RESUMEN
Prenylflavonoids are promising candidates for food additives and functional foods due to their diverse biological activities and potential health benefits. However, natural prenylflavonoids are generally present in low abundance and are limited to specific plant species. Here, we report the biosynthesis of licoflavanone from naringenin and prenol by recombinant Escherichia coli. By investigating the activities of seven different sources of prenyltransferases overexpressed in E. coli toward various flavonoid substrates, the prenyltransferase AnaPT exhibits substrate preference when naringenin serves as the prenyl acceptor. Furthermore, licoflavanone production was successfully achieved by coupling the isopentenol utilization pathway and AnaPT in recombinant E. coli. In addition, the effects of fermentation temperatures, induction temperatures, naringenin concentrations, and substrate feeding strategies were investigated on the biosynthesis of licoflavanone in recombinant E. coli. Consequently, the recombinant E. coli strain capable of improved dimethylallyl diphosphate (DMAPP) supply and suitable for prenylflavonoid biosynthesis increased licoflavanone titers to 142.1 mg/L in a shake flask and to 537.8 mg/L in a 1.3 L fermentor, which is the highest yield for any prenylflavonoids reported to date. These strategies proposed in this study provide a reference for initiating the production of high-value prenylflavonoids.
Asunto(s)
Dimetilaliltranstransferasa , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Dimetilaliltranstransferasa/metabolismo , Dimetilaliltranstransferasa/genética , Pentanoles/metabolismo , Ingeniería Metabólica , Flavonoides/metabolismo , Flavonoides/biosíntesis , Hemiterpenos/metabolismo , FermentaciónRESUMEN
Isoamyl fatty acid esters (IAFEs) are widely used as fruity flavor compounds in the food industry. In this study, various IAFEs were synthesized from isoamyl alcohol and various fatty acids using a cutinase enzyme (Rcut) derived from Rhodococcus bacteria. Rcut was immobilized on methacrylate divinylbenzene beads and used to synthesize isoamyl acetate, butyrate, hexanoate, octanoate, and decanoate. Among them, Rcut synthesized isoamyl butyrate (IAB) most efficiently. Docking model studies showed that butyric acid was the most suitable substrate in terms of binding energy and distance from the active site serine (Ser114) γ-oxygen. Up to 250 mM of IAB was synthesized by adjusting reaction conditions such as substrate concentration, reaction temperature, and reaction time. When the enzyme reaction was performed by reusing the immobilized enzyme, the enzyme activity was maintained at least six times. These results demonstrate that the immobilized Rcut enzyme can be used in the food industry to synthesize a variety of fruity flavor compounds, including IAB.
Asunto(s)
Hidrolasas de Éster Carboxílico , Enzimas Inmovilizadas , Aromatizantes , Simulación del Acoplamiento Molecular , Rhodococcus , Enzimas Inmovilizadas/metabolismo , Enzimas Inmovilizadas/química , Rhodococcus/enzimología , Rhodococcus/metabolismo , Aromatizantes/metabolismo , Aromatizantes/química , Hidrolasas de Éster Carboxílico/metabolismo , Hidrolasas de Éster Carboxílico/química , Ésteres/metabolismo , Ésteres/química , Pentanoles/metabolismo , Pentanoles/química , Ácidos Grasos/metabolismo , Ácidos Grasos/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Temperatura , Especificidad por Sustrato , Ácido Butírico/metabolismo , Ácido Butírico/química , Dominio CatalíticoRESUMEN
Bacterial isoprenoids are necessary for many biological processes, including maintaining membrane integrity, facilitating intercellular communication, and preventing oxidative damage. All bacterial isoprenoids are biosynthesized from two five carbon structural isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are cell impermeant. Herein, we demonstrate exogenous delivery of IPP and DMAPP into Bacillus subtilis by utilizing a self-immolative ester (SIE)-caging approach. We initially evaluated native B. subtilis esterase activity, which revealed a preference for short straight chain esters. We then examined the viability of the SIE-caging approach in B. subtilis and demonstrate that the released caging groups are well tolerated and the released IPP and DMAPP are bioavailable, such that isoprenoid biosynthesis can be rescued in the presence of pathway inhibitors. We further show that IPP and DMAPP are both toxic and inhibit growth of B. subtilis at the same concentration. Lastly, we establish the optimal ratio of IPP to DMAPP (5 : 1) for B. subtilis growth and find that, surprisingly, DMAPP alone is insufficient to rescue isoprenoid biosynthesis under high concentrations of fosmidomycin. These findings showcase the potential of the SIE-caging approach in B. subtilis and promise to both aid in novel isoprenoid discovery and to inform metabolic engineering efforts in bacteria.
Asunto(s)
Bacillus subtilis , Hemiterpenos , Compuestos Organofosforados , Terpenos , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Hemiterpenos/metabolismo , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Terpenos/metabolismo , Terpenos/química , Pentanoles/metabolismo , Pentanoles/químicaRESUMEN
2-Methyl-1-butanol (2MB) and 3-Methyl-1-butanol (3MB) are microbial volatile organic compounds (VOCs) and found in indoor air. Here, we applied rice as a bioindicator to investigate the effects of these indoor microbial volatile pollutants. A remarkable decrease in germination percentage, shoot and root elongation, as well as lateral root numbers were observed in 3MB. Furthermore, ROS production increased by 2MB and 3MB, suggesting that pentanol isomers could induce cytotoxicity in rice seedlings. The enhancement of peroxidase (POD) and catalase (CAT) activity provided evidence that pentanol isomers activated the enzymatic antioxidant scavenging systems, with a more significant effect observed in 3MB. Furthermore, 3MB induced higher activity levels of glutathione (GSH), oxidized glutathione (GSSG), and the GSH/GSSG ratio in rice compared to the levels induced by 2MB. Additionally, qRT-PCR analysis showed more up-regulation in the expression of glutaredoxins (GRXs), peroxiredoxins (PRXs), thioredoxins (TRXs), and glutathione S-transferases (GSTUs) genes in 3MB. Taking the impacts of pentanol isomers together, the present study suggests that 3MB exhibits more cytotoxic than 2MB, as such has critical effects on germination and the early seedling stage of rice. Our results provide molecular insights into how isomeric indoor microbial volatile pollutants affect plant growth through airborne signals.
Asunto(s)
Contaminantes Ambientales , Oryza , Antioxidantes/metabolismo , Plantones , Oryza/metabolismo , Pentanoles/metabolismo , Pentanoles/farmacología , 1-Butanol/metabolismo , 1-Butanol/farmacología , Contaminantes Ambientales/metabolismo , Disulfuro de Glutatión/metabolismo , Estrés Oxidativo , Glutatión/metabolismo , Raíces de Plantas/metabolismoRESUMEN
The isopentenol utilization pathway (IUP) is potential in terpenoids synthesis. This study aimed to construct IUP-employed Escherichia coli chassis for stably synthesizing terpenoids. As to effectiveness, promotor engineering strategy was employed to regulate IUP expression level, while ribosome-binding site (RBS) library of the key enzyme was constructed for screening the optimal RBS, followed by optimization of concentration of inducer and substrates, the titer of reporting production, lycopene, from 0.087 to 8.67 mg OD600 -1 . As about stability, the IUP expression cassette was integrated into the genome through transposition tool based on CRISPR-associated transposases. Results showed that the strain with 13 copies produced 1.78-fold lycopene titer that of the controlled strain with IUP-harbored plasmid, and it exhibited stable expression after ten successions while the plasmid loss was observed in the controlled strain in the 3rd succession. This strategy provides valuable information for rapid construction of highly effective and stable chassis employing IUP for terpenoids production.
Asunto(s)
Escherichia coli , Terpenos , Escherichia coli/genética , Escherichia coli/metabolismo , Terpenos/metabolismo , Licopeno/metabolismo , Pentanoles/metabolismo , Ingeniería MetabólicaRESUMEN
Alnus cremastogyne Burkill (Betulaceae), an actinorhizal plant, can enter a mutualistic symbiosis with Frankia species that leads to the formation of nitrogen fixing root nodules. Some primary metabolites (carbohydrates, dicarboxylic acids, amino acids, citrulline and amides) involved in carbon and nitrogen metabolism in actinorhizal nodules have been identified, while specialized metabolites in A. cremastogyne root nodules are yet to be characterized. In this study, we isolated and identified three undescribed 3-pentanol glycosides, i.e., 3-pentyl α-l-arabinofuranosyl-(1''â6')-ß-d-glucopyranoside, 3-pentyl α-l-rhamnopyranosyl-(1''â6')-ß-d-glucopyranoside, and 3-pentyl 6'-(3-hydroxy3-methylglutaryl)-ß-d-glucopyranoside, as well as seventeen known compounds from A. cremastogyne root nodules. 3-Pentanol glycosides are abundantly distributed in root nodules, while they are distributed in stems, roots, leaves and fruits at low/zero levels. A. cremastogyne plants treated by root nodule suspension emit 3-pentanol. This study enriches the knowledge about specialized metabolites in the actinorhizal host, and provides preliminarily information on the signal exchange in the actinorhizal symbiosis between A. cremastogyne and Frankia.
Asunto(s)
Alnus , Frankia , Pentanoles/metabolismo , Glicósidos/metabolismo , Raíces de Plantas , Frankia/metabolismo , Simbiosis , Plantas , Nitrógeno/metabolismo , Fijación del Nitrógeno , Nódulos de las Raíces de las PlantasRESUMEN
BACKGROUND: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). RESULTS: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. CONCLUSION: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.
Asunto(s)
Butanoles/metabolismo , Ingeniería Metabólica , Pentanoles/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Luz , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Fotosíntesis , Regiones Promotoras Genéticas , Synechocystis/crecimiento & desarrolloRESUMEN
Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Técnicas Biosensibles , Butanoles/metabolismo , Pentanoles/metabolismo , Saccharomyces cerevisiae/metabolismo , 2-Isopropilmalato Sintasa/genética , 2-Isopropilmalato Sintasa/metabolismo , Vías Biosintéticas , Etanol/metabolismo , Ensayos Analíticos de Alto Rendimiento , Leucina/metabolismo , Ingeniería Metabólica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biología SintéticaRESUMEN
Coumarins are a group of natural compounds commonly found in the families of Rutaceae and Umbelliferae. 7-Isopentenyloxycoumarin (ISC), auraptene (AUR), and umbelliprenin (UM) belong to prenyloxycoumarins (PYCs), which link isopentenyl, geranyl, and farnesyl group at C7 position, respectively. The substituent of 7-ethoxycoumarin (ETC) is the ethyl group. In this study, UPLC-ESI-QTOF-MS (ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-MS)-based metabolomics was used to evaluate the in vivo and in vitro metabolism of PYCs. Results showed that ETC produced 10 known metabolites, and ISC was transformed into 17 metabolites in vivo and in vitro, which were undescribed compounds. A total of 35 AUR metabolites, including 34 undescribed metabolites were identified, and 21 metabolites were reported for the first time in UM. The results indicated that hydroxylation and N-acetylcysteine conjugation were the common metabolic reactions for PYCs. The metabolic rates of ETC, ISC, AUR and UM were 26%, 36%, 81%, and 38%, respectively, in human liver microsome, while they were 24%, 40%, 80%, and 37%, respectively, in mouse liver microsomes. In addition, recombinant cytochrome P450s (CYPs) screening showed that CYP1A1, 2C19, 3A4, and 3A5 were the major metabolic enzymes involved in the formation of hydroxylation metabolites. Together, these results suggest that the isopentenyl group plays an important role in the metabolism of PYCs.
Asunto(s)
Cumarinas , Metabolómica/métodos , Pentanoles , Animales , Cromatografía Líquida de Alta Presión , Cumarinas/análisis , Cumarinas/química , Cumarinas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Pentanoles/análisis , Pentanoles/química , Pentanoles/metabolismo , Espectrometría de Masas en TándemRESUMEN
Microbial production of α-farnesene from renewable raw materials is a feasible alternative to traditional petroleum craft. Recently, the research on improving α-farnesene production in Pichia pastoris mainly focused on cytoplasmic engineering, while comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we first sought to confirm that the isopentenol utilization pathway (IUP) could act as a two-step shortcut for IPP synthesis in P. pastoris peroxisomes. In addition, we proposed dual regulation of cytoplasm and peroxisomes to boost α-farnesene synthesis in P. pastoris X33, thus the resultant strain produced 2.18 ± 0.04 g/L, which was 1.3 times and 2.1 times than that of the strain only with peroxisomal or cytoplasmic engineering, respectively. The α-farnesene production achieved 2.56 ± 0.04 g/L in shake flasks after carbon source cofeeding, which was the highest reported production in worldwide literatures to the best of my knowledge. Therefore, we propose these strategies as efficient approaches to enhancing α-farnesene production in P. pastoris, which might bring new ideas for the biosynthesis of high-value compounds.
Asunto(s)
Ingeniería Metabólica/métodos , Peroxisomas/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Sesquiterpenos/metabolismo , Medios de Cultivo/química , Citosol/metabolismo , Hemiterpenos/biosíntesis , Ácidos Oléicos/metabolismo , Compuestos Organofosforados , Pentanoles/metabolismo , Transducción de Señal/genéticaRESUMEN
As the effects of climate change become increasingly severe, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. The design and optimization of microorganisms to produce gasoline, diesel, and jet fuel compounds from renewable feedstocks can significantly reduce dependence on fossil fuels and thereby produce fewer emissions. Over the past two decades, a tremendous amount of research has contributed to the development of microbial strains to produce advanced fuel compounds, including branched-chain higher alcohols (BCHAs) such as isopentanol (3-methyl-1-butanol; 3M1B) and isobutanol (2-methyl-1-propanol). In this review, we provide an overview of recent advances in the development of microbial strains for the production of isopentanol in both conventional and non-conventional hosts. We also highlight metabolic engineering strategies that may be employed to enhance product titers, reduce end-product toxicity, and broaden the substrate range to non-sugar carbon sources. Finally, we offer glimpses into some promising future directions in the development of isopentanol producing microbial strains.
Asunto(s)
Biocombustibles/microbiología , Pentanoles/metabolismo , Ingeniería Metabólica , Energía Renovable , Biología SintéticaRESUMEN
In previous research, we modeled the ethanol production by certain bacteria under controlled experimental conditions in an attempt to quantify the production of microbial postmortem ethanol in cases where other alcohols were co-detected. This contribution on the modeling of postmortem ethanol production by Candida albicans is complementary to these previous studies. Τhis work aimed to study ethanol, higher alcohols (1-propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol), and 1-butanol production by Candida albicans: (i) in different culture media (Brain Heart Infusion, BHI and, Sabouraud Dextrose Broth, SDB), (ii) under mixed aerobic/anaerobic or strict anaerobic conditions, and (iii) at different temperatures (37 °C, 25 °C and, 4 °C), and develop simple mathematical models, resulted from fungal cultures at 25 °C, to predict the microbially produced ethanol in correlation with the other alcohols. The applicability of the models was tested in the C. albicans cultures in BHI and SDB media at 37 °C, in denatured human blood at 25 °C, acidic and neutral with different concentrations of additional glucose, in acidic denatured blood diluted with dextrose solution and in blood from autopsy cases. The received results indicated that the C. albicans models could apply in cases where yeasts have been activated in blood with elevated glucose levels. Overall, the in vitro ethanol production by C. albicans in blood depended on temperature, time, glucose (or carbohydrate) content, pH of the medium and endogenous changes in the medium composition through time. Our results showed that methyl-butanol is the most significant indicator of fungal ethanol production, followed by the equally important isobutanol and 1-propanol in qualitative and quantitative terms.
Asunto(s)
Candida albicans/metabolismo , Etanol/metabolismo , Modelos Teóricos , Cambios Post Mortem , 1-Butanol/metabolismo , 1-Propanol/metabolismo , Glucemia , Butanoles/metabolismo , Técnicas de Cultivo , Humanos , Pentanoles/metabolismo , Manejo de Especímenes , TemperaturaRESUMEN
Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.
Asunto(s)
Monoterpenos Acíclicos/química , Pentanoles/química , Sesquiterpenos/metabolismo , Transferasas/metabolismo , Monoterpenos Acíclicos/metabolismo , Secuencia de Aminoácidos , Escherichia coli/genética , Hemiterpenos/metabolismo , Ácido Mevalónico/metabolismo , Pentanoles/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Transducción de Señal , Streptomyces/enzimología , Terpenos/metabolismo , Transferasas/genéticaRESUMEN
Ambrosia beetles (Coleoptera: Scolytinae) cultivate their fungal symbiont within host substrates as the sole source of nutrition on which the larvae and adults must feed. To investigate a possible role for semiochemicals in this interaction, we characterized electrophysiological and behavioral responses of Xylosandrus germanus to volatiles associated with its fungal symbiont Ambrosiella grosmanniae. During still-air walking bioassays, X. germanus exhibited an arrestment response to volatiles of A. grosmanniae, but not antagonistic fungi Beauveria bassiana, Metarhizium brunneum, Trichoderma harzianum, the plant pathogen Fusarium proliferatum, or malt extract agar. Solid phase microextraction-gas chromatography-mass spectrometry identified 2-ethyl-1-hexanol, 2-phenylethanol, methyl benzoate and 3-methyl-1-butanol in emissions from A. grosmanniae; the latter two compounds were also detected in emissions from B. bassiana. Concentration-responses using electroantennography documented weak depolarizations to A. grosmanniae fungal volatiles, unlike the comparatively strong response to ethanol. When tested singly in walking bioassays, volatiles identified from A. grosmanniae elicited relatively weak arrestment responses, unlike the responses to ethanol. Xylosandrus germanus also exhibited weak or no long-range attraction to the fungal volatiles when tested singly during field trials in 2016-2018. None of the fungal volatiles enhanced attraction of X. germanus to ethanol when tested singly; in contrast, 2-phenylethanol and 3-methyl-1-butanol consistently reduced attraction to ethanol. Volatiles emitted by A. grosmanniae may represent short-range olfactory cues that could aid in distinguishing their nutritional fungal symbiont from other fungi, but these compounds are not likely to be useful as long-range attractants for improving detection or mass trapping tactics.
Asunto(s)
Feromonas/química , Compuestos Orgánicos Volátiles/química , Animales , Ascomicetos/metabolismo , Conducta Animal , Benzoatos/química , Benzoatos/metabolismo , Evolución Biológica , Fenómenos Electrofisiológicos , Etanol/química , Etanol/metabolismo , Femenino , Fusarium/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Hexanoles/química , Hexanoles/metabolismo , Control de Insectos , Pentanoles/química , Pentanoles/metabolismo , Feromonas/metabolismo , Microextracción en Fase Sólida , Simbiosis , Compuestos Orgánicos Volátiles/metabolismo , GorgojosRESUMEN
The soil bacterium Bacillus subtilis forms beneficial biofilms that induce plant defences and prevent the growth of pathogens. It is naturally found in the rhizosphere, where microorganisms coexist in an extremely competitive environment, and thus have evolved a diverse arsenal of defence mechanisms. In this work, we found that volatile compounds produced by B. subtilis biofilms inhibited the development of competing biofilm colonies, by reducing extracellular matrix gene expression, both within and across species. This effect was dose-dependent, with the structural defects becoming more pronounced as the number of volatile-producing colonies increased. This inhibition was mostly mediated by organic volatiles, and we identified the active molecules as 3-methyl-1-butanol and 1-butanol. Similar results were obtained with biofilms formed by phylogenetically distinct bacterium sharing the same niche, Escherichia coli, which produced the biofilm-inhibiting 3-methyl-1-butanol and 2-nonanon. The ability of established biofilms to inhibit the development and spreading of new biofilms from afar might be a general mechanism utilized by bacterial biofilms to protect an occupied niche from the invasion of competing bacteria.
Asunto(s)
Biopelículas/efectos de los fármacos , Interacciones Microbianas/efectos de los fármacos , Compuestos Orgánicos Volátiles/farmacología , 1-Butanol/metabolismo , 1-Butanol/farmacología , Bacillus subtilis/fisiología , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Escherichia coli/fisiología , Matriz Extracelular de Sustancias Poliméricas/efectos de los fármacos , Matriz Extracelular de Sustancias Poliméricas/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Cetonas/metabolismo , Cetonas/farmacología , Microbiota , Pentanoles/metabolismo , Pentanoles/farmacología , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
NFAT-133 is a Streptomyces-derived aromatic polyketide compound with immunosuppressive, antidiabetic, and antitrypanosomal activities. It inhibits transcription mediated by nuclear factor of activated T cells (NFAT), leading to the suppression of interleukin-2 expression and T cell proliferation. It also activates the AMPK pathway in L6 myotubes and increases glucose uptake. In addition to NFAT-133, a number of its congeners, e.g., panowamycins and benwamycins, have been identified. However, little is known about their modes of formation in the producing organisms. Through genome sequencing of Streptomyces pactum ATCC 27456, gene inactivation, and genetic complementation experiments, the biosynthetic gene cluster of NFAT-133 and its congeners has been identified. The cluster contains a highly disordered genetic organization of type I modular polyketide synthase genes with several genes that are necessary for the formation of the aromatic core unit and tailoring processes. In addition, a number of new analogs of NFAT-133 were isolated and their chemical structures elucidated. It is suggested that the heptaketide NFAT-133 is derived from an octaketide intermediate, TM-123. The current study shows yet another unusual biosynthetic pathway involving a noncanonical polyketide synthase assembly line to produce a group of small molecules with valuable bioactivities.
Asunto(s)
Pentanoles/metabolismo , Pentanonas/metabolismo , Streptomyces/metabolismo , Biología Computacional , Genes Bacterianos , Genoma Bacteriano , Espectroscopía de Resonancia Magnética , Familia de Multigenes , Sintasas Poliquetidas/genética , Streptomyces/enzimología , Streptomyces/genéticaRESUMEN
Soy (tofu) whey is a liquid by-product generated from tofu (soybean curd) production and it is often discarded off as a waste liquid by the tofu manufacturers. Previous studies have demonstrated that soy whey can be biotransformed into a soy alcoholic beverage by using Saccharomyces and non-Saccharomyces yeasts even though soy whey is low in yeast assimilable nitrogen (YAN) content. In this study, the initial YAN of the soy whey was estimated to be 46.6 mg N/L and Torulaspora delbrueckii Biodiva was used to ferment soy whey supplemented with either isoleucine only or isoleucine paired with valine, leucine or phenylalanine (each amino acid supplemented at a dosage of 30 mg N/L). Amino acid supplementation was found to enhance sugar utilization by the yeast, which led to higher ethanol production (7.49% v/v in control versus 8.35-8.80% v/v in supplemented samples). Samples supplemented with isoleucine only experienced slower sugar utilization during the fermentation as compared to the paired amino acid samples, but the yeast was still able to utilize the sugar to low levels at the end of the fermentation. The presence of leucine supplementation counteracted the "inhibition" induced by the presence of isoleucine at the first day of the fermentation. Amino acid supplementation slowed down glutamic acid utilization and resulted in higher levels of residual glutamic acid and alanine. Amino acid supplementation increased the corresponding fusel alcohol production and the presence of other amino acids reduced the active amyl alcohol production. Therefore, interactions between amino acids can impact the metabolism of the yeast as well as the flavor modulation during soy whey fermentation.
Asunto(s)
Bebidas Alcohólicas/microbiología , Fermentación/fisiología , Isoleucina/metabolismo , Alimentos de Soja , Torulaspora/metabolismo , Bebidas Alcohólicas/análisis , Etanol/metabolismo , Pentanoles/metabolismo , Leche de Soja/química , Gusto , Suero Lácteo/metabolismo , Proteína de Suero de Leche/metabolismo , VinoRESUMEN
The objective of this study was to investigate the role of yeasts in the wet fermentation of coffee beans and their contribution to coffee quality using a novel approach. Natamycin (300 ppm) was added to the fermentation mass to suppress yeast growth and their metabolic activities, and the resultant microbial ecology, bean chemistry and sensory quality were analyzed and compared to non-treated spontaneous fermentation we reported previously. The yeast community was dominated by Hanseniaspora uvarum and Pichia kudriavzevii and grew to a maximum population of about 5.5 log CFU/g in the absence of Natamycin, while when Natamycin was added yeasts were suppressed. The major bacterial species in both the spontaneous and yeast-suppressed fermentations included the lactic acid bacteria Leuconostoc mesenteroides and Lactococcus lactis, the acetic acid bacteria Gluconobacter cerinus and Acetobacter persici and the Enterobacteriaceae Enterobacter, Citrobacter and Erwinia. For both fermentations, the mucilage layers were completely degraded by the end of the process and the absence of yeast activities had no significant impact on mucilage degradation. During fermentation, reducing sugars were consumed while lactic acid was accumulated inside the beans, and its concentration was significantly higher in the spontaneous fermentation (3 times) than that where yeasts were suppressed by Natamycin. Glycerol was detected with a concentration of 0.08% in the absence of Natamycin and was not identified when Natamycin was added. Green beans fermented with yeast growth contained a higher amount of isoamyl alcohol (21 times), ethanol (3.7 times), acetaldehyde (8 times), and ethyl acetate (25 times) compared to beans fermented in the absence of yeast activities, which remained higher in the former after roasting. Beans fermented without yeast activities had a mild fruity aroma, and lower sensory scores of fragrances (7.0), flavor (6.5), acidity (6.3), body (7.0) and overall score (6.5) compared to the former. These findings demonstrated the crucial roles of yeasts in wet fermentation of coffee beans and for producing high quality coffee.
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Bacterias/metabolismo , Café/metabolismo , Fermentación/fisiología , Hanseniaspora/metabolismo , Pichia/metabolismo , Levaduras/metabolismo , Acetaldehído/metabolismo , Acetatos/metabolismo , Ácido Acético/metabolismo , Antiinfecciosos/farmacología , Bacterias/clasificación , Reactores Biológicos/microbiología , Café/microbiología , Etanol/metabolismo , Ácido Láctico/metabolismo , Natamicina/farmacología , Odorantes/análisis , Pentanoles/metabolismo , GustoRESUMEN
The abundant supply of biosynthetic precursors and product compatibility with the intracellular environment play important roles for microbial isoprenoid production. In this study, we tailor to both of these requirements by introducing the two-step isopentenol utilization pathway (IUP) to augment the native pathway in the oleaginous yeast Yarrowia lipolytica. With shortcut access to the common isoprenoid precursor, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), IUP is capable of elevating IPP + DMAPP levels by 15.7-fold compared to the mevalonate pathway alone. The increase in IPP + DMAPP levels can directly lead to better isoprenoid synthesis, which is illustrated using lycopene as a model compound. Moreover, we also demonstrate that higher lipid contents in the cells correlate with improved intracellular lycopene production, suggesting the importance of having a substantial hydrophobic environment to sequester isoprenoids. Combining these strategies with further genetic and fermentation optimizations, we achieved a final lycopene titer of 4.2 g/L. Overall, these strategies hold great potential for strengthening the synthesis of long-chain isoprenoids and fat-soluble natural products in microbes.
Asunto(s)
Ingeniería Metabólica , Pentanoles/metabolismo , Terpenos/metabolismo , Yarrowia , Interacciones Hidrofóbicas e Hidrofílicas , Yarrowia/genética , Yarrowia/metabolismoRESUMEN
Chemosensation plays a role in the behaviors and life cycles of numerous organisms, including nematodes. Many guilds of nematodes exist, ranging from the free-living Caenorhabditis elegans to various parasitic species such as entomopathogenic nematodes (EPNs), which are parasites of insects. Despite ecological differences, previous research has shown that both EPNs and C. elegans respond to prenol (3-methyl-2-buten-1-ol), an odor associated with EPN infections. However, it is unclear how C. elegans responds to prenol. By utilizing natural variation and genetic neuron ablation to investigate the response of C. elegans to prenol, we found that the AWC neurons are involved in the detection of prenol and that several genes (including dcap-1, dcap-2, and clec-39) influence response to this odorant. Furthermore, we identified that the response to prenol is mediated by the canonically proposed pathway required for other AWC-sensed attractants. However, upon testing genetically diverse isolates, we found that the response of some strains to prenol differed from their response to isoamyl alcohol, suggesting that the pathways mediating response to these two odorants may be genetically distinct. Further, evaluations leveraging natural variation and genome wide association revealed specific genes that influence nematode behavior and provide a foundation for future studies to better understand the role of prenol in nematode behavioral ecology.