategies provide a novel approach for the efficient production of RK, taking into account cell viability and growth.

We report a significantly more rapid method for quantitation of the urinary mercapturic acids of the isomeric toxicants crotonaldehyde, methacrolein, and methyl vinyl ketone. The major innovation of this novel method is detection by liquid chromatography-negative atmospheric pressure chemical ionization-tandem mass spectrometry-selected reaction monitoring as opposed to detection by negative electrospray ionization in our previously reported method. The new method also uses an improved Raptor Biphenyl HPLC column. The total chromatographic analysis time was reduced to about 8 min compared to 35 min in our previously published method. Accuracy, precision, and ruggedness of the new method were established, and its suitability for the analysis of urine samples from 2500 cigarette smokers and non-smokers was confirmed. The improved method is practical for quantitation of these important toxicants in clinical studies requiring analysis of thousands of urine samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Raspberry ketone accounts for the characteristic aroma of the raspberry fruit. In order to explore the genes involved in raspberry ketone synthesis, the transcriptome in fruit tissues of two red raspberry varieties "Polka" and "Orange legend", were sequenced and 24213 single genes were obtained. As the red raspberry fruit ripening, genes involved in flavonoid and anthocyanin synthesis were up-regulated, while those associated with lignin synthesis were down-regulated. A gene (RinPKS4) highly related to raspberry ketone synthesis was identified by transcriptome analysis, and RinPKS4 gene was over-expressed in raspberry in order to further understand the function of RinPKS4 gene in raspberry ketone synthesis. The results showed that the gene expression level of RinPKS4 in the leaf tissues of a transgenic lines increased by about 4-fold and the content of raspberry ketone increased by 42.64% compared with the wide type. This study lays a theoretical foundation for further study on the synthesis and regulation of raspberry ketone in red raspberry., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Early detection of biomarkers in lung cancer is one of the best preventive strategies. Although many attempts have been made to understand the early events of lung carcinogenesis including cigarette smoking (CS) induced lung carcinogenesis, the integrative metabolomics and next-generation sequencing approaches are lacking. In this study, we treated the female A/J mice with CS carcinogen 4-[methyl(nitroso)amino]-1-(3-pyridinyl)-1-butanone (NNK) and naturally occurring organosulphur compound, diallyl sulphide (DAS) for 2 and 4 weeks after NNK injection and examined the metabolomic and DNA CpG methylomic and RNA transcriptomic profiles in the lung tissues. NNK drives metabolic changes including mitochondrial tricarboxylic acid (TCA) metabolites and pathways including Nicotine and its derivatives like nicotinamide and nicotinic acid. RNA-seq analysis and Reactome pathway analysis demonstrated metabolism pathways including Phase I and II drug metabolizing enzymes, mitochondrial oxidation and signaling kinase activation pathways modulated in a sequential manner. DNA CpG methyl-seq analyses showed differential global methylation patterns of lung tissues from week 2 versus week 4 in A/J mice including Adenylate Cyclase 6 (ADCY6), Ras-related C3 botulinum toxin substrate 3 (Rac3). Oral DAS treatment partially reversed some of the mitochondrial metabolic pathways, global methylation and transcriptomic changes during this early lung carcinogenesis stage. In summary, our result provides insights into CS carcinogen NNK's effects on driving alterations of metabolomics, epigenomics and transcriptomics and the chemopreventive effect of DAS in early stages of sequential lung carcinogenesis in A/J mouse model., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Novel food and food compounds interventions have attracted a lot of attention nowadays for the prevention and treatment of metabolic diseases. Raspberry ketone (RK) is aromatic compound found within red fruits and berries, has been used as an over-the-counter product for weight loss. However, actually, the effect of RK on weight loss is still controversial, and the mechanism is largely unknown. Besides, in vivo and in vitro studies have demonstrated the beneficial effect of RK on the development of other metabolic diseases. In this review, we comprehensively highlighted the synthesis, bioavailability, and metabolism of RK, and summarized the progress made in our understanding of the potential biological activities of RK, including antiobesity, antidiabetes, cardioprotection, and hepatoprotection, as well as their underlying mechanisms. This paper provides a critical overview about the current findings and proposes the future studies in the area of RK on human health. PRACTICAL APPLICATIONS: Raspberry ketone (RK) has been used for weight control for years, but this effect is controversial considering food intake. Additionally, RK is beneficial for T2DM, liver and heart injury. The underlying mechanisms of the protective effect of RK including accelerating fatty acid oxidation, balancing serum glucose level, anti-inflammation, antioxidant process, and so on. In this context, we provide a comprehensive analysis of the benefits of RK against many metabolic diseases and discuss the underlying molecular mechanisms. We hope our work will be helpful for further researches on RK and improve its public recognition., (© 2021 Wiley Periodicals LLC.)

There is a considerable interest in the asymmetric production of chiral allylic alcohols, the main building blocks of many functional molecules. The asymmetric reduction of α,β-unsaturated ketones is difficult with traditional chemical protocols in a regioselective and stereoselective manner. In this study, the reductive capacity of whole cell of Leuconostoc mesenteroides N6, Weissella paramesenteroides N7, Weissella cibaria N9, and Leuconostoc pseudomesenteroides N13 was investigated as whole-cell biocatalysts in the enantioselective reduction of (E)-4-phenylbut-3-en-2-one (1). The biocatalytic reduction of 1 to (S,E)-4-phenylbut-3-en-2-ol ((S,E)-2) using the whole cell of W. cibaria N9 isolated from Turkish sourdough was developed in a regioselective fashion, occurring with excellent conversion and recovering the product in good yield. In biocatalytic reduction reactions, the conversion of the substrate and the enantiomeric excess (ee) of the product are significantly affected by optimization parameters such as temperature, agitation rate, pH, and incubation time. Effects of these parameters on ee and conversion were investigated comprehensively. In addition, to our knowledge, this is the first report on production of (S,E)-2 using whole-cell biocatalyst in excellent yield, conversion with enantiopure form and at gram scale. These findings pave the way for the use of whole cell of W. cibaria N9 for challenging higher substrate concentrations of different α,β-unsaturated ketones for regioselective reduction at industrial scale., (© 2021 Wiley Periodicals LLC.)

Background:  A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg -1 ) fine chemical farmed from raspberry (Rubeus rubrum) fruit., Results:  By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture., Conclusions:  Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

Background: Phenylpropanoid including raspberry ketone, is a kind of important natural plant product and widely used in pharmaceuticals, chemicals, cosmetics, and healthcare products. Bioproduction of phenylpropanoid in Escherichia coli and other microbial cell factories is an attractive approach considering the low phenylpropanoid contents in plants. However, it is usually difficult to produce high titer phenylpropanoid production when fermentation using glucose as carbon source. Developing novel bioprocess using alternative sources might provide a solution to this problem. In this study, typical phenylpropanoid raspberry ketone was used as the target product to develop a biosynthesis pathway for phenylpropanoid production from fatty acids, a promising alternative low-cost feedstock., Results: A raspberry ketone biosynthesis module was developed and optimized by introducing 4-coumarate-CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone reductase (RZS) in Escherichia coli strains CR1-CR4. Then strain CR5 was developed by introducing raspberry ketone biosynthesis module into a fatty acids-utilization chassis FA09 to achieve production of raspberry ketone from fatty acids feedstock. However, the production of raspberry ketone was still limited by the low biomass and unable to substantiate whole-cell bioconversion process. Thus, a process by coordinately using fatty-acids and glycerol was developed. In addition, we systematically screened and optimized fatty acids-response promoters. The optimized promoter Pfrd3 was then successfully used for the efficient expression of key enzymes of raspberry ketone biosynthesis module during bioconversion from fatty acids. The final engineered strain CR8 could efficiently produce raspberry ketone repeatedly using bioconversion from fatty acids feedstock strategy, and was able to produce raspberry ketone to a concentration of 180.94 mg/L from soybean oil in a 1-L fermentation process., Conclusion: Metabolically engineered Escherichia coli strains were successfully developed for raspberry ketone production from fatty acids using several strategies, including optimization of bioconversion process and fine-tuning key enzyme expression. This study provides an essential reference to establish the low-cost biological manufacture of phenylpropanoids compounds.

In this study we describe the crystal structures of the apoform, the binary and the ternary complexes of a double bond reductase from Malus domestica L. (MdDBR) and explore a range of potential substrates. The overall fold of MdDBR is similar to that of the medium chain reductase/dehydrogenase/zinc-dependent alcohol dehydrogenase-like family. Structural comparison of MdDBR with Arabidopsis thaliana DBR (AtDBR), Nicotiana tabacum DBR (NtDBR) and Rubus idaeus DBR (RiDBR) allowed the identification of key amino acids involved in cofactor and ligands binding and shed light on how these residues may guide the orientation of the substrates. The enzyme kinetic for the substrate trans-4-phenylbuten-2-one has been analyzed, and MdDBR activity towards a variety of substrates was tested. This enzyme has been reported to be involved in the phenylpropanoid pathway where it would catalyze the NADPH-dependent reduction of the α, β-unsaturated double bond of carbonyl metabolites. Our study provides new data towards the identification of MdDBR natural substrate and the biosynthetic pathway where it belongs. Furthermore, the originally proposed involvement in dihydrochalcone biosynthesis in apple must be questioned., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Raspberry ketone (RK) (4-(4-hydroxyphenyl)-2-butanone) is the major compound responsible for the characteristic aroma of red raspberries, and has long been used commercially as a flavoring agent and recently as a weight loss supplement. A targeted UHPLC-QqQ-MS/MS method was developed and validated for analysis of RK and 25 associated metabolites in mouse plasma and brain. Dispersion and projection analysis and central composite design were used for method optimization. Random effect analysis of variance was applied for validation inference and variation partition. Within this framework, repeatability, a broader sense of precision, was calculated as fraction of accuracy variance, reflecting instrumental imprecision, compound degradation and carry-over effects. Multivariate correlation analysis and principle component analysis were conducted, revealing underlying association among the manifold of method traits. R programming was engaged in streamlined statistical analysis and data visualization. Two particular phenomena, the analytes' background existence in the enzyme solution used for phase II metabolites deconjugation, and the noted lability of analytes in pure solvent at 4 ℃ vs. elevated stability in biomatrices, were found critical to method development and validation. The approach for the method development and validation provided a foundation for experiments that examine RK metabolism and bioavailability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The phenylbutanoid 4-(4-hydroxyphenyl)butan-2-one, commonly known as raspberry ketone, is responsible for the typical scent and flavor of ripe raspberries. Chemical production of nature-identical raspberry ketone is well established as this compound is frequently used to flavor food, beverages and perfumes. However, high demand for natural raspberry ketone, but low natural abundance in raspberries, render raspberry ketone one of the most expensive natural flavoring components., Results: In this study, Corynebacterium glutamicum was engineered for the microbial synthesis of the character impact compound raspberry ketone from supplemented p-coumaric acid. In this context, the NADPH-dependent curcumin/dihydrocurcumin reductase CurA from Escherichia coli was employed to catalyze the final step of raspberry ketone synthesis as it provides a hitherto unknown benzalacetone reductase activity. In combination with a 4-coumarate: CoA ligase from parsley (Petroselinum crispum) and a monofunctional benzalacetone synthase from Chinese rhubarb (Rheum palmatum), CurA constitutes the synthetic pathway for raspberry ketone synthesis in C. glutamicum. The resulting strain accumulated up to 99.8 mg/L (0.61 mM) raspberry ketone. In addition, supplementation of other phenylpropanoids allowed for the synthesis of two other naturally-occurring and flavoring phenylbutanoids, zingerone (70 mg/L, 0.36 mM) and benzylacetone (10.5 mg/L, 0.07 mM)., Conclusion: The aromatic product portfolio of C. glutamicum was extended towards the synthesis of the flavoring phenylbutanoids raspberry ketone, zingerone and benzylacetone. Key to success was the identification of CurA from E. coli having a benzalacetone reductase activity. We believe, that the constructed C. glutamicum strain represents a versatile platform for the production of natural flavoring phenylbutanoids at larger scale.

Objectives: Raspberry ketone (RK) is the primary aroma compound in red raspberries and a dietary supplement for weight loss. This work aims to 1) compare RK bioavailability in male versus female, normal-weight versus obese mice; 2) characterize RK metabolic pathways., Methods: Study 1: C57BL/6J male and female mice fed a low-fat diet (LFD; 10% fat) receive a single oral gavage dose of RK (200 mg kg -1 ). Blood, brain, and white adipose tissue (WAT) are collected over 12 h. Study 2: Male mice are fed a LFD or high-fat diet (45% fat) for 8 weeks before RK dosing. Samples collected are analyzed by UPLC-MS/MS for RK and its metabolites., Results: RK is rapidly absorbed (T max  ≈ 15 min), and bioconverted into diverse metabolites in mice. Total bioavailability (AUC 0-12 h ) is slightly lower in females than males (566 vs 675 nmol mL -1 min -1 ). Total bioavailability in obese mice is almost doubled that of control mice (1197 vs 679 nmol mL -1 min -1 ), while peaking times and elimination half-lives are delayed. Higher levels of RK and major metabolites are found in WAT of the obese than normal-weight animals., Conclusions: RK is highly bioavailable, rapidly metabolized, and exhibits significantly different pharmacokinetic behaviors between obese and control mice. Lipid-rich tissues, especially WAT, can be a direct target of RK., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Raspberry ketone is a primary aroma component of the red raspberry. The glycosylation of this compound is a potential approach used to improve its pharmaceutical properties. In this work, raspberry ketone glycosides are produced in bacteria for the first time. Bacillus licheniformis PI15, an organic solvent-tolerant glycosyltransferase-producing strain, was isolated from chemically polluted soil. The cloning and heterologous expression of a glycosyltransferase, which was designated PI-GT1, in Escherichia coli BL21 resulted in the expression of an active and soluble protein that accounted for 15% of the total cell protein content. Purified PI-GT1 was highly active and stable over a broad pH range (6.0-10.0) and showed excellent pH stability. PI-GT1 maintained almost 60% of its maximal activity after 3 H of incubation at 20-40 °C and demonstrated optimal activity at 30 °C. Additionally, PI-GT1 displayed high stability and activity in the presence of hydrophilic solvents with log P ≤ -0.2 and retained more than 80% of its activity after 3 H of treatment. Supplementation with 10% DMSO markedly improved the glycosylation of raspberry ketone, resulting in a value 26 times higher than that in aqueous solution. The organic solvent-tolerant PI-GT1 may have potential uses in industrial chemical and pharmaceutical synthesis applications., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Common strategies for conversion of lignocellulosic biomass to chemical products center on deconstructing biomass polymers into fermentable sugars. Here, we demonstrate an alternative strategy, a growth-coupled, high-yield bioconversion, by feeding cells a non-sugar substrate, by-passing central metabolism, and linking a key metabolic step to generation of acetyl-CoA that is required for biomass and energy generation. Specifically, we converted levulinic acid (LA), an established degradation product of lignocellulosic biomass, to butanone (a.k.a. methyl-ethyl ketone - MEK), a widely used industrial solvent. Our strategy combines a catabolic pathway from Pseudomonas putida that enables conversion of LA to 3-ketovaleryl-CoA, a CoA transferase that generates 3-ketovalerate and acetyl-CoA, and a decarboxylase that generates 2-butanone. By removing the ability of E. coli to consume LA and supplying excess acetate as a carbon source, we built a strain of E. coli that could convert LA to butanone at high yields, but at the cost of significant acetate consumption. Using flux balance analysis as a guide, we built a strain of E. coli that linked acetate assimilation to production of butanone. This strain was capable of complete bioconversion of LA to butanone with a reduced acetate requirement and increased specific productivity. To demonstrate the bioconversion on real world feedstocks, we produced LA from furfuryl alcohol, a compound readily obtained from biomass. These LA feedstocks were found to contain inhibitors that prevented cell growth and bioconversion of LA to butanone. We used a combination of column chromatography and activated carbon to remove the toxic compounds from the feedstock, resulting in LA that could be completely converted to butanone. This work motivates continued collaboration between chemical and biological catalysis researchers to explore alternative conversion pathways and the technical hurdles that prevent their rapid deployment., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Polyhydroxylated compounds are building blocks for the synthesis of carbohydrates and other natural products. Their synthesis is mainly achieved by different synthetic versions of aldol-coupling reactions, catalyzed either by organocatalysts, enzymes, or metal-organic catalysts. We have investigated the formation of 1,4-substituted 2,3-dihydroxybutan-1-one derivatives from para- and meta-substituted phenylacetaldehydes by three distinctly different strategies. The first involved a direct aldol reaction with hydroxyacetone, dihydroxyacetone, or 2-hydroxyacetophenone, catalyzed by the cinchona derivative cinchonine. The second was reductive cross-coupling with methyl- or phenylglyoxal promoted by SmI 2 , resulting in either 5-substituted 3,4-dihydroxypentan-2-ones or 1,4 bis-phenyl-substituted butanones, respectively. Finally, in the third case, aldolase catalysis was employed for synthesis of the corresponding 1,3,4-trihydroxylated pentan-2-one derivatives. The organocatalytic route with cinchonine generated distereomerically enriched syn-products (de = 60-99%), with moderate enantiomeric excesses (ee = 43-56%) but did not produce aldols with either hydroxyacetone or dihydroxyacetone as donor ketones. The SmI 2 -promoted reductive cross-coupling generated product mixtures with diastereomeric and enantiomeric ratios close to unity. This route allowed for the production of both 1-methyl- and 1-phenyl-substituted 2,3-dihydroxybutanones at yields between 40-60%. Finally, the biocatalytic approach resulted in enantiopure syn-(3 R,4 S) 1,3,4-trihydroxypentan-2-ones.

Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N -heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O 2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo , into 3-succinoyl-semialdehyde-pyridine. NAD(P) + , O 2 , and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H 2 O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria. IMPORTANCE Nicotine has been studied as a model for toxic N -heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O 2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N -heterocyclic aromatic compounds in microorganisms., (Copyright © 2019 American Society for Microbiology.)

Raspberry ketone is an important ingredient in the flavor and fragrance industries. Due to its low content in fruits and vegetables, the production of natural raspberry ketone using heterologous synthesis in microbial strains is recently attracting increased attention. In this work, a heterologous pathway to produce raspberry ketone from p-coumaric acid, including 4-coumarate: CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone/zingerone synthase (RZS1) from plants, was successfully assembled in Escherichia coli. When the RZS1 gene was introduced into E. coli and co-expressed with two other genes, the intermediate 4-hydroxybenzylidene acetone in the pathway was almost completely transformed into a raspberry ketone. Substituting TB medium for M9 medium increased raspberry ketone titers by 3-4 times. Furthermore, the heterologous pathway was partitioned into two modules; module one produced p-coumaroyl-CoA from p-coumaric acid by 4CL, and module two produced raspberry ketone from coumaroyl-CoA by the action of BAS and RZS1. Optimizing the balanced expression of the two modules, it was shown that moderate expression of module one and high expression of module two was the best combination to enhance raspberry ketone production. The engineered strain CZ-8 reached 90.97 mg/l of raspberry ketone, which was 12 times higher than previously reported. In addition, the preferred approach of the heterologous pathway was related to the heterologous genes from different sources; for example, 4CL from Arabidopsis thaliana seemed to be more suitable for raspberry ketone production than that from Petroselinum crispum. This work paves an alternative way for future economic production of natural raspberry ketone.

The peroxisome proliferator-activated receptor-α (PPAR-α) controls the lipid and glucose metabolism and also affects inflammation, cell proliferation and apoptosis during cardiovascular disease. Raspberry ketone (RK) is a red raspberry (Rubusidaeus, Family-Rosaceae) plant constituent, which activates PPAR-α. This study was conducted to assess the cardioprotective action of RK against isoproterenol (ISO)-induced cardiotoxicity. Wistar rats were randomly divided into six groups (six rats/group). Rats were orally administered with RK (50, 100 and 200 mg/kg, respectively) and fenofibrate (standard, 80 mg/kg) for 28 days and ISO was administered (85 mg/kg, subcutaneously) on 27th and 28th day. Administration of ISO in rats significantly altered hemodynamic and electrocardiogram patterns, total antioxidant capacity, PPAR-α, and apolipoprotein C-III levels. These myocardial aberrations were further confirmed during infarct size, heart weight to body weight ratio and immunohistochemical assessments (caspase-3 and nuclear factor-κB). RK pretreatment (100 and 200 mg/kg) significantly protected rats against oxidative stress, inflammation, and dyslipidemia caused by ISO as demonstrated by change in hemodynamic, biochemical and histological parameters. The results so obtained were quite comparable with fenofibrate. Moreover, RK was found to have binding affinity with PPAR-α, as confirmed by docking analysis. PPAR-α expression and concentration was also found increased in presence of RK which gave impression that RK probably showed cardioprotection via PPAR-α activation, however direct binding study of RK with PPAR-α is needed to confirm this assumption., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Microorganisms release a plethora of volatile secondary metabolites. Up to now, it has been widely accepted that these volatile organic compounds are produced and emitted as a final product by a single organism e.g. a bacterial cell. We questioned this commonly assumed perspective and hypothesized that in diversely colonized microbial communities, bacterial cells can passively interact by emitting precursors which non-enzymatically react to form the active final compound. This hypothesis was inspired by the discovery of the bacterial metabolite schleiferon A. This bactericidal volatile compound is formed by a non-enzymatic reaction between acetoin and 2-phenylethylamine. Both precursors are released by Staphylococcus schleiferi cells. In order to provide evidence for our hypothesis that these precursors could also be released by bacterial cells of different species, we simultaneously but separately cultivated Serratia plymuthica 4Rx13 and Staphylococcus delphini 20771 which held responsible for only one precursor necessary for schleiferon A formation, respectively. By mixing their headspace, we demonstrated that these two species were able to deliver the active principle schleiferon A. Such a joint formation of a volatile secondary metabolite by different bacterial species has not been described yet. This highlights a new aspect of interpreting multispecies interactions in microbial communities as not only direct interactions between species might determine and influence the dynamics of the community. Events outside the cell could lead to the appearance of new compounds which could possess new community shaping properties.

The limited supply of fossil fuels and the establishment of new environmental policies shifted research in industry and academia toward sustainable production of the second generation of biofuels, with methyl ethyl ketone (MEK) being one promising fuel candidate. MEK is a commercially valuable petrochemical with an extensive application as a solvent. However, as of today, a sustainable and economically viable production of MEK has not yet been achieved despite several attempts of introducing biosynthetic pathways in industrial microorganisms. We used BNICE.ch as a retrobiosynthesis tool to discover all novel pathways around MEK. Out of 1325 identified compounds connecting to MEK with one reaction step, we selected 3-oxopentanoate, but-3-en-2-one, but-1-en-2-olate, butylamine, and 2-hydroxy-2-methylbutanenitrile for further study. We reconstructed 3 679 610 novel biosynthetic pathways toward these 5 compounds. We then embedded these pathways into the genome-scale model of E. coli, and a set of 18 622 were found to be the most biologically feasible ones on the basis of thermodynamics and their yields. For each novel reaction in the viable pathways, we proposed the most similar KEGG reactions, with their gene and protein sequences, as candidates for either a direct experimental implementation or as a basis for enzyme engineering. Through pathway similarity analysis we classified the pathways and identified the enzymes and precursors that were indispensable for the production of the target molecules. These retrobiosynthesis studies demonstrate the potential of BNICE.ch for discovery, systematic evaluation, and analysis of novel pathways in synthetic biology and metabolic engineering studies.

Many chemical carcinogens require metabolic activation via xenobiotic-metabolizing enzymes in order to exert their genotoxic effects. Evidence from numerous in-vitro studies, utilizing reconstituted systems, microsomal fractions and cultured cells, implicates cytochrome P450 enzymes as being the predominant enzymes responsible for the metabolic activation of many procarcinogens. With the development of targeted gene disruption methodologies, knockout mouse models have been generated that allow investigation of the in-vivo roles of P450 enzymes in the metabolic activation of carcinogens. This review covers studies in which five procarcinogens representing different chemical classes, benzo[a]pyrene, 4-aminobiphenyl (4-ABP), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-9H-pyrido[2,3-b]indole and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, have been administered to different P450 knockout mouse models. Paradoxically, while in-vitro studies using subcellular fractions enriched with P450 enzymes and their cofactors have been widely used to determine the pathways of activation of carcinogens, there is evidence from the in-vivo studies of cases where these same enzyme systems appear to have a more predominant role in carcinogen detoxication rather than activation.

Inhibition of α-glucosidase is directly associated with treatment of type 2 diabetes. In this regard, we conducted enzyme kinetics integrated with computational docking simulation to assess the inhibitory effect of raspberry ketone (RK) on α-glucosidase. RK bound to the active site of α-glucosidase and interacted with several key residues such as ASP68, TYR71, HIS111, PHE157, PHE158, PHE177, GLN181, ASP214, THR215, ASP349, ASP408, and ARG439, as detected by protein-ligand docking simulation. Subsequently, we confirmed the action of RK on α-glucosidase as the non-competitive type of inhibition in a reversible and rapidly binding manner. The relevant kinetic parameters were IC 50 =6.17±0.46mM and K i =7.939±0.211mM. Regarding the structure-activity relationship, the higher concentration of RK induced slight modulation of the shape of the active site as monitored by hydrophobic exposure. The tertiary conformational change was linked to RK inhibition, and mostly involved regional changes of the active site. Our study provides insight into the functional role of RK due to its structural property of a hydroxyphenyl ring that interacts with the active site of α-glucosidase. We suggest that similar hydroxyphenyl ring compounds targeting the key residues of the active site might be potential α-glucosidase inhibitors., (Copyright © 2018. Published by Elsevier B.V.)

1. Multiple exposures are ubiquitous in industrial environments. In this article, we highlight the risks faced by workers and complete the data available on the metabolic impact of a common mixture: toluene (TOL) and methylethylketone (MEK). 2. Rats were exposed by inhalation under controlled conditions either to each solvent individually, or to mixtures of the two. How the interaction between the two solvents affected their fate in the blood and brain, their main relevant urinary metabolites (o-cresol, benzylmercapturic acid for TOL and 2,3-butanediols for MEK) and their hepatic metabolism were investigated. 3. Although the cytochrome P450 concentration was unchanged, and the activities of CYP1A2 and CYP2E1 isoforms were not additively or synergistically induced by co-exposure, TOL metabolism was inhibited by the presence of MEK (and vice versa). Depending on the relative proportions of each compound in the mixture, this sometimes resulted in a large increase in blood and brain concentrations. Apart from extreme cases (unbalanced mixtures), the amount of o-cresol and benzylmercapturic acid (and to a lesser extent 2,3-butanediols) excreted were proportional to the blood solvent concentrations. 4. In a co-exposure context, ortho-cresol and benzylmercapturic acid can be used as urinary biomarkers in biomonitoring for employees to relatively accurately assess TOL exposure.

Highly reactive α,β-unsaturated carbonyl compounds, such as acrolein (ACR), crotonaldehyde (CA) and methyl vinyl ketone (MVK), are environmental pollutants present in high concentrations in cigarette smoke. We have previously found that these carbonyl compounds in cigarette smoke extract (CSE) react with intracellular glutathione (GSH) to produce the corresponding GSH-ACR, GSH-CA and GSH-MVK adducts via Michael addition reaction. These adducts are then further reduced to the corresponding alcohol forms by intracellular aldo-keto reductases in highly metastatic mouse melanoma (B16-BL6) cells and then excreted into the extracellular fluid. This time, we conducted a similar study using sheep erythrocytes and found analogous changes in the sheep erythrocytes after exposure to CSE as those with B16-BL6 cells. This indicates similarity of the detoxification pathways of the α,β-unsaturated carbonyl compounds in sheep blood cells and B16-BL6 cells. Also, we found that the GSH-MVK adduct was reduced by aldose reductase in a cell-free solution to generate its alcohol form, and its reduction reaction was completely suppressed by pretreatment with epalrestat, an aldose reductase inhibitor, a member of the aldo-keto reductase family. In the presence of sheep blood cells, however, reduction of the GSH-MVK adduct was partially inhibited by epalrestat. This revealed that some member of the aldo-keto reductase superfamily other than aldose reductase is involved in reduction of the GSH-MVK adduct in sheep blood. These results suggest that blood cells, mainly erythrocytes are involved in reducing the inhalation toxicity of cigarette smoke via an aldo-keto reductase pathway other than that of aldose reductase.

There is substantial interest in engineering microorganisms to produce industrial chemicals that are currently derived from petroleum. One of these petrochemicals is butanone, which could be produced from microbially synthesized 2,3-butanediol through the action of a suitable dehydratase enzyme. Unfortunately, however, there are no known enzymes that natively catalyze this reaction. In this work, the authors set out to engineer the B 12 -dependent glycerol dehydratase from Klebsiella pneumoniae (KpGDHt), in order to increase its activity for the conversion of meso-2,3-butanediol into butanone. The authors began by fusing the α and β subunits of the enzyme, to simplify downstream high-throughput screening protocols. Serendipitously, the fusion protein showed a 20°C increase in its temperature optimum. Using this stabilized scaffold as a starting point, the authors employed the combinatorial active site saturation test and consensus-guided mutagenesis to randomize 28 residues within 12 Å of the KpGDHt active site. By screening over 5500 variants, the authors discovered a single point mutation (T200S) that increased the catalytic efficiency of meso-2,3-butanediol dehydration by four-fold, to a value of k cat /K M  = 5.1 × 10 3 M -1 s -1 . Thus the authors report what is, to date, the most comprehensive mutagenesis and the largest engineered increase in catalytic efficiency on the B 12 -dependent glycerol dehydratase scaffold., (© 2017 The Authors. Biotechnology Journal Published by Wiley-VCHVerlag GmbH & Co. KGaA.)

Flavin-containing monooxygenases (FMOs) are emerging as effective players in oxidative drug metabolism. Until recently, the functions of the five human FMO isoforms were mostly linked to their capability of oxygenating molecules containing soft N- and S-nucleophiles. However, the human FMO isoform 5 was recently shown to feature an atypical activity as Baeyer-Villiger monooxygenase. With the aim of evaluating such an alternative entry point in the metabolism of active pharmaceutical ingredients, we selected and tested drug molecules bearing a carbonyl group on an aliphatic chain. Nabumetone and pentoxifylline, two widely used pharmaceuticals, were thereby demonstrated to be efficiently oxidized in vitro by FMO5 to the corresponding acetate esters with high selectivity. The proposed pathways explain the formation of a predominant plasma metabolite of pentoxifylline as well as the crucial transformation of the pro-drug nabumetone into the pharmacologically active compound. Using the recombinant enzyme, the ester derivatives of both drugs were obtained in milligram amounts, purified, and fully characterized. This protocol can potentially be extended to other FMO5 candidate substrates as it represents an effective and robust bench-ready platform applicable to API screening and metabolite synthesis.

It took several millennia to fully understand the scientific intricacies of the process through which grape juice is turned into wine. This yeast-driven fermentation process is still being perfected and advanced today. Motivated by ever-changing consumer preferences and the belief that the 'best' wine is yet to be made, numerous approaches are being pursued to improve the process of yeast fermentation and the quality of wine. Central to recent enhancements in winemaking processes and wine quality is the development of Saccharomyces cerevisiae yeast strains with improved robustness, fermentation efficiencies and sensory properties. The emerging science of Synthetic Biology - including genome engineering and DNA editing technologies - is taking yeast strain development into a totally new realm of possibility. The first example of how future wine strain development might be impacted by these new 'history-making' Synthetic Biology technologies, is the de novo production of the raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one, in a wine yeast containing a synthetic DNA cassette. This article explores how this breakthrough and the imminent outcome of the international Yeast 2.0 (or Sc2.0) project, aimed at the synthesis of the entire genome of a laboratory strain of S. cerevisiae, might accelerate the design of improved wine yeasts., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The presence of nicotine and nicotinic acid (NA) in the marine environment has caused great harm to human health and the natural environment. Therefore, there is an urgent need to use efficient and economical methods to remove such pollutants from the environment. In this study, a nicotine and NA-degrading bacterium-strain JQ581-was isolated from sediment from the East China Sea and identified as a member of Pseudomonas putida based on morphology, physio-biochemical characteristics, and 16S rDNA gene analysis. The relationship between growth and nicotine/NA degradation suggested that strain JQ581 was a good candidate for applications in the bioaugmentation treatment of nicotine/NA contamination. The degradation intermediates of nicotine are pseudooxynicotine (PN) and 3-succinoyl-pyridine (SP) based on UV, high performance liquid chromatography, and liquid chromatography-mass spectrometry analyses. However, 6-hydroxy-3-succinoyl-pyridine (HSP) was not detected. NA degradation intermediates were identified as 6-hydroxynicotinic acid (6HNA). The whole genome of strain JQ581 was sequenced and analyzed. Genome sequence analysis revealed that strain JQ581 contained the gene clusters for nicotine and NA degradation. This is the first report where a marine-derived Pseudomonas strain had the ability to degrade nicotine and NA simultaneously., Competing Interests: The authors declare no conflict of interest.

The purpose of this work was to study the potential of gas chromatography-ion mobility spectrometry (GC-IMS) to differentiate lactic acid bacteria (LAB) through target identification and fingerprints of volatile metabolites. The LAB selected were used as reference strains for their influence in the flavour of cheese. The four strains of LAB can be distinguished by the fingerprints generated by the volatile organic compounds (VOCs) emitted. 2-butanone, 2-pentanone, 2-heptanone and 3-methyl-1-butanol were identified as relevant VOCs for Lactobacillus casei and Lactobacillus paracasei subsp. paracasei. 2-Butanone and 3-methyl-1-butanol were identified in Lactococcus lactis subsp. lactis and Lactococcus cremoris subsp. cremoris. The IMS signals monitoring during a 24-30h period showed the growth of the LAB in vitro. The results demonstrated that GC-IMS is a useful technology for bacteria recognition and also for screening the aromatic potential of new isolates of LAB., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Methyl ethyl ketone (MEK) is a common groundwater contaminant often present with more toxic compounds of primary interest. Because of this, few studies have been performed to determine the effect of microbial community structure on MEK biodegradation rates in aquifer sediments. Here, microcosms were prepared with aquifer sediments containing MEK following a massive spill event and compared to laboratory-spiked sediments, with MEK biodegradation rates quantified under mixed aerobic/anaerobic conditions. Biodegradation was achieved in MEK-contaminated site sediment microcosms at about half of the solubility (356 mg/L) with largely Firmicutes population under iron-reducing conditions. MEK was biodegraded at a higher rate [4.0 ± 0.74 mg/(L days)] in previously exposed site samples compared to previously uncontaminated sediments [0.51 ± 0.14 mg/(L days)]. Amplicon sequencing and denaturing gradient gel electrophoresis of 16S rRNA genes were combined to understand the relationship between contamination levels, biodegradation, and community structure across the plume. More heavily contaminated sediments collected from an MEK-contaminated field site had the most similar communities than less contaminated sediments from the same site despite differences in sediment texture. The more diverse microbial community observed in the laboratory-spiked sediments reduced MEK concentration 47 % over 92 days. Results of this study suggest lower rates of MEK biodegradation in iron-reducing aquifer sediments than previously reported for methanogenic conditions and biodegradation rates comparable to previously reported nitrate- and sulfate-reducing conditions.

A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP + binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate. The resulting libraries were screened using whole cell biotransformations, and headspace gas chromatography-mass spectrometry was used to identify improved AcCHMO variants. This revealed that the I491A AcCHMO mutant exhibits a significant improvement over the wild type enzyme in the desired regioselectivity using 2-butanone as a substrate (40% vs 26% methyl propanoate, respectively). Another interesting mutant is the T56S AcCHMO mutant, which exhibits a higher conversion yield (92%) and k cat (0.5 s -1 ) than wild type AcCHMO (52% and 0.3 s -1 , respectively). Interestingly, the uncoupling rate for the T56S AcCHMO mutant is also significantly lower than that for the wild type enzyme. The T56S/I491A double mutant combined the beneficial effects of both mutations leading to higher conversion and improved regioselectivity. This study shows that even for a relatively small aliphatic substrate (2-butanone), catalytic efficiency and regioselectivity can be tuned by structure-inspired enzyme engineering.

N'-Nitrosonornicotine (NNN) is considered to be one of the most carcinogenic compounds of the four conventionally measured tobacco-specific N-nitrosamines (TSNAs). In order to evaluate the significance of metabolic activation for the carcinogenic potential of NNN, its catalysis by different phase I enzymes and its interaction with nicotine and nicotine-derived TSNAs need to be investigated. Using an in vitro model system, NNN was found to interact with various cytochrome P450 enzymes, predominantly CYP2A13. Mass-spectrometric analysis confirmed the presence of various predicted NNN metabolites, including 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and 4-hydroxy-4-(3-pyridyl)-butyric acid (hydroxy acid) but little amount of 4-oxo-4-(3-pyridyl) butanal (OPB), which was somewhat different from in vitro NNK metabolism. Addition of nicotine, N'-Nitrosoanatabine (NAT), N'-Nitrosoanabasine (NAB) resulted in a competitive inhibition for NNN metabolism. The inhibition constant Ki value was calculated as 0.98 μM (nicotine), 1.37 μM (NAT), 0.71 μM (NAB) for HPB formation, 1.35 μM (nicotine), 1.35 μM (NAT), 1.01 μM (NAB) for hydroxy acid formation and 8.40 μM (nicotine), 3.40 μM (NAT), 3.04 μM (NAB) for OPB formation, respectively. These results implied that CYP2A13 is the most efficient enzyme to metabolize NNN in vitro and structurally similar tobacco constitutes including nicotine, NAT and NAB influence the metabolic activation of NNN, which may further interfere in its carcinogenicity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

1-Chloro-3-buten-2-one (CBO) is an in vitro metabolite of 1,3-butadiene (BD), a carcinogenic air pollutant. CBO exhibited potent cytotoxicity and genotoxicity that have been attributed in part to its reactivity toward DNA. Previously, we have characterized the CBO adducts with 2'-deoxycytidine and 2'-deoxyguanosine. In the present study, we report on the reaction of CBO with 2'-deoxyadenosine (dA) under in vitro physiological conditions (pH 7.4, 37 °C). We used the synthesized standards and their decomposition and acid-hydrolysis products to characterize the CBO-DNA adducts formed in human cells. The fused-ring dA adducts (dA-1 and dA-2) were readily synthesized and were structurally characterized as 1,N(6)-(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine and 1,N(6)-(1-hydroxy-1-chloromethylpropan-1,3-diyl)-2'-deoxyadenosine, respectively. dA-1 exhibited a half-life of 16.0 ± 0.7 h and decomposed to dA at pH 7.4 and 37 °C. At similar conditions, dA-2 decomposed to dA-1 and dA, and had a half-life of 0.9 ± 0.1 h. These results provide strong evidence for dA-1 being a degradation product of dA-2. dA-1 is formed by replacement of the chlorine atom of dA-2 by a hydroxyl group. The slow decomposition of dA-1 to dA, along with the detection of hydroxymethyl vinyl ketone (HMVK) as another degradation product, suggested equilibrium between dA-1 and a ring-opened carbonyl-containing intermediate that undergoes a retro-Michael reaction to yield dA and HMVK. Acid hydrolysis of dA-1 and dA-2 yielded the corresponding deribosylated products A-1D and A-2D, respectively. In the acid-hydrolyzed reaction mixture of CBO with calf thymus DNA, both A-1D and A-2D could be detected; however, the amount of A-2D was significantly larger than that of A-1D. Interestingly, only A-2D could be detected by LC-MS analysis of acid-hydrolyzed DNA from cells incubated with CBO, suggesting that dA-2 was stable in DNA and thus may play an important role in the genotoxicity and carcinogenicity of BD. In addition, A-2D could be developed as a biomarker of CBO formation in human cells.

To expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway in Escherichia coli for heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producing E. coli host strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohol genesis under selected fermentative culture conditions were investigated. Using the engineered E. coli strain for batch cultivation with 30 g liter(-1)glycerol as the carbon source, we achieved coproduction of 1.3 g liter(-1)butanone and 2.9 g liter(-1)acetone. The results suggest that approximately 42% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Flurbiprofen axetil (FPA) is an injection product and a prodrug of a non-steroidal anti-inflammatory drug (NSAID). After injection, it is rapidly hydrolyzed to the active form, flurbiprofen (FP). Since frequent injections of FPA can lead to abnormal physiology, an administration strategy is necessary to ensure there is enhancement of the analgesic efficiency of FP after a single dose and to reduce the total number of doses. FP strongly binds to site II of albumin, and thus the free (unbound) FP concentration is low. This study focused on 6-methoxy-2-naphthylacetic acid (6-MNA), the active metabolite of nabumetone (a prodrug of NSAID). We performed ultrafiltration experiments and pharmacokinetics analysis in rats to investigate whether the inhibitory effect of 6-MNA on FP binding to albumin increased the free FP concentration in vitro and in vivo. Results indicated that 6-MNA inhibited the binding of FP to albumin competitively. When 6-MNA was injected in rats, there was a significant increase in the free FP concentration and the area under concentration-time curve (AUC) calculated from the free FP concentration, while there was a significant decrease in the total (bound + free) FP concentration and the AUC calculated from the total FP concentration. These findings indicate that 6-MNA inhibits the protein binding of FP in vivo. This suggests that the frequency of FPA injections can be reduced when administered with nabumetone, as there is increase in the free FP concentration associated with pharmacological effect.

Background: Raspberry ketone is the primary aroma compound found in raspberries and naturally derived raspberry ketone is a valuable flavoring agent. The economic incentives for the production of raspberry ketone, combined with the very poor yields from plant tissue, therefore make this compound an excellent target for heterologous production in synthetically engineered microbial strains., Methods: A de novo pathway for the production of raspberry ketone was assembled using four heterologous genes, encoding phenylalanine/tyrosine ammonia lyase, cinnamate-4-hydroxlase, coumarate-CoA ligase and benzalacetone synthase, in an industrial strain of Saccharomyces cerevisiae. Synthetic protein fusions were also explored as a means of increasing yields of the final product., Results: The highest raspberry ketone concentration achieved in minimal media exceeded 7.5 mg/L when strains were fed with 3 mM p-coumaric acid; or 2.8 mg/L for complete de novo synthesis, both of which utilized a coumarate-CoA ligase, benzalacetone synthase synthetic fusion protein that increased yields over fivefold compared to the native enzymes. In addition, this strain was shown to be able to produce significant amounts of raspberry ketone in wine, with a raspberry ketone titer of 3.5 mg/L achieved after aerobic fermentation of Chardonnay juice or 0.68 mg/L under anaerobic winemaking conditions., Conclusions: We have shown that it is possible to produce sensorially-relevant quantities of raspberry ketone in an industrial heterologous host. This paves the way for further pathway optimization to provide an economical alternative to raspberry ketone derived from plant sources.

Queensland fruit fly, Bactrocera tryoni (Q-fly), is a major pest of horticultural crops in eastern Australia. Lures that attract male Q-fly are important for detection of incursions and outbreaks, monitoring of populations, and control by mass trapping and male annihilation. Cuelure, an analog of naturally occurring raspberry ketone, is the standard Q-fly lure, but it has limited efficacy compared with lures that are available for some other fruit flies such as methyl eugenol for B. dorsalis. Melolure is a more recently developed raspberry ketone analog that has shown better attraction than cuelure in some field studies but not in others. A novel fluorinated analog of raspberry ketone, raspberry ketone trifluoroacetate (RKTA), has been developed as a potential improvement on cuelure and melolure. RKTA placed on laboratory cages containing 2-week-old Q-flies elicited strong behavioral responses from males. Quantification of Q-fly responses in these cages, using digital images to estimate numbers of flies aggregated near different lures, showed RKTA attracted and arrested significantly more flies than did cuelure or melolure. RKTA shows good potential as a new lure for improved surveillance and control of Q-fly.

Pseudooxynicotine amine oxidase (Pnao) is essential to the pyrrolidine pathway of nicotine degradation of Pseudomonas putida strain S16, which is significant for the detoxification of nicotine, through removing the CH3NH2 group. However, little is known about biochemical mechanism of this enzyme. Here, we characterized its properties and biochemical mechanism. Isotope labeling experiments provided direct evidence that the newly introduced oxygen atom in 3-succinoylsemialdehyde-pyridine is derived from H2O, but not from O2. Pnao was very stable at temperatures below 50 °C; below this temperature, the enzyme activity increased as temperature rose. Site-directed mutagenesis studies showed that residue 180 is important for its thermal stability. In addition, tungstate may enhance the enzyme activity, which has rarely been reported before. Our findings make a further understanding of the crucial Pnao in nicotine degradation.

2-Butanone is an important commodity chemical of wide application in different areas. In this study, Klebsiella pneumoniae was engineered to directly produce 2-butanone from glucose by extending its native 2, 3-butanediol synthesis pathway. To identify the potential enzyme for the efficient conversion of 2, 3-butanediol to 2-butanone, we screened different glycerol dehydratases and diol dehydratases. By introducing the diol dehydratase from Lactobacillus brevis and deleting the ldhA gene encoding lactate dehydrogenase, the engineered K. pneumoniae was able to accumulate 246 mg/L of 2-butanone in shake flask. With further optimization of culture condition, the titer of 2-butanone was increased to 450 mg/L. This study lays the basis for developing an efficient biological process for 2-butanone production.

Mycobacteria such as Mycobacterium smegmatis strain mc(2)155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E. coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The metabolic reduction of nabumetone was examined by inhibition and correlation studies using human liver microsomes and cytosol. This reduction was observed in both fractions, with the V(max) values for reduction activity being approximately fourfold higher, and the V(max)/K(m) values approximately three-fold higher, in the microsomes than in the cytosol. The reduction of nabumetone was inhibited by 18β-glycyrrhetinic acid, an 11β-hydroxysteroid dehydrogenase (11β-HSD) inhibitor, in the microsomal fraction. The reduction activity was also inhibited by quercetin and menadione [carbonyl reductase (CBR) inhibitors], and by phenolphthalein and medroxyprogesterone acetate [potent inhibitors of aldo-keto reductase (AKR) 1C1, 1C2 and 1C4] in the cytosol. A good correlation (r² = 0.93) was observed between the reduction of nabumetone and of cortisone, as a marker of 11β-HSD activity, in the microsomal fractions. There was also an excellent relationship between reduction of nabumetone and of the AKR1C substrates, acetohexamide, and ethacrynic acid (r 2 = 0.92 and 0.93, respectively), in the cytosol fractions. However, a poor correlation was observed between the formation of 4-(6-methoxy-2-naphthyl)-butan-2-ol (MNBO) from nabumetone and CBR activity (with 4-benzoyl pyridine reduction as a CBR substrate) in the cytosol fractions (r² = 0.24). These findings indicate that nabumetone may be metabolized by 11β-HSD in human liver microsomes, and primarily by AKR1C4 in human liver cytosol, although multiple enzymes in the AKR1C subfamily may be involved. It cannot be completely denied that CBR is involved to some extent in the formation of MNBO from nabumetone in the cytosol fraction.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen. We used an air-liquid interface epithelial cell culture system to model changes associated with NNK exposure relative to pathologies documented in human tobacco-related illnesses. Although in vitro systems exhibit certain limitations, they often offer accentuation of subtle pathologies. While the distribution of cell types in control cultures typically favors the ciliated cell phenotype, NNK-exposed cultures transitioned to non-ciliated cell phenotypes as well as reflecting features consistent with squamous metaplasia. We conclude that NNK impacts normal growth and differentiation of human airway epithelium in a short interval of time in vitro.

An online solid-phase microextraction coupled liquid chromatography-electrospray ionization-ion trap mass spectrometry was developed for the analysis of trace R- and S-4-phenyl-2-butanol (R- and S-pbol) in salt rich cell culture of Saccharomyces cerevisiae catalyzed stereoselective reduction of 4-pheny-2-butanone (pbone). A Supel-Q PLOT capillary column was used for the extraction and deionized distilled water was used as the extraction mobile phase. The extraction flow rate and extraction time were at 0.1 mL min(-1) and 0.95 min, respectively. The three target analytes, pbone, R-pbol, and S-4-pbol, were desorbed and eluted by the mobile phase of water/methanol/isopropanol (55/25/20, v/v/v) with a flow rate of 0.5 mL min(-1) and analyzed by a chiral column. The mass spectrometric detection of the three target analytes was in positive ion mode with the signal [M+Na](+). The matrix-matched external standard calibration curves with linear concentration range between 0 and 50 μg mL(-1) were used for quantitative analysis. The linear regression correlation coefficients (r(2)) of the standard calibration curves were between 0.9950 and 0.9961. The yeast mediated reduction was performed with a recation culture of yeast incubation culture/glycerol (70/30, v/v) for 4 days. This biotransformation possessed 82.3% yield and 92.9% S-enantomeric excess. The limit of detection (LOD)/limit of quantification (LOQ) for pbone, R-pbol, and S-pbol was 0.02/0.067, 0.01/0.033, and 0.01/0.033 μg mL(-1), respectively. The intra-day and inter-day precisions from repeated measurements were 10.8-21.1% and 11.6-18.7%, respectively. The analysis accuracy from spike recovery was 84-91%., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1.

In this study, a novel strain of Pichia jadinii, HBY61, capable of the biocatalysis of 4-hydroxy-2-butanone (4H2B) to (R)-1,3-BD was isolated. HBY61 produced (R)-1,3-BD with high activity and absolute stereochemical selectivity (100 % e.e). Glucose and beef extract were found to be the key factors governing the fermentation, and their optimal concentrations were determined to be 84.2 and 43.7 g/L, respectively. The optimal bioconversion conditions of 4H2B catalyzed by HBY61 were pH 7.4, 30 °C, and 250 rpm with 6 % (v/v) glucose as the co-substrate. Accordingly, when 45 g/L of 4H2B was divided into three equal parts and added successively into the system at set time intervals, the maximum (R)-1,3-BD concentration reached 38.3 g/L with high yield (85.1 %) and strict 100 % enantioselectivity. Compared with previously reported yields for the biocatalytic production of (R)-1,3-BD, the use of strain HBY61 provided a high yield with excellent stereoselectivity.

In tephritid fruit flies of the genus Bactrocera Macquart, a group of plant derived compounds (sensu amplo 'male lures') enhance the mating success of males that have consumed them. For flies responding to the male lure methyl eugenol, this is due to the accumulation of chemicals derived from the male lure in the male rectal gland (site of pheromone synthesis) and the subsequent release of an attractive pheromone. Cuelure, raspberry ketone and zingerone are a second, related group of male lures to which many Bactrocera species respond. Raspberry ketone and cuelure are both known to accumulate in the rectal gland of males as raspberry ketone, but it is not known if the emitted male pheromone is subsequently altered in complexity or is more attractive to females. Using Bactrocera tryoni as our test insect, and cuelure and zingerone as our test chemicals, we assess: (i) lure accumulation in the rectal gland; (ii) if the lures are released exclusively in association with the male pheromone; and (iii) if the pheromone of lure-fed males is more attractive to females than the pheromone of lure-unfed males. As previously documented, we found cuelure was stored in its hydroxyl form of raspberry ketone, while zingerone was stored largely in an unaltered state. Small but consistent amounts of raspberry ketone and β-(4-hydroxy-3-methoxyphenyl)-propionic acid were also detected in zingerone-fed flies. Males released the ingested lures or their analogues, along with endogenous pheromone chemicals, only during the dusk courtship period. More females responded to squashed rectal glands extracted from flies fed on cuelure than to glands from control flies, while more females responded to the pheromone of calling cuelure-fed males than to control males. The response to zingerone treatments in both cases was not different from the control. The results show that male B. tryoni release ingested lures as part of their pheromone blend and, at least for cuelure, this attracts more females., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

2-Butanol and its chemical precursor butanone (methyl ethyl ketone--MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuteri), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions.

Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). Despite worldwide use of nabumetone as an anti-inflammatory agent, the mechanism of its carbon-carbon bond cleavage reaction remains obscure. With the help of authentic synthetic standards, we report here that the reaction involves 3-hydroxylation, carbon-carbon cleavage to the aldehyde, and oxidation of the aldehyde to the acid, all catalyzed by CYP1A2 or, less effectively, by other P450 enzymes. The data indicate that the carbon-carbon bond cleavage is mediated by the ferric peroxo anion rather than the ferryl species in the P450 catalytic cycle. CYP1A2 also catalyzes O-demethylation and alcohol to ketone transformations of nabumetone and its analogs.

Tobacco wastes that contain nicotine alkaloids are harmful to human health and the environment. In the investigation, a novel nicotine-biodegrading bacterium TND35 was isolated and identified as Pseudomonas plecoglossicida on the basis of phenotypic, biochemical characteristics and 16S rRNA sequence homology. We have studied the nicotine biodegradation potential of strain TND35 by detecting the intermediate metabolites using an array of approaches such as HPLC, GC-MS, NMR and FT-IR. Biotransformation metabolites, N-methylmyosmine, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and other three new intermediate metabolites namely, 3,5-bis (1-methylpyrrolidin-2-yl) pyridine, 2,3-dihydro-1-methyl-5-(pyridin-3-yl)-1H-pyrrol-2-ol and 5-(pyridin-3-yl)-1H-pyrrol-2(3H)-one have been identified. Interestingly, these intermediate metabolites suggest that the strain TND35 employs a novel nicotine biodegradation pathway, which is different from the reported pathways of Aspergillus oryzae 112822, Arthrobacter nicotinovorans pAO1, Agrobacterium tumefaciens S33 and other species of Pseudomonas. The metabolite, HPB reported in this study can also be used as biochemical marker for tobacco related cancer studies.

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches., (© 2013.)