RESUMO
The balance between bacterial colonization and its containment in the intestine is indispensable for the symbiotic relationship between humans and their bacteria. One component to maintain homeostasis at the mucosal surfaces is immunoglobulin A (IgA), the most abundant immunoglobulin in mammals1,2. Several studies have revealed important characteristics of poly-reactive IgA3,4, which is produced naturally without commensal bacteria. Considering the dynamic changes within the gut environment, however, it remains uncertain how the commensal-reactive IgA pool is shaped and how such IgA affects the microbial community. Here we show that acetate-one of the major gut microbial metabolites-not only increases the production of IgA in the colon, but also alters the capacity of the IgA pool to bind to specific microorganisms including Enterobacterales. Induction of commensal-reactive IgA and changes in the IgA repertoire by acetate were observed in mice monocolonized with Escherichia coli, which belongs to Enterobacterales, but not with the major commensal Bacteroides thetaiotaomicron, which suggests that acetate directs selective IgA binding to certain microorganisms. Mechanistically, acetate orchestrated the interactions between epithelial and immune cells, induced microbially stimulated CD4 T cells to support T-cell-dependent IgA production and, as a consequence, altered the localization of these bacteria within the colon. Collectively, we identified a role for gut microbial metabolites in the regulation of differential IgA production to maintain mucosal homeostasis.
Assuntos
Acetatos/farmacologia , Bactérias/imunologia , Microbioma Gastrointestinal/imunologia , Imunoglobulina A/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Colo/imunologia , Dieta , Ácidos Graxos Voláteis/metabolismo , Homeostase/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , SimbioseRESUMO
Hydroxyequols are promising analogues of the biologically active flavonoid, equol. We recently found that the flavin-dependent monooxygenase HpaBro-3 of Rhodococcus opacus regioselectively synthesizes 3'-hydroxyequol from equol, whereas HpaBpl-1 of Photorhabdus luminescens synthesizes 6-hydroxyequol. In this study, we investigated the cascade synthesis of a dihydroxyequol compound from equol using these two enzymes. When Escherichia coli cells expressing HpaBro-3 and cells expressing HpaBpl-1 were simultaneously incubated with equol, the cells efficiently synthesized 6,3'-dihydroxyequol (8.7 mM, 2.4 g/L) via 3'- and 6-hydroxyequols in one pot. The antioxidant activity of the equol derivatives increased with an increase in the number of hydroxyl groups on the equol scaffold. 6,3'-Dihydroxyequol exhibited potent antioxidant activity. In addition, 6-hydroxyequol significantly inhibited the growth of E. coli. Cell survival studies suggested that 6-hydroxyequol is a bactericidal rather than bacteriostatic compound. To our knowledge, this is the first report describing the antibacterial activity of hydroxyequols.
Assuntos
Equol , Isoflavonas , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Biocatálise , Equol/farmacologia , Escherichia coli , Isoflavonas/farmacologiaRESUMO
BACKGROUND: Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst. RESULTS: The two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts. CONCLUSIONS: Asymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing heterologous alcohol dehydrogenase showed advantages for practical usage relative to the in vitro coupling system. The results suggest a hopeful perspective of the hydrogen-driven bioprocess as an environmentally outstanding method to achieve industrial green innovation. Hydrogen-oxidizing bacteria can be useful hosts for the development of hydrogen-driven microbial cell factories.
Assuntos
Acetona/análogos & derivados , Álcool Desidrogenase/metabolismo , Proteínas de Bactérias/metabolismo , Cupriavidus necator/enzimologia , Kluyveromyces/metabolismo , Propilenoglicol/metabolismo , Acetona/química , Acetona/metabolismo , Álcool Desidrogenase/genética , Proteínas de Bactérias/genética , Técnicas de Cultura Celular por Lotes , Biocatálise , Hidrogênio/química , Oxirredução , Propilenoglicol/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , EstereoisomerismoRESUMO
Monooxygenases exhibiting high activity and differing regioselectivity for the dietary isoflavone metabolite equol were discovered among enzymes in the HpaBC family by a genome mining approach. These enzymes enabled the one-step product-selective synthesis of 3'- and 6-hydroxyequols from equol and molecular oxygen.
RESUMO
A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.
Assuntos
1-Propanol/farmacologia , Etanol/farmacologia , Genoma Fúngico , Pentanóis/farmacologia , Saccharomyces cerevisiae/genética , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/fisiologiaRESUMO
The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-ECHB) from ethyl 4-chloroacetoacetate was studied using whole recombinant cells of Escherichia coli expressing a secondary alcohol dehydrogenase of Candida parapsilosis. Using 2-propanol as an energy source to regenerate NADH, the yield of (R)-ECHB reached 36.6 g/l (more than 99% ee, 95.2% conversion yield) without addition of NADH to the reaction mixture.
Assuntos
Álcool Desidrogenase/metabolismo , Butiratos/metabolismo , Candida/enzimologia , Escherichia coli/metabolismo , Recombinação Genética , Sequência de Bases , Primers do DNA , Escherichia coli/genéticaRESUMO
The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation. Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4% from a racemate of 15% and an optical purity of 95% ee.
Assuntos
Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Butileno Glicóis/síntese química , Escherichia coli/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMO
A novel carbonyl reductase (KLCR1) that reduced ethyl 4-chloroacetoacetate (ECAA) to synthesize ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-ECHB) was purified from Kluyveromyces lactis. KLCR1 catalyzed the NADPH-dependent reduction of ECAA enantioselectively but not the oxidation of (S)-ECHB. From partial amino acid sequences, KLCR1 was suggested to be an alpha subunit of fatty acid synthase (FAS) but did not have FAS activity.
Assuntos
Oxirredutases do Álcool/isolamento & purificação , Butiratos/metabolismo , Kluyveromyces/enzimologia , Acetoacetatos/metabolismo , Oxirredutases do Álcool/química , Aldeído Redutase , Aldo-Ceto Redutases , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Kluyveromyces/metabolismo , Conformação Molecular , Peso Molecular , Protaminas/química , Especificidade por Substrato , UltrafiltraçãoRESUMO
To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.