RESUMEN
Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo-inositol into scyllo-inositol at 30°C. In a scaled-up reaction, 10 g of myo-inositol was converted to 1.8 g of scyllo-inositol, which was further purified to yield 970 mg of pure powder. Notably, myo-inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.
Asunto(s)
Proteínas Bacterianas , Geobacillus , Inositol , Inositol/metabolismo , Geobacillus/genética , Geobacillus/enzimología , Geobacillus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regiones Promotoras GenéticasRESUMEN
Glycerol dehydrogenase (GDH) catalyzes glycerol oxidation to dihydroxyacetone in a NAD+-dependent manner. As an initiator of the oxidative pathway of glycerol metabolism, a variety of functional and structural studies of GDH have been conducted previously. Structural studies revealed intriguing features of GDH, like the flexible ß-hairpin and its significance. Another commonly reported structural feature is the enzyme's octameric oligomerization, though its structural details and functional significance remained unclear. Here, with a newly reported GDH structure, complexed with both NAD+ and glycerol, we analyzed the octamerization of GDH. Structural analyses revealed that octamerization reduces the structural dynamics of the N-domain, which contributes to more consistently maintaining a distance required for catalysis between the cofactor and substrate. This suggests that octamerization may play a key role in increasing the likelihood of the enzyme reaction by maintaining the ligands in an appropriate configuration for catalysis. These findings expand our understanding of the structure of GDH and its relation to the enzyme's activity.
Asunto(s)
NAD , Deshidrogenasas del Alcohol de Azúcar , NAD/metabolismo , Glicerol/metabolismo , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Oxidación-Reducción , Glutamato Deshidrogenasa/metabolismoRESUMEN
Gluconobacter strains perform incomplete oxidation of various sugars and alcohols, employing regio- and stereoselective membrane-bound dehydrogenases oriented toward the periplasmic space. This oxidative fermentation process is utilized industrially. The ketogluconate production pathway, characteristic of these strains, begins with the conversion of d-glucose to d-gluconate, which then diverges and splits into 2 pathways producing 5-keto-d-gluconate and 2-keto-d-gluconate and subsequently 2,5-diketo-d-gluconate. These transformations are facilitated by membrane-bound d-glucose dehydrogenase, glycerol dehydrogenase, d-gluconate dehydrogenase, and 2-keto-d-gluconate dehydrogenase. The variance in end products across Gluconobacter strains stems from the diversity of enzymes and their activities. This review synthesizes biochemical and genetic knowledge with biotechnological applications, highlighting recent advances in metabolic engineering and the development of an efficient production process focusing on enzymes relevant to the ketogluconate production pathway in Gluconobacter strains.
Asunto(s)
Biotecnología , Gluconatos , Gluconobacter , Deshidrogenasas del Alcohol de Azúcar , Gluconatos/metabolismo , Gluconobacter/metabolismo , Gluconobacter/enzimología , Gluconobacter/genética , Biotecnología/métodos , Fermentación , Ingeniería Metabólica/métodos , Glucosa/metabolismo , Glucosa 1-Deshidrogenasa/metabolismo , Glucosa 1-Deshidrogenasa/genéticaRESUMEN
INTRODUCTION: Ethylene glycol (EG) is a frequently considered toxicant in poisoned patients. Definitive diagnosis relies on gas chromatography (GC), but this is unavailable at most hospitals. A glycerol dehydrogenase (GDH)-based assay rapidly detects EG. A rapid turnaround time and wide availability of necessary instrumentation suggest this method could facilitate the rapid detection of EG. METHODS: This is a prospective, observational analysis of banked, remnant serum samples submitted to the laboratory of a large, multi-hospital healthcare system. Samples were submitted over a 12-month period for the explicit purpose of testing for suspected EG ingestion. All samples underwent GC and the GDH-based assay. RESULTS: Of the 118 analyzed samples, 88 had no EG detected by GC, and 30 were "positive." At the manufacturer's threshold of 6 mg/dL EG, there was 100% (95%CI; 88.7-100) positive percent agreement (PPA) and 98% (92.1-99.6) negative percent agreement (NPA). Adjusted to a threshold of 9 mg/dL, both the PPA and NPA were 100%. Deming regression of the observed concentrations revealed a slope of 1.16 (1.01 to 1.32) and intercept of -5.3 (-8.9 to -1.7). CONCLUSIONS: The GDH assay provides a sensitive and specific method for the detection and quantification of EG that is comparable to a GC-based method. More widespread use of this rapid, inexpensive assay could improve the care of patients with suspected toxic alcohol exposure. Further study is needed to evaluate the test performance in real-time patient treatment decisions.
Asunto(s)
Sustancias Peligrosas , Deshidrogenasas del Alcohol de Azúcar , Humanos , NonoxinolRESUMEN
During glycerol metabolism, the initial step of glycerol oxidation is catalysed by glycerol dehydrogenase (GDH), which converts glycerol to dihydroxyacetone in a NAD+ -dependent manner via an ordered Bi-Bi kinetic mechanism. Structural studies conducted with GDH from various species have mainly elucidated structural details of the active site and ligand binding. However, the structure of the full GDH complex with both cofactor and substrate bound is not determined, and thus, the structural basis of the kinetic mechanism of GDH remains unclear. Here, we report the crystal structures of Escherichia coli GDH with a substrate analogue bound in the absence or presence of NAD+ . Structural analyses including molecular dynamics simulations revealed that GDH possesses a flexible ß-hairpin, and that during the ordered progression of the kinetic mechanism, the flexibility of the ß-hairpin is reduced after NAD+ binding. It was also observed that this alterable flexibility of the ß-hairpin contributes to the cofactor binding and possibly to the catalytic efficiency of GDH. These findings suggest the importance of the flexible ß-hairpin to GDH enzymatic activity and shed new light on the kinetic mechanism of GDH.
Asunto(s)
NAD , Deshidrogenasas del Alcohol de Azúcar , NAD/metabolismo , Glicerol/metabolismo , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/química , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Oxidación-Reducción , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Glutamato Deshidrogenasa/metabolismoRESUMEN
Glycerol dehydrogenase (GldA) from Escherichia coli BW25113, naturally catalyzes the oxidation of glycerol to dihydroxyacetone. It is known that GldA exhibits promiscuity towards short-chain C2-C4 alcohols. However, there are no reports regarding the substrate scope of GldA towards larger substrates. Herein we demonstrate that GldA can accept bulkier C6-C8 alcohols than previously anticipated. Overexpression of the gldA gene in the knockout background, E. coli BW25113 ΔgldA, was strikingly effective converting 2 mM of the compounds: cis-dihydrocatetechol, cis-(1 S,2 R)- 3-methylcyclohexa-3,5-diene-1,2-diol and cis-(1 S,2 R)- 3-ethylcyclohexa-3,5-diene-1,2-diol, into 2.04 ± 0.21 mM of catechol, 0.62 ± 0.11 mM 3-methylcatechol, and 0.16 ± 0.02 mM 3-ethylcatechol, respectively. In-silico studies on the active site of GldA enlightened the decrease in product formation as the steric substrate demand increased. These results are of high interests for E. coli-based cell factories expressing Rieske non-heme iron dioxygenases, producing cis-dihydrocatechols, since such sough-after valuable products can be immediately degraded by GldA, substantially hampering the expected performance of the recombinant platform.
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Dioxigenasas , Deshidrogenasas del Alcohol de Azúcar , Escherichia coli/genética , Escherichia coli/metabolismo , Deshidrogenasas del Alcohol de Azúcar/genética , Dioxigenasas/metabolismo , Oxidación-Reducción , Glicerol/metabolismoRESUMEN
The function of human dicarbonyl/Lxylulose reductase (DCXR) in the pathophysiology of breast cancer is yet to be elucidated. The present study aimed to investigate the function of DCXR in glycolysis and the cell cycle of breast cancer cells with respect to cell proliferation. Differential expressed DCXR was identified in The Cancer Genome Atlas (TCGA) database and verified in clinical breast cancer tissue. DCXR silencing and overexpression were induced by RNA interference and lentiviral vectors, respectively. Cell cycle progression, proliferation and glycolytic activity of breast cancer cells were detected by flow cytometry, Cell Counting Kit8 assay and chemical methods, respectively. Tumorigenicity was detected using nude mice xenograft models. The expression of DCXR was increased in TCGA breast cancer database and the function of DCXR was enriched in 'glycolysis' and 'cell cycle'. Further analysis using clinical breast cancer samples confirmed upregulation of DCXR. The silencing of DCXR suppressed proliferation and cell cycle progression of breast cancer cells and significantly decreased the capacity for glycolysis, thereby demonstrating the effect of DCXR on the function of breast cancer cells. Similar conclusions were obtained in DCXR overexpressing cells; notably, DCXR overexpression promoted proliferation, cell cycle progression at S phase and glycolysis. 2DeoxyDglucose inhibited the effect of DCXR on the proliferation and cell cycle progression of breast cancer cells. The present study revealed that DCXR regulated breast cancer cell cycle progression and proliferation by increasing glycolysis activity and thus may serve as an oncogene for breast cancer.
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Neoplasias de la Mama , Deshidrogenasas del Alcohol de Azúcar , Efecto Warburg en Oncología , Animales , Femenino , Humanos , Ratones , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/genética , Ratones Desnudos , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/metabolismoRESUMEN
Numerous studies reveal that metabolism dysfunction contributes to the development of pathological cardiac hypertrophy. While the abnormal lipid and glucose utilization in cardiomyocytes responding to hypertrophic stimuli have been extensively studied, the alteration and implication of glutaminolysis are rarely discussed. In the present work, we provide the first evidence that glutamate dehydrogenase (GDH), an enzyme that catalyzes conversion of glutamate into É-ketoglutarate (AKG), participates in isoprenaline (ISO)-induced cardiac hypertrophy through activating mammalian target of rapamycin (mTOR) signaling. The expression and activity of GDH were enhanced in cultured cardiomyocytes and rat hearts following ISO treatment. Overexpression of GDH, but not its enzymatically inactive mutant, provoked cardiac hypertrophy. In contrast, GDH knockdown could relieve ISO-triggered hypertrophic responses. The intracellular AKG level was elevated by ISO or GDH overexpression, which led to increased phosphorylation of mTOR and downstream effector ribosomal protein S6 kinase (S6K). Exogenous supplement of AKG also resulted in mTOR activation and cardiomyocyte hypertrophy. However, incubation with rapamycin, an mTOR inhibitor, attenuated hypertrophic responses in cardiomyocytes. Furthermore, GDH silencing protected rats from ISO-induced cardiac hypertrophy. These findings give a further insight into the role of GDH in cardiac hypertrophy and suggest it as a potential target for hypertrophy-related cardiomyopathy.
Asunto(s)
Glutamato Deshidrogenasa , Ácidos Cetoglutáricos , Animales , Cardiomegalia/metabolismo , Glucosa/metabolismo , Glutamato Deshidrogenasa/metabolismo , Glutamatos/metabolismo , Isoproterenol/farmacología , Ácidos Cetoglutáricos/metabolismo , Lípidos , Miocitos Cardíacos/metabolismo , Ratas , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sirolimus/farmacología , Deshidrogenasas del Alcohol de Azúcar , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helixloophelix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.
Asunto(s)
Acinetobacter baumannii , Secuencias Hélice-Asa-Hélice , Manitol , Deshidrogenasas del Alcohol de Azúcar , Acinetobacter baumannii/enzimología , Manitol/metabolismo , Presión Osmótica , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Estrés Salino , Deshidrogenasas del Alcohol de Azúcar/química , Deshidrogenasas del Alcohol de Azúcar/metabolismoRESUMEN
Wheat allergens are responsible for symptoms in 60-70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21-27 kDa that were recognized by the pooled baker's IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.
Asunto(s)
Alérgenos/inmunología , Glucosa 1-Deshidrogenasa/inmunología , Proteínas de Choque Térmico Pequeñas/inmunología , Proteínas de Plantas/inmunología , Deshidrogenasas del Alcohol de Azúcar/inmunología , Hipersensibilidad al Trigo/inmunología , Adulto , Anciano , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Masculino , Persona de Mediana Edad , Unión Proteica , Pruebas de Función Respiratoria , Pruebas Cutáneas , Espectrometría de Masas en Tándem , Hipersensibilidad al Trigo/diagnósticoRESUMEN
Glycerol is an abundant byproduct of biodiesel production that has significant industrial value and can be converted into dihydroxyacetone (DHA). DHA is widely used for the production of various chemicals, pharmaceuticals, and food additives. Gluconobacter can convert glycerol to DHA through two different pathways, including membrane-bound dehydrogenases with pyrroloquinoline quinone (PQQ) and NAD(P)+ -dependent enzymes. Previous work has indicated that membrane-bound dehydrogenases are present in Gluconobacter oxydans and Gluconobacter frateurii, but the metabolic mechanism of Gluconobacter thailandicus's glycerol conversion is still not clear. Through in-depth analysis of the G. thailandicus genome and annotation of its metabolic pathways, we revealed the existence of both PQQ and NAD(P)+ -dependent enzymes in G. thailandicus. In addition, this study provides important information related to the tricarboxylic acid cycle, glycerol dehydrogenase level, and phylogenetic relationships of this important species.
Asunto(s)
Genoma Bacteriano , Gluconobacter , Glicerol , Microorganismos Modificados Genéticamente , Ciclo del Ácido Cítrico/genética , Dihidroxiacetona/metabolismo , Ingeniería Genética , Genoma Bacteriano/genética , Gluconobacter/genética , Gluconobacter/metabolismo , Glicerol/metabolismo , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , NAD/metabolismo , NADP/metabolismo , Cofactor PQQ/metabolismo , Filogenia , Deshidrogenasas del Alcohol de Azúcar/análisisRESUMEN
The combined cross-linked enzyme aggregates (combi-CLEAs) containing galactitol dehydrogenase (Gdh) and NADH oxidase (Nox) were prepared for L-tagatose synthesis. To prevent the excess consumption of cofactor, Nox in the combi-CLEAs was used to in situ regenerate NAD+. In the immobilization process, ammonia sulfate and glutaraldehyde were used as the precipitant and cross-linking reagent, respectively. The preparation conditions were optimized as follows: 60% ammonium sulfate, 1:1 (molar ratio) of Gdh to Nox, 20:1 (molar ratio) of protein to glutaraldehyde, and 6 h of cross-linking time at 35 °C. Under these conditions, the activity of the combi-CLEAs was 210 U g-1. The combi-CLEAs exhibited higher thermostability and preserved 51.5% of the original activity after eight cycles of reuses at 45 °C. The combi-CLEAs were utilized for the preparation of L-tagatose without by-products. Therefore, the combi-CLEAs have the industrial potential for the bioconversion of galactitol to L-tagatose.
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Enzimas Inmovilizadas , Hexosas , Regeneración , Reactivos de Enlaces Cruzados , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Hexosas/biosíntesis , Hexosas/química , Complejos Multienzimáticos , NADH NADPH Oxidorreductasas , Deshidrogenasas del Alcohol de AzúcarRESUMEN
This study investigated the occurrence of Giardia duodenalis and Cryptosporidium spp. in rodents and marsupials from the Atlantic Forest in southern Bahia, northeastern Brazil. Two hundred and four fecal samples were collected from different forest areas in the municipalities of Ilhéus, Una, Belmonte, and Mascote. Identifications were performed using PCR and nested PCR followed by sequencing of the gdh and tpi genes for G. duodenalis, and the gp60 and Hsp-70 genes for Cryptosporidium. The total frequency of positive PCR samples for both G. duodenalis and Cryptosporidium spp. was 5.4% (11/204). Giardia duodenalis occurred in 2.94% (4/136) of rodents and 2.94% (2/68) of marsupials. The prevalence of Cryptosporidium in rodents and marsupials was 1.47% (2/136) and 4.41% (3/68), respectively. In the areas sampled, the frequency of parasitism was 50% (7/14), while the Mascote region alone had no parasitized animals. The G. duodenalis subgenotype AI was identified in the rodent species Hylaeamys laticeps, Oecomys catherinae, Oligoryzomys nigripes and Akodon cursor, and in the marsupials Gracilinanus agilis and Monodelphis americana. In the rodents Rhipidomys mastacalis, H. laticeps and in the marsupial Marmosa murina the protozoa Cryptosporidium fayeri, Cryptosporidium parvum and Cryptosporidium ubiquitum with subtypes IIa and IVg by the gp60 gene were found. In conclusion, this study provides the genetic characterization of Giardia and Cryptosporidium species and genotypes in rodents and marsupials. And, these findings reinforce that the rodent and marsupial species mentioned above play a role as new hosts for Giardia and Cryptosporidium.
Asunto(s)
Criptosporidiosis/epidemiología , Cryptosporidium/genética , Giardia lamblia/genética , Giardiasis/epidemiología , Giardiasis/veterinaria , Zoonosis/epidemiología , Animales , Brasil/epidemiología , Criptosporidiosis/parasitología , Cryptosporidium/aislamiento & purificación , ADN-Topoisomerasas/genética , ADN Protozoario/genética , Heces/parasitología , Giardia lamblia/aislamiento & purificación , Giardiasis/parasitología , Glicoproteínas/genética , Proteínas HSP70 de Choque Térmico/genética , Marsupiales/parasitología , Reacción en Cadena de la Polimerasa , Prevalencia , Proteínas Protozoarias/genética , Roedores/parasitología , Análisis de Secuencia de ADN , Deshidrogenasas del Alcohol de Azúcar/genética , Zoonosis/parasitologíaRESUMEN
From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.
Asunto(s)
Albinismo/terapia , Alcaptonuria/terapia , Cistinuria/terapia , Errores Innatos del Metabolismo/terapia , Albinismo/genética , Albinismo/metabolismo , Albinismo/patología , Alcaptonuria/genética , Alcaptonuria/metabolismo , Alcaptonuria/patología , Errores Innatos del Metabolismo de los Carbohidratos/genética , Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Errores Innatos del Metabolismo de los Carbohidratos/patología , Errores Innatos del Metabolismo de los Carbohidratos/terapia , Cistinuria/genética , Cistinuria/metabolismo , Cistinuria/patología , Humanos , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/patología , Fenilcetonurias/genética , Fenilcetonurias/metabolismo , Fenilcetonurias/patología , Fenilcetonurias/terapia , Deshidrogenasas del Alcohol de Azúcar/deficiencia , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Xilulosa/genética , Xilulosa/metabolismoRESUMEN
According to various reports, current methods of sperm freezing destroy the integrity of the sperm plasma membrane and acrosome. This study aimed to determine the changes in the existence and location of three proteins, namely fertilin ß, IZUMO1, and P34H, in ram spermatozoa. By using frozen-thawed spermatozoa, ejaculated fresh spermatozoa, and testicular and epididymal spermatozoa (obtained from caput, corpus, and caudal regions), the localizations of the mentioned proteins were performed using signal labeling with indirect immunofluorescence, and the quantification of these proteins was compared using Western blot analyses. Moreover, protein localization and signal labeling in fresh and frozen-thawed spermatozoa subjected to in vitro capacitation and acrosome reaction were compared. Using chlortetracycline (CTC) staining, as expected, it was detected that after incubating for 4 hours under capacitating conditions related to the control sample (0 hour), capacitated and acrosome-reacted sperm were increased (p < 0.001). Frozen-thawed samples had a lower density and expression than the ejaculate samples. Expression was not obtained, except for IZUMO1, from samples that underwent in vitro capacitation/acrosome reactions. Expression of IZUMO1 was seen as an increasing band formation from the equatorial region through the acrosome, after in vitro capacitation. However, after the acrosome reaction, the band formation was only on the equatorial region. Region-specific differences of proteins at the kDa level were obtained using Western blot analysis and possible isoforms specific to ram spermatozoa or proteins with similar epitopes were expressed. Considering the changes in surface proteins in frozen-thawed sperm, it is suggested that fertilin ß and P34H can be used as fertility or freezability markers.
Asunto(s)
Fertilinas , Proteínas de la Membrana , Capacitación Espermática , Espermatozoides , Deshidrogenasas del Alcohol de Azúcar , Acrosoma , Reacción Acrosómica , Animales , Inmunoglobulinas , Masculino , OvinosRESUMEN
Aspergillus nidulans produces cleistothecia as sexual reproductive organs in a process affected by genetic and external factors. To gain a deeper insight into A. nidulans sexual development, we performed comparative proteome analyses based on the wild type developmental periods. We identified sexual development-specific proteins with a more than twofold increase in production during hypoxia or the sexual period compared to the asexual period. Among the sexual development-specific proteins analyzed by gene-deletion experiments and functional assays, MpdA, a putative mannitol-1-phosphate 5-dehydrogenase, plays multiple roles in growth and differentiation of A. nidulans. The most distinct mpdA-deletion phenotype was ascosporogenesis failure. Genetic mpdA deletion resulted in small cleistothecia with no functional ascospores. Transcriptional analyses indicated that MpdA modulates the expression of key development- and meiosis-regulatory genes during sexual development. The mpdA deletion increased hyphal branching and decreased conidial heat resistance. Mannitol production in conidia showed no difference, whereas it was decreased in mycelia and sexual cultures. Addition of mannitol during vegetative growth recovered the defects in conidial heat resistance and ascospore genesis. Taken together, these results indicate that MpdA plays an important role in sexual development, hyphal branching, and conidial heat resistance in Aspergillus nidulans.
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Aspergillus nidulans/genética , Hifa/genética , Esporas Fúngicas/genética , Deshidrogenasas del Alcohol de Azúcar/genética , Aspergillus nidulans/crecimiento & desarrollo , Aspergillus nidulans/patogenicidad , Regulación Fúngica de la Expresión Génica/genética , Hifa/crecimiento & desarrollo , Manitol/metabolismo , Meiosis/genética , Desarrollo Sexual/genética , Esporas Fúngicas/metabolismoRESUMEN
L-Tagatose, a promising building block in the production of many value-added chemicals, is generally produced by chemical routes with a low yield, which may not meet the increasing demands. Synthesis of l-tagatose by enzymatic oxidation of d-galactitol has not been applied on an industrial scale because of the high cofactor costs and the lack of efficient cofactor regeneration methods. In this work, an efficient and environmentally friendly enzymatic method containing a galactitol dehydrogenase for d-galactitol oxidation and a water-forming NADH oxidase for regeneration of NAD+ was first designed and used for l-tagatose production. Supplied with only 3 mM NAD+, subsequent reaction optimization facilitated the efficient transformation of 100 mM of d-galactitol into l-tagatose with a yield of 90.2 % after 12 h (obtained productivity: 7.61 mM.h-1). Compared with the current chemical and biocatalytic methods, the strategy developed avoids by-product formation and achieves the highest yield of l-tagatose with low costs. It is expected to become a cleaner and more promising route for industrial biosynthesis of l-tagatose.
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Hexosas/biosíntesis , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Hexosas/química , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación Molecular , TemperaturaRESUMEN
The compound cis-1,2-dihydrocatechol (DHC) is highly valuable since it finds wide application in the production of fine chemicals and bioactive compounds with medical relevance. The biotechnological process to generate DHC involves a dearomatizing dihydroxylation reaction catalyzed by toluene dioxygenase (TDO) from P. putida F1, employing benzene as substrate. We aimed to enhance the biotechnological E. coli BW25113 platform for DHC production by identifying the key operational parameters positively influencing the final isolated yield. Thereby, we observed an unreported downstream reaction, generating catechol from DHC, affecting, in a negative manner, the final titer for the product. Expression temperature for the TDO-system showed to have the highest influence in terms of final isolated yield. A KEIO-collection-based screening approach highlighted glycerol dehydrogenase (GldA) as the main responsible enzyme for the undesired reaction. We transferred the TDO-system to E. coli BW25113 ΔgldA and applied the enhanced operational set-up on it. This enhanced platform enabled the production of 1.41â¯g L-1 DHC in isolated yield, which represents a two-fold increase compared with the starting working conditions. To our knowledge, this is the highest DHC production accomplished in recombinant E. coli at semi-preparative scale, providing a robust and accessible biotechnological platform for DHC synthesis.
Asunto(s)
Pseudomonas putida , Catecoles , Escherichia coli/genética , Oxigenasas , Deshidrogenasas del Alcohol de AzúcarRESUMEN
Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h-1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h-1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.
Asunto(s)
Aldehído-Liasas/genética , Vías Biosintéticas/genética , Dihidroxiacetona/biosíntesis , Escherichia coli K12/enzimología , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Glicerol/metabolismo , Alelos , Fructosafosfatos/metabolismo , Eliminación de Gen , Glucosa/metabolismo , Glucosafosfato Deshidrogenasa/genética , Glicerol Quinasa/genética , Isoenzimas/genética , Vía de Pentosa Fosfato/genética , Fosfofructoquinasas/química , Fosfofructoquinasas/genética , Deshidrogenasas del Alcohol de Azúcar/genéticaRESUMEN
Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes ß-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-D-glucose by LgdB2; next, LgdC reduces 3-keto-D-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.