RESUMEN
Algae are potential candidates for biodiesel production; thus, it is important to gain insight into the molecular mechanism of their lipid metabolism. Time-course transcriptome analyses were carried out during the lipid biosynthesis and accumulation processes of the model green alga Chlamydomonas reinhardtii using the Illumina RNA-seq platform. Transcriptome results indicated that over 2500 genes are upregulated or during lipid accumulation compared to log phase growth. As a proof of principle, two of the enzymes required for lipid metabolism that were significantly up-regulated during lipid accumulation, Lyso-Phosphatidic Acid Acyltransferase (LPAAT), diacylglycerol acyltransferase (DAGAT) were knocked down using artificial microRNAs. Neutral lipid production decreased in strains knocked down in expression of the lpaat and dagat genes. In addition, forty-one transcription factors were up- or down-regulated during the lipid accumulation process. This transcriptome data will be useful for engineering economic algae species aimed at biodiesel production.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Metabolismo de los Lípidos , Transcriptoma , Aciltransferasas/genética , Aciltransferasas/metabolismo , Chlamydomonas reinhardtii/genética , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Interferente Pequeño , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Targeting the important enzyme in human glucose metabolic pathway, we established a high throughput screening model for human pancreatic alpha-amylase inhibitors. METHODS: Pichia pastoris expression system was used to clone and express the human pancreatic alpha-amylase; we established the alpha-amylase inhibitor screening model using the catalytic properties of enzyme; this model was applied in screening of actinomycete' metabolites; the taxonomic status of positive strains were analyzed by constructing 16S rRNA phylogenetic tree. RESULTS: We cloned and expressed the intact gene of human pancreatic alpha-amylase successfully; the high-throughput screening model of alpha-amylase inhibitors was established; nearly 2000 actinomycete' metabolites were screened, 14 alpha-amylase inhibitor producing strains were obtained finally, and showed taxonomically rich diversity. CONCLUSION: The alpha-amylase inhibitor high-throughput screening model had high practical value for developing new hypoglycemic drugs.
Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , alfa-Amilasas Pancreáticas/antagonistas & inhibidores , Actinobacteria/clasificación , Actinobacteria/metabolismo , Humanos , alfa-Amilasas Pancreáticas/genética , Filogenia , Pichia/genética , Proteínas Recombinantes/biosíntesisRESUMEN
Lung infections caused by Pseudomonas aeruginosa in cystic fibrosis (CF) patients cause progressive airway obstruction and tissue damage, which is the predominant cause of morbidity and mortality in patients with CF. This paper describes the functional characterization of the pfm gene (open reading frame PA2950) of P. aeruginosa. Using DNA microarrays, we found that the transcriptional levels of type II secretory system genes were significantly reduced in the pfm mutant strain. The type-II-dependent exoprotein LasB could not be secreted normally. The pfm gene was identified as a gene involved in bacterial protein secretion that was critical for the extracellular release of elastase in P. aeruginosa. The abilities to induce lung injury by wild-type and pfm mutant P. aeruginosa were evaluated in a murine acute lung infection model. The results showed that the pathogenicity and virulence of the pfm mutant strain was significantly reduced compared with that of the wild-type strain. The pfm gene and its expression product, as potential new drug targets against P. aeruginosa infection, have important research significance.
Asunto(s)
Bronconeumonía/microbiología , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Proteínas de Transporte de Membrana/metabolismo , Infecciones por Pseudomonas/microbiología , Animales , Bronconeumonía/mortalidad , Bronconeumonía/patología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Humanos , Pulmón/patología , Proteínas de Transporte de Membrana/genética , Ratones , Análisis por Micromatrices , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Virulencia , Factores de Virulencia/metabolismoRESUMEN
OBJECTIVE: Targeted at the important enzyme in human glucose metabolic pathway, the purpose of this paper is to establish alpha-glucosidase inhibitors high throughput screening model. METHODS: Pichia pastoris expression system was used to clone and express the human alpha-maltase glucosidase. Using the catalytic properties of enzyme to establish alpha-glucosidase inhibitor screening model. This model was applied in screening of actinomycete metabolites library. The taxonomic status of positive strains were analyzed by constructing 16S rRNA phylogenetic tree. RESULTS: The N-terminal catalytic domain of human alpha-maltase glucosidase was successfully cloned and expressed for the first time. The high-throughput screening model of alpha-glucosidase inhibitors was established. A natural product library containing metabolites from nearly 2000 actinomycetes was screened, 20 alpha-maltase glucosidase inhibitor producing strains were obtained finally, of which, 19 strains initially identified as Streptomyces, and showed taxonomically rich diversity. CONCLUSION: The alpha-glucosidase inhibitor high-throughput screening model has high practical value, this work laid the foundation for developing new hypoglycemic drugs.