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1.
Sci Rep ; 11(1): 17632, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34480052

RESUMO

Sterile male Queensland fruit fly, Bactrocera tryoni (Froggatt), fed as immature adults on the plant compound raspberry ketone (RK), show a reduced attraction to cuelure, a synthetic analogue of RK used as an attractant in Male Annihilation Technique. We hypothesized the reduced attraction of RK-fed adult males to cuelure may be a consequence of altered expression of chemoreception genes. A Y-tube olfactometer assay with RK-fed and RK-unfed sterile B. tryoni males tested the subsequent behavioural response to cuelure. Behavioral assays confirmed a significant decrease in attraction of RK-fed sterile males to cuelure. RK-fed, non-responders (to cue-lure) and RK-unfed, responders (to cue-lure) males were sampled and gene expression compared by de novo RNA-seq analysis. A total of 269 genes in fly heads were differentially expressed between replicated groups of RK-fed, cuelure non-responders and RK-unfed, cuelure responders. Among them, 218 genes including 4 chemoreceptor genes were up regulated and 51 genes were down regulated in RK-fed, cuelure non-responders. De novo assembly generated many genes with unknown functions and no significant BLAST hits to homologues in other species. The enriched and suppressed genes reported here, shed light on the transcriptional changes that affect the dynamics of insect responses to chemical stimuli.


Assuntos
Butanonas , Células Quimiorreceptoras/metabolismo , Suplementos Nutricionais , Regulação da Expressão Gênica , Infertilidade Masculina/metabolismo , Tephritidae/metabolismo , Animais , Masculino
2.
Talanta ; 235: 122716, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517584

RESUMO

Raspberry ketone (RK) is a major flavor compound in red raspberries, and it has been marketed as a popular weight-loss dietary supplement with high potential in accumulating in fatty tissues. However, challenges in extracting and characterizing RK and its associated phenolic compounds in fatty tissues persist due to the complex matrix effect. In this work, we reported a high-throughput sample preparation method for RK and 25 related phenolic compounds in white adipose tissues using an improved micro-scale QuEChERS (quick, efficient, cheap, easy, rugged and safe) approach with enhanced matrix removal (EMR)-lipid cleanup in 96-well plates, followed by UHPLC-QqQ-MS/MS analysis. The absolute recovery was 73-105% at the extraction step, and achieved 71-96% at the EMR cleanup step. The EMR cleanup removed around 66% of total lipids in the acetonitrile extract as profiled by UHPLC-QTOF-MS/MS. The innovative introduction of a reversed-phase C18 sorbent into the extract significantly improved the analytes' recovery during SpeedVac drying. The final accuracy achieved 80-120% for most analytes. Overall, this newly developed and validated method could serve as a powerful tool for analyzing RK and related phenolic compounds in fatty tissues.


Assuntos
Butanonas , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Fenóis , Extração em Fase Sólida
3.
Chemosphere ; 283: 131183, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34467940

RESUMO

Air-water interfaces are ubiquitous in nature, as manifested in the form of the surfaces of oceans, lakes, and atmospheric aqueous aerosols. The aerosol droplets interface, in particular, plays a critical role in numerous atmospheric chemistry processes. Methyl vinyl ketone (MVK) and methacrolein (MACR), two abundant volatile organic compounds, are the significant precursors of Criegee intermediates and secondary organic aerosol. In this work, the physicochemical properties of MVK and MACR at the air-water interface are studied from a theoretical perspective. The free energy wells of MVK and MACR occur at the air-water interface, and the absorption probabilities of them are 71% and 67%, respectively. Repulsion dominates the interactions between MVK/MACR and water molecules in the bulk region, while attraction is dominant at the interface. The two molecules tend to tilt at the interface, with the CC bond exposed at the outer interface. The most likely reaction scenario of O3-initiated MVK/MACR reaction in the troposphere is also determined for the first time. Based on the molecular dynamics simulation results, the activity sequence of MVK + O3 is given at four different environments by the density functional theory method: air-water interface, mineral clusters interface, bulk solution, and homogeneous gas. The interfacial water molecule can catalyze the reaction of MVK with O3, and the rate constant at the air-water interface is ~6 times larger than that on the mineral surface model. Compared with mineral particles, aqueous particles play a more significant role in modifying the reaction properties of atmospheric organic species.


Assuntos
Poluentes Atmosféricos , Água , Acroleína/análogos & derivados , Adsorção , Poluentes Atmosféricos/análise , Butanonas , Meio Ambiente , Água/análise
4.
Angew Chem Int Ed Engl ; 60(43): 23232-23240, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34339587

RESUMO

The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.


Assuntos
Acetaldeído/análise , Acetona/análise , Aldeídos/análise , Butanonas/análise , Di-Hidroxiacetona/análise , Metabolômica/métodos , Acetaldeído/sangue , Acetaldeído/química , Acetaldeído/urina , Acetamidas/química , Acetona/sangue , Acetona/química , Acetona/urina , Aldeídos/sangue , Aldeídos/química , Aldeídos/urina , Butanonas/sangue , Butanonas/química , Butanonas/urina , Carbono/química , Isótopos de Carbono/química , Di-Hidroxiacetona/sangue , Di-Hidroxiacetona/química , Di-Hidroxiacetona/urina , Fezes/química , Microbioma Gastrointestinal , Humanos , Indicadores e Reagentes/química , Limite de Detecção , Urina/química
5.
J Econ Entomol ; 114(5): 2147-2154, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34324680

RESUMO

The sterile insect technique (SIT) is a sustainable pest management tool based on the release of millions of sterile insects that suppress reproduction in targeted populations. Success of SIT depends on survival, maturation, dispersal, and mating of released sterile insects. Laboratory and field cage studies have demonstrated that dietary supplements of methoprene and raspberry ketone (RK) promote sexual maturation of adult Queensland fruit fly, Bactrocera tryoni (Froggatt), and may hence shorten the delay between release and maturity in the field. We investigated the effects of methoprene and RK dietary supplements on field abundance of sexually mature sterile Q-flies relative to untreated flies fed only sugar and yeast hydrolysate before release at 2 d of age. Compared with untreated flies, more methoprene- and RK-treated flies were recaptured in cuelure traps to which only sexually mature males are attracted. At distances of 100 and 200 m from the release point, recapture rates were higher for methoprene- and RK-treated flies than for untreated flies, but at 300 m recapture rates were low and were similar for treated and untreated flies. Rainfall, relative humidity, wind speed, and wind direction did not affect recapture rates, but temperature was positively correlated with recapture rates for all treatments. There was a strong correlation between the number of sterile and wild flies caught in traps, indicating co-location in the field. Dietary supplements of methoprene and RK can substantially increase abundance of sexually mature sterile male Q-flies in the field following release as 2-d-old immature adults.


Assuntos
Tephritidae , Animais , Butanonas , Suplementos Nutricionais , Masculino , Metoprene , Saccharomyces cerevisiae
6.
Sheng Wu Gong Cheng Xue Bao ; 37(7): 2495-2502, 2021 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-34327914

RESUMO

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 µg/g (fresh weight) and 5.9 µg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 µg/g).


Assuntos
Chlamydomonas reinhardtii , Policetídeo Sintases , Acil Coenzima A , Butanonas , Chlamydomonas reinhardtii/genética , Ligases
7.
Molecules ; 26(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205848

RESUMO

In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates' molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles.


Assuntos
Butanonas/química , Lipase/metabolismo , Xilose/química , Biocatálise , Esterificação , Temperatura Alta , Ácido Oleico/química
8.
Biomed Res Int ; 2021: 9981815, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34307685

RESUMO

Background: Colorectal cancer (CRC) is among the top three gastrointestinal malignancy in morbidity and mortality. The abnormal activation of Wnt/ß-catenin pathway is considered to be a key factor in the occurrence and development of CRC. Novel inhibitor discovery against key factor in WNT pathway is important for CRC treatment and prevention. Methods: Cell proliferation was detected after hydroxyphenyl butanone treatment in human colorectal cancer HCT116, LOVO, and normal colonic epithelial NCM460 cells. Colony formation, cell invasion ability, and cell cycle were detected with and without GSK-3ß knockdown. Results: Hydroxyphenyl butanone induces cycle arresting on G1-S phase of colorectal cancer cell line through GSK3ß in Wnt/ß-catenin pathway and inhibits malignant biological manifestations of cell proliferation, colony formation, and invasion. The inhibition in the high concentration group is stronger than that in the low concentration group, and the antitumor effect is different for different tumor cells. Under the same concentration of natural hydroxyphenyl butanone, the inhibition on normal colonic epithelial cells is significantly lower than that on tumor cells. The natural hydroxyphenyl butanone with medium and low concentration could promote the proliferation of normal colonic epithelial cells. Conclusion: This study illustrated natural hydroxyphenyl butanone as new inhibitor of GSK3ß and revealed the mechanisms underlying the inhibitory effects in colorectal cancer.


Assuntos
Butanonas/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias Colorretais/enzimologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Invasividade Neoplásica , Extratos Vegetais/farmacologia , Rubus/química , Fase S/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Via de Sinalização Wnt/efeitos dos fármacos
9.
J Org Chem ; 86(13): 8797-8804, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34142838

RESUMO

Herein, an interesting palladium-catalyzed procedure for the direct carbonylative thiomethylation of aromatic amine derivatives with 4-methylthio-2-butanone is developed. Using 4-methylthio-2-butanone as (methylthio) transfer agent, a variety of corresponding thioesters are obtained with moderate to good yields under base-free condition. In addition, good functional group tolerance can be observed.


Assuntos
Aminas , Paládio , Butanonas , Catálise , Sais
10.
Spectrochim Acta A Mol Biomol Spectrosc ; 261: 120032, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111836

RESUMO

Although Dietary supplements are readily accessible and extensively used worldwide, they are inadequately regulated and consumers are victims of manufacturers' fraud. Thus, quality regulations are required to ensure safety of products available to the public. We propose the first native spectrofluorimetric quality control assay of raspberry ketone, a popular dietary supplement ingredient for weight loss. This work relies on the constant wavelength synchronous scan of the Raspberry Ketone native fluorescence, overcoming the demerits of conventional excitation/ emission spectra. For the best measurement conditions, several parameters were optimized including Δλ value, diluting solvent, medium pH and the effect of surfactants/ macromolecules. In aqueous medium (Δλ = 110 nm), a linear relationship exists between synchronous fluorescence intensity at peak maximum 405.6 nm and solution concentration in the range 300-1500 ng/mL. Method sensitivity was recorded with LOD and LOQ values 60.63 and 183.72 ng/mL; respectively. Validation was done in accordance to International Conference on Harmonization (ICH) guidelines. This simple procedure was successfully applied to the analysis of Raspberry Ketone in commercially available dietary supplement capsules with average recovery 98.67% ± 1.74 and further extended to weight variation testing following the official United States Pharmacopeial (USP) guidelines. Finally, green assessment was done using the ''Analytical Eco-scale'' tool. The total score was 89/100 points revealing excellent greenness of our proposal. Our proposal is simple, eco-friendly and cheap. It can be conveniently adopted for routine quality control practices especially in developing countries.


Assuntos
Butanonas , Suplementos Nutricionais , Controle de Qualidade , Espectrometria de Fluorescência
11.
Plant Cell Rep ; 40(9): 1631-1646, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34146141

RESUMO

KEY MESSAGE: Studying RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes) in 5ß-cardenolide formation. Progesterone 5ß-reductases (P5ßR) are assumed to catalyze the reduction of progesterone to 5ß-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5ß-cardenolides. P5ßRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5ßRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5ßR1 (AY585867.1) and DlP5ßR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5ß-cardenolide formation, we here established several RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5ßR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5ßR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5ßR2-RNAi lines but not in Dl P5ßR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5ßR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).


Assuntos
Cardenolídeos/metabolismo , Digitalis/genética , Digitalis/metabolismo , Oxirredutases/genética , Proteínas de Plantas/genética , Butanonas/farmacologia , Butionina Sulfoximina/farmacologia , Digitalis/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Glutationa/farmacologia , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/genética , Plantas Geneticamente Modificadas , Progesterona/farmacologia , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Microb Cell Fact ; 20(1): 116, 2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112158

RESUMO

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 DH10ß, as a routine cloning host. The use of E. coli DH10ß 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.


Assuntos
Vias Biossintéticas , Butanonas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Poliésteres/metabolismo , Tirosina/metabolismo , Clonagem Molecular/métodos , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Microbiologia Industrial , Regiões Promotoras Genéticas , Biologia Sintética
13.
Appl Microbiol Biotechnol ; 105(10): 4189-4197, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33988735

RESUMO

Raspberry ketone is a widely used flavor compound in food and cosmetic industry. Several processes for its biocatalytic production have already been described, but either with the use of genetically modified organisms (GMOs) or incomplete conversion of the variety of precursors that are available in nature. Such natural precursors are rhododendrol glycosides with different proportions of (R)- and (S)-rhododendrol depending on the origin. After hydrolysis of these rhododendrol glycosides, the formed rhododendrol enantiomers have to be oxidized to obtain the final product raspberry ketone. To be able to achieve a high conversion with different starting material, we assembled an alcohol dehydrogenase toolbox that can be accessed depending on the optical purity of the intermediate rhododendrol. This is demonstrated by converting racemic rhododendrol using a combination of (R)- and (S)-selective alcohol dehydrogenases together with a universal cofactor recycling system. Furthermore, we conducted a biocatalytic cascade reaction starting from naturally derived rhododendrol glycosides by the use of a glucosidase and an alcohol dehydrogenase to produce raspberry ketone in high yield. KEY POINTS: • LB-ADH, LK-ADH and LS-ADH oxidize (R)-rhododendrol • RR-ADH and ADH1E oxidize (S)-rhododendrol • Raspberry ketone production via glucosidase and alcohol dehydrogenases from a toolbox.


Assuntos
Álcool Desidrogenase , Butanonas , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Biocatálise , Recursos Naturais
14.
Toxicol Lett ; 346: 34-46, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33872747

RESUMO

Tobacco exposure is well known to induce genetic and epigenetic changes that contribute to the pathogenesis of lung cancer. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a significant tobacco-specific carcinogen, but the oncogenic mechanisms of NNK have not been thoroughly elucidated. In this study we found that DNA methyltransferase 1 (DNMT1) was overexpressed in malignantly transformed human bronchial epithelial Beas-2B cells induced by NNK (2B-NNK cells), by treatment with NNK (400 µg/mL) for 7 days. An Arraystar Human noncoding RNA Promoter Microarray was used to detect the DNA methylation status of the promoter region of long noncoding RNAs (lncRNAs). The result showed that 1010 differentially methylated fragments were present in the lncRNA promoter region. QRT-PCR revealed that the expression of lncRNA AC007255.8 was remarkably downregulated in 2B-NNK cells and lung cancer tissues. Furthermore, Methylation-specific PCR showed that the methylation of the lncRNA AC007255.8 promoter was increased in 2B-NNK cells and lung cancer tissues. The reduced expression of lncRNA AC007255.8 was significantly associated with hypermethylation of lncRNA AC007255.8 promoter region. LncRNA AC007255.8 overexpression could result in decreased cell proliferation and increased cell apoptosis in 2B-NNK cells. In conclusion, NNK induced lncRNA AC007255.8 promoter hypermethylation via upregulation of DNMT1 in Beas-2B cells, leading to downregulation of lncRNA AC007255.8, and ultimately the enhancement of cell proliferation and the inhibition of apoptosis. This research affords novel insights into the epigenetic mechanisms of lung cancer, and will stimulate further research into the involvement of aberrant DNA methylation of non-coding regions of the genome in the pathogenesis of lung cancer.


Assuntos
Butanonas/toxicidade , DNA/metabolismo , Nitrosaminas/toxicidade , RNA Longo não Codificante/metabolismo , Brônquios/citologia , Linhagem Celular , Transformação Celular Neoplásica , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Células Epiteliais , Regulação da Expressão Gênica/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Pulmonares/metabolismo , Metilação , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Mucosa Respiratória/citologia , Regulação para Cima
15.
Microb Cell Fact ; 20(1): 68, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33706766

RESUMO

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.


Assuntos
Butanonas/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Engenharia Metabólica , Vias Biossintéticas , Escherichia coli/genética , Fermentação , Glicerol/metabolismo , Regiões Promotoras Genéticas , Óleo de Soja/metabolismo
16.
J Agric Food Chem ; 69(8): 2549-2556, 2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33593064

RESUMO

Raspberry ketone (RK), the main aroma compound of raspberry fruit, has applications in cosmetics, food industry, and pharmaceutics. In this study, we biosynthesized RK via the catalytic reduction of 4-hydroxybenzylidenacetone using a whole-cell biocatalyst. Reductase RiRZS1 from Rubus idaeus and glucose dehydrogenase SyGDH from Thermoplasma acidophilum were expressed in Escherichia coli to regenerate NADPH for the whole-cell catalytic reaction. Following the optimization of balancing the coexpression of two enzymes in pRSFDuet-1, we obtained 9.89 g/L RK with a conversion rate of 98% and a space-time yield of 4.94 g/(L·h). The optimum conditions are 40 °C, pH 5.5, and a molar ratio of substrate to auxiliary substrate of 1:2.5. Our study findings provide a promising method of biosynthesizing RK.


Assuntos
Escherichia coli , Glucose 1-Desidrogenase , Butanonas , Escherichia coli/genética , Guaiacol/análogos & derivados
17.
Int J Biol Macromol ; 171: 89-99, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33412202

RESUMO

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, ß-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.


Assuntos
Apoproteínas/química , Butanonas/química , Malus/química , NADP/química , Oxirredutases/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Arabidopsis/química , Arabidopsis/enzimologia , Sítios de Ligação , Butanonas/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Malus/enzimologia , Modelos Moleculares , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rubus/química , Rubus/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , Tabaco/química , Tabaco/enzimologia
18.
Cancer Res ; 81(1): 144-157, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33122306

RESUMO

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease that is associated with increased risk of lung cancer. Pseudomonas aeruginosa (PA) infections are frequent in patients with COPD, which increase lung inflammation and acute exacerbations. However, the influences of PA-induced inflammation on lung tumorigenesis and the efficacy of immune checkpoint blockade remain unknown. In this study, we initiated a murine model of lung cancer by treating FVB/NJ female mice with tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alone or in combination with PA-lipopolysaccharide (LPS). LPS-mediated chronic inflammation induced T-cell exhaustion, increased the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis, and enhanced NNK-induced lung tumorigenesis through an immunosuppressive microenvironment characterized by accumulation of myeloid-derived suppressive cells (MDSC) and regulatory T cells. Anti-PD-1 antibody treatment reduced tumors in NNK/LPS-treated mice with a 10-week LPS treatment but failed to inhibit tumor growth when LPS exposure was prolonged to 16 weeks. Anti-Ly6G antibody treatment coupled with depletion of MDSC alone reduced tumor growth; when combined with anti-PD-1 antibody, this treatment further enhanced antitumor activity in 16-week NNK/LPS-treated mice. Immune gene signatures from a human lung cancer dataset of PD-1 blockade were identified, which predicted treatment responses and survival outcome and overlapped with those from the mouse model. This study demonstrated that LPS-mediated chronic inflammation creates a favorable immunosuppressive microenvironment for tumor progression and correlates with the efficacy of anti-PD-1 treatment in mice. Immune gene signatures overlap with human and mouse lung tumors, providing potentially predictive markers for patients undergoing immunotherapy. SIGNIFICANCE: This study identifies an immune gene signature that predicts treatment responses and survival in patients with tobacco carcinogen-induced lung cancer receiving immune checkpoint blockade therapy.


Assuntos
Butanonas/toxicidade , Carcinógenos/toxicidade , Inibidores de Checkpoint Imunológico/farmacologia , Inflamação/complicações , Lipopolissacarídeos/toxicidade , Neoplasias Pulmonares/patologia , Nitrosaminas/toxicidade , Tabaco/toxicidade , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/induzido quimicamente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Camundongos , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Taxa de Sobrevida , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
19.
Mar Drugs ; 19(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374505

RESUMO

Age-related macular degeneration (AMD) is a progressive eye disease that causes irreversible impairment of central vision, and effective treatment is not yet available. Extracellular accumulation of amyloid-beta (Aß) in drusen that lie under the retinal pigment epithelium (RPE) has been reported as one of the early signs of AMD and was found in more than 60% of Alzheimer's disease (AD) patients. Extracellular deposition of Aß can induce the expression of inflammatory cytokines such as IL-1ß, TNF-α, COX-2, and iNOS in RPE cells. Thus, finding a compound that can effectively reduce the inflammatory response may help the treatment of AMD. In this research, we investigated the anti-inflammatory effect of the coral-derived compound 4-(phenylsulfanyl) butan-2-one (4-PSB-2) on Aß1-42 oligomer (oAß1-42) added to the human adult retinal pigment epithelial cell line (ARPE-19). Our results demonstrated that 4-PSB-2 can decrease the elevated expressions of TNF-α, COX-2, and iNOS via NF-κB signaling in ARPE-19 cells treated with oAß1-42 without causing any cytotoxicity or notable side effects. This study suggests that 4-PSB-2 is a promising drug candidate for attenuation of AMD.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Anti-Inflamatórios/farmacologia , Butanonas/farmacologia , Mediadores da Inflamação/metabolismo , Degeneração Macular/tratamento farmacológico , Fragmentos de Peptídeos/toxicidade , Epitélio Pigmentado da Retina/efeitos dos fármacos , Sulfetos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Humanos , Interleucina-1beta/metabolismo , Degeneração Macular/metabolismo , Degeneração Macular/patologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Fator de Necrose Tumoral alfa/metabolismo
20.
Molecules ; 25(21)2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33153022

RESUMO

The influence of distant London dispersion forces on the docking preference of alcohols of different size between the two lone electron pairs of the carbonyl group in pinacolone was explored by infrared spectroscopy of the OH stretching fundamental in supersonic jet expansions of 1:1 solvate complexes. Experimentally, no pronounced tendency of the alcohol to switch from the methyl to the bulkier tert-butyl side with increasing size was found. In all cases, methyl docking dominates by at least a factor of two, whereas DFT-optimized structures suggest a very close balance for the larger alcohols, once corrected by CCSD(T) relative electronic energies. Together with inconsistencies when switching from a C4 to a C5 alcohol, this points at deficiencies of the investigated B3LYP and in particular TPSS functionals even after dispersion correction, which cannot be blamed on zero point energy effects. The search for density functionals which describe the harmonic frequency shift, the structural change and the energy difference between the docking isomers of larger alcohols to unsymmetric ketones in a satisfactory way is open.


Assuntos
Butanonas/química , Etanol/química , Modelos Químicos , Espectrofotometria Infravermelho
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