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Applied Microbiology and Biotechnology Apr 2023Biobased polymers derived from plant oils are sustainable alternatives to petro based polymers. In recent years, multienzyme cascades have been developed for the...
Biobased polymers derived from plant oils are sustainable alternatives to petro based polymers. In recent years, multienzyme cascades have been developed for the synthesis of biobased ω-aminocarboxylic acids, which serve as building blocks for polyamides. In this work, we have developed a novel enzyme cascade for the synthesis of 12-aminododeceneoic acid, a precursor for nylon-12, starting from linoleic acid. Seven bacterial ω-transaminases (ω-TAs) were cloned, expressed in Escherichia coli and successfully purified by affinity chromatography. Activity towards the oxylipin pathway intermediates hexanal and 12-oxododecenoic acid in their 9(Z) and 10(E) isoforms was demonstrated for all seven transaminases in a coupled photometric enzyme assay. The highest specific activities were obtained with ω-TA from Aquitalea denitrificans (TR), with 0.62 U mg for 12-oxo-9(Z)-dodecenoic acid, 0.52 U mg for 12-oxo-10(E)-dodecenoic acid and 1.17 U mg for hexanal. A one-pot enzyme cascade was established with TR and papaya hydroperoxide lyase (HPL), reaching conversions of 59% according to LC-ELSD quantification. Starting from linoleic acid, up to 12% conversion to 12-aminododecenoic acid was achieved with a 3-enzyme cascade comprising soybean lipoxygenase (LOX-1), HPL and TR. Higher product concentrations were achieved by the consecutive addition of enzymes compared to simultaneous addition at the beginning. KEY POINTS: • Seven ω-transaminases converted 12-oxododecenoic acid into its corresponding amine. • A three-enzyme cascade with lipoxygenase, hydroperoxide lyase, and ω-transaminase was established for the first time. • A one-pot transformation of linoleic acid to 12-aminododecenoic acid, a precursor of nylon-12 was achieved.
Topics: Transaminases; Oxylipins; Linoleic Acid; Lipoxygenase; Polymers
PubMed: 36807735
DOI: 10.1007/s00253-023-12422-6 -
Applied Biochemistry and Biotechnology Dec 2022Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid,...
Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid, which is not yet used industrially. Here, HPL from Carica papaya (HPL) was cloned and functionally expressed in Escherichia coli to investigate synthesis of 12-oxo-9(Z)-dodecenoic acid in detail. To improve the low catalytic activity of full-length HPL, the hydrophobic, non-conserved N-terminal sequence was deleted. This enhanced enzyme activity from initial 10 to 40 U/l. With optimization of solubilization buffer, expression media enzyme activity was increased to 2700 U/l. The tetrameric enzyme was produced in a 1.5 l fermenter and enriched by affinity chromatography. The enzyme preparation possesses a slightly acidic pH optimum and a catalytic efficiency (k/K) of 2.73 × 10 s·M towards 13S-HPODE. Interestingly, HPL could be applied for the synthesis of 12-oxo-9(Z)-dodecenoic acid, and 1 mM of 13S-HPODE was transformed in just 10 s with a yield of 90%. At protein concentrations of 10 mg/ml, the slow formation of the 10(E)-isomer traumatin was observed, pointing to a non-enzymatic isomerization process. Bearing this in mind, a one-pot enzyme cascade starting from safflower oil was developed with consecutive addition of Pseudomonas fluorescens lipase, Glycine max lipoxygenase (LOX-1), and HPL. A yield of 43% was obtained upon fast extraction of the reaction mixtures after 1 min of HPL reaction. This work provides first insights into an enzyme cascade synthesis of 12-oxo-9(Z)-dodecenoic acid, which may serve as a bifunctional precursor for bio-based polymer synthesis.
Topics: Carica; Polymers; Aldehyde-Lyases; Cytochrome P-450 Enzyme System
PubMed: 35904676
DOI: 10.1007/s12010-022-04095-0 -
Microbiology Spectrum Aug 2022Burkholderia cenocepacia is a human opportunistic pathogen that mostly employs two types of quorum-sensing (QS) systems to regulate its various biological functions and...
Burkholderia cenocepacia is a human opportunistic pathogen that mostly employs two types of quorum-sensing (QS) systems to regulate its various biological functions and pathogenicity: the -2-dodecenoic acid (BDSF) system and the -acyl homoserine lactone (AHL) system. In this study, we reported that oridonin, which was screened from a collection of natural products, disrupted important B. cenocepacia phenotypes, including motility, biofilm formation, protease production, and virulence. Genetic and biochemical analyses showed that oridonin inhibited the production of BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Furthermore, we revealed that oridonin directly binds to the regulator RqpR of the two-component system RqpSR that dominates the above-mentioned QS systems to inhibit the expression of the BDSF and AHL signal synthase-encoding genes. Oridonin also binds to the transcriptional regulator CepR of the AHL system to inhibit its binding to the promoter of . These findings suggest that oridonin could potentially be developed as a new QS inhibitor against pathogenic B. cenocepacia. Burkholderia cenocepacia is an important human opportunistic pathogen that can cause life-threatening infections in susceptible individuals. It employs quorum-sensing (QS) systems to regulate biological functions and virulence. In this study, we have identified a lead compound, oridonin, that is capable of interfering with B. cenocepacia QS signaling and physiology. We demonstrate that oridonin suppressed -2-dodecenoic acid (BDSF) and -acyl homoserine lactone (AHL) signal production and attenuated virulence in B. cenocepacia. Oridonin also impaired QS-regulated phenotypes in various Burkholderia species. These results suggest that oridonin could interfere with QS signaling in many Burkholderia species and might be developed as a new antibacterial agent.
Topics: Acyl-Butyrolactones; Bacterial Proteins; Burkholderia cenocepacia; Diterpenes, Kaurane; Gene Expression Regulation, Bacterial; Humans; Quorum Sensing; Virulence
PubMed: 35856676
DOI: 10.1128/spectrum.01787-22 -
PloS One 2020In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell...
In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell lines (MCF-7, SK-BR-3, and YMB-1) using a solid-phase Porapak Q extraction revealed that two compounds of moderate volatility, 1-hexadecanol and 5-(Z)-dodecenoic acid, were detected with markedly higher amount than those in the medium of fibroblast cell line (KMST-6). Furthermore, LC-TOF/MS analysis of the extracts clarified that in addition to the above two fatty acids, the amounts of five unsaturated fatty acids [decenoic acid (C10:1), decadienoic acid (C10:2), 5-(Z)-dodecenoic acid (C12:1), 5-(Z)-tetradecenoic acid (C14:1), and tetradecadienoic acid (C14:2)] in MCF-7 medium were higher than those in medium of KMST-6. Interestingly, H2O2-oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid produced volatile aldehydes that were reported as specific volatiles in breath from various cancer patients, such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal. Thus, we concluded that these identified compounds over-produced in breast cancer cells in this study could serve as potential precursors producing reported cancer-specific volatiles.
Topics: Breast Neoplasms; Fatty Acids; Female; Gas Chromatography-Mass Spectrometry; Humans; Oxidation-Reduction; Solid Phase Microextraction; Tumor Cells, Cultured; Volatile Organic Compounds
PubMed: 32598404
DOI: 10.1371/journal.pone.0235442 -
PLoS Pathogens Jul 2015Many pathogenic bacteria use cell-cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell... (Review)
Review
Many pathogenic bacteria use cell-cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell density or confinement to niches. Bacteria produce signals of diverse structural classes. Signal molecules of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids. The paradigm is cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris pv. campestris (Xcc), which controls virulence in this plant pathogen. Although DSF synthesis was thought to be restricted to the xanthomonads, it is now known that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia and Pseudomonas aeruginosa. Furthermore, signaling involving these DSF family members contributes to bacterial virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Here we review the recent advances in understanding DSF signaling and its regulatory role in different bacteria. These advances include the description of the pathway/mechanism of DSF biosynthesis, identification of novel DSF synthases and new members of the DSF family, the demonstration of a diversity of DSF sensors to include proteins with a Per-Arnt-Sim (PAS) domain and the description of some of the signal transduction mechanisms that impinge on virulence factor expression. In addition, we address the role of DSF family signals in interspecies signaling that modulates the behavior of other microorganisms. Finally, we consider a number of recently reported approaches for the control of bacterial virulence through the modulation of DSF signaling.
Topics: Animals; Bacterial Proteins; Cell Communication; Gene Expression Regulation, Bacterial; Humans; Signal Transduction; Virulence; Xanthomonas campestris
PubMed: 26181439
DOI: 10.1371/journal.ppat.1004986 -
European Journal of Biochemistry Jan 1999Chemical communication in scarab beetles (Coleoptera: Scarabaeidae) is achieved with a wide variety of pheromones, but one typical structure is the gamma-lactone having...
Chemical communication in scarab beetles (Coleoptera: Scarabaeidae) is achieved with a wide variety of pheromones, but one typical structure is the gamma-lactone having a long unsaturated hydrocarbon chain. Several species utilize (R, Z)-5-(-)-(oct-1-enyl)-oxacyclopentan-2-one (buibuilactone), (R, Z)-5-(-)-(dec-1-enyl)-oxacyclopentan-2-one and (S, Z)-5-(+)-(dec-1-enyl)-oxacyclopentan-2-one [(R)-japonilure and (S)-japonilure]. Using deuterated precursors, we have demonstrated that these compounds are biosynthesized from fatty acids. (9, 10-d4)-Palmitic acid, (9,10-d4)-stearic acid, (9,10-d2)-palmitoleic acid, (9,10-d2)-oleic acid, (9,10-d2)-8-hydroxypalmitoleic acid and (9,10-d2)-8-hydroxyoleic acid were readily incorporated by female Anomala cuprea into the pheromone molecules, while (Z)-(5, 6-d2)-5-dodecenoic acid and (Z)-(5,6-d2)-5-tetradecenoic acid were not. Therefore, the reaction pathway starts from saturated fatty acids, involves their desaturation, followed by 8-hydroxylation, chain shortening and cyclization. The products obtained from racemic (9,10-d2)-8-hydroxypalmitoleic acid and (9,10-d2)-8-hydroxyoleic acid were also racemic, implying that the steps following hydroxylation were not stereospecific. Perdeuterated palmitic acid was applied to disclose the mechanism of the unique hydroxylation reaction. Retention of all deuterium atoms implied that this reaction was a direct process mediated by a specific fatty acid hydroxylase, and preceding desaturation or epoxidation was not involved.
Topics: 4-Butyrolactone; Animals; Coleoptera; Fatty Acids; Female; Hydroxylation; Lactones; Male; Mass Spectrometry; Models, Chemical; Pheromones; Sex Attractants; Stereoisomerism
PubMed: 9914490
DOI: 10.1046/j.1432-1327.1999.00018.x -
Biomedical and Environmental Sciences :... Mar 2019To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid (BDSF) and fluconazole (FLU) or itraconazole (ITRA) against two azole-resistant C....
OBJECTIVE
To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid (BDSF) and fluconazole (FLU) or itraconazole (ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo.
METHODS
Minimum inhibitory concentrations (MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction (qRT-PCR).
RESULTS
BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ⪕ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 μg/mL. The qRT-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mRNA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis.
CONCLUSION
The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.
Topics: Antifungal Agents; Burkholderia cenocepacia; Candida albicans; Candidiasis; Drug Resistance, Fungal; Fatty Acids, Monounsaturated; Fluconazole; Humans; Microbial Sensitivity Tests; Triazoles
PubMed: 30987694
DOI: 10.3967/bes2019.027 -
Applied and Environmental Microbiology Oct 2023Outer membrane vesicle (OMV)-delivered quinolone signal (PQS) plays a critical role in cell-cell communication in . However, the functions and mechanisms of...
Outer membrane vesicle (OMV)-delivered quinolone signal (PQS) plays a critical role in cell-cell communication in . However, the functions and mechanisms of membrane-enclosed PQS in interspecies communication in microbial communities are not clear. Here, we demonstrate that PQS delivered by both OMVs from and liposome reduces the competitiveness of , which usually shares the same niche in the lungs of cystic fibrosis patients, by interfering with quorum sensing (QS) in through the LysR-type regulator ShvR. Intriguingly, we found that ShvR regulates the production of the QS signals cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) by directly binding to the promoters of signal synthase-encoding genes. Perception of PQS influences the regulatory activity of ShvR and thus ultimately reduces QS signal production and virulence in . Our findings provide insights into the interspecies communication mediated by the membrane-enclosed QS signal among bacterial species residing in the same microbial community.IMPORTANCEQuorum sensing (QS) is a ubiquitous cell-to-cell communication mechanism. Previous studies showed that mainly employs cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) QS systems to regulate biological functions and virulence. Here, we demonstrate that quinolone signal (PQS) delivered by outer membrane vesicles from or liposome attenuates virulence by targeting the LysR-type regulator ShvR, which regulates the production of the QS signals BDSF and AHL in a. Our results not only suggest the important roles of membrane-enclosed PQS in interspecies and interkingdom communications but also provide a new perspective on the use of functional nanocarriers loaded with QS inhibitors for treating pathogen infections.
Topics: Humans; Quorum Sensing; Virulence; Acyl-Butyrolactones; Liposomes; Bacterial Proteins; Burkholderia cenocepacia; Pseudomonas aeruginosa; Gene Expression Regulation, Bacterial
PubMed: 37796010
DOI: 10.1128/aem.01184-23 -
Journal of Chemical Ecology Dec 2021Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and...
Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional ∆9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone.
Topics: Animals; Brassicaceae; Dodecanol; Metabolic Engineering; Moths; Sex Attractants
PubMed: 34762210
DOI: 10.1007/s10886-021-01316-4 -
Plant Physiology Mar 197912-Oxo-trans-10-dodecenoic acid (trans-10-ODA) is an oxidation product of polyunsaturated fatty acids in plant tissues. The structural similarity of trans-10-ODA and...
12-Oxo-trans-10-dodecenoic acid (trans-10-ODA) is an oxidation product of polyunsaturated fatty acids in plant tissues. The structural similarity of trans-10-ODA and traumatic acid, a compound considered to be a wound hormone, suggested that trans-10-ODA might be a precursor of traumatic acid. Both trans-10-ODA and traumatic acid were active in the Wehnelt bean assay. The results were more consistent with trans-10-ODA than with traumatic acid. Cucumber (Cucumis sativus L. var. National Pickling) hypocotyls also showed a growth increase following treatment with trans-10-ODA, which suggested that trans-10-ODA has a more general influence on plant development than previously ascribed to traumatic acid.Runner beans (Phaseolus vulgaris L. var. Kentucky Wonder) were analyzed for the presence of endogenous trans-10-ODA and traumatic acid. These are the beans from which traumatic acid was originally isolated in 1939. They contained trans-10-ODA but no traumatic acid. Young beans were a better source of trans-10-ODA than older beans and an increase in the esterified form of trans-10-ODA with age may have been due to a conversion of the free acid to the esterified form. The amount of endogenous trans-10-ODA increased when bean pod tissue was sliced and wounded. Rapid stirring and the presence of oxygen increased autooxidation of trans-10-ODA to traumatic acid in runner beans, which indicated that the compound identified as traumatic acid is formed by autooxidation of trans-10-ODA and that trans-10-ODA is a natural compound with growth-regulating properties.Enzyme extracts of runner beans synthesized trans-10-ODA from linoleic acid. No enzymic synthesis of traumatic acid was observed even when cofactors were added to the reaction mixture. This confirmed the conclusion that traumatic acid is formed by autooxidation of trans-10-ODA.
PubMed: 16660762
DOI: 10.1104/pp.63.3.536