RESUMO
Halomonas bluephagenesis, a robust and contamination-resistant microorganism has been developed as a chassis for "Next Generation Industrial Biotechnology". The non-model H. bluephagenesis requires efficient tools to fine-tune its metabolic fluxes for enhanced production phenotypes. Here we report a highly efficient gene expression regulation system (PrrF1-2-HfqPa) in H. bluephagenesis, small regulatory RNA (sRNA) PrrF1 scaffold from Pseudomonas aeruginosa and a target-binding sequence that downregulate gene expression, and its cognate P. aeruginosa Hfq (HfqPa), recruited by the scaffold to facilitate the hybridization of sRNA and the target mRNA. The PrrF1-2-HfqPa system targeting prpC in H. bluephagenesis helps increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to 21 mol% compared to 3.1 mol% of the control. This sRNA system repressed phaP1 and minD simultaneously, resulting in large polyhydroxybutyrate granules. Further, an sRNA library targeting 30 genes was employed for large-scale target identification to increase mevalonate production. This work expands the study on using an sRNA system not based on Escherichia coli MicC/SgrS-Hfq to repress gene expression, providing a framework to exploit new powerful genome engineering tools based on other sRNAs.
Assuntos
Halomonas , Pequeno RNA não Traduzido , Biotecnologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Halomonas/genética , Halomonas/metabolismo , Hidroxibutiratos/metabolismo , Engenharia Metabólica/métodos , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismoRESUMO
BACKGROUND: Dai-Huang-Fu-Zi-Tang (DHFZT) is a famous traditional Chinese prescription with intestinal obstruction, acute pancreatitis and cholecystalgia for thousands of years. Our previous work found that DHFZT could act against pulmonary and intestinal pathological injury in rats with severe acute pancreatitis (SAP). But the underlying mechanism has not been fully elucidated. The aim of present study was to investigate whether DHFZT could relieve pulmonary and intestinal injury by regulating aquaporins after SAP induced by sodium taurocholate in rats. METHODS: Forty of SD rats were used for dose dependant experiments of DHFZT.Accurate-mass Time-of-flight liquid chromatography-mass spectrometry was used for qualitative screening of chemical compositions of DHFZT. Twenty-four rats were randomly divided into 3 groups: sham group (n = 8), model group (SAP, n = 8), DHFZT group (SAP with DHFZT treatment, n = 8). SAP models were established by retrograde injections of 5% sodium taurocholate solutions into rat pancreaticobiliary ducts. Blood samples were taken at 0, 12, 24, 48 h post-operation for detecting serum amylase, lipase, endotoxin, TNF-α, IL-6 and IL-10. Protein expression and location of aquaporin (AQP)1, 5, 8 and 9 were assessed by immunohistochemistry, western blot and immunofluorescence respectively. RESULTS: The study showed that 27 kinds of chemical composition were identified, including 10 kinds in positive ion mode and 17 kinds in negative ion mode. The results showed that AQP1, AQP5 of lung, and AQP1, AQP5, AQP8 of intestine in model group were significantly lower than that of sham group (P < 0.05), and which were obviously reversed by treatment with DHFZT. In addition, protein levels of pro-inflammatory cytokines such as TNF-α, IL-6 and endotoxin in peripheral blood were significantly suppressed by DHFZT, and that anti-inflammatory cytokine like IL-10 was just opposite. Finally, we also noted that DHFZT reduced serum levels of amylase, lipase and endotoxin, and also improved edema and pathological scores of lung and intestine after SAP. CONCLUSIONS: DHFZT ameliorated the pulmonary and intestinal edema and injury induced by SAP via the upregulation of different AQPs in lung and intestine, and suppressed TNF-α, IL-6 expression and enhanced IL-10 expression.
Assuntos
Aquaporinas/genética , Medicamentos de Ervas Chinesas/administração & dosagem , Enteropatias/tratamento farmacológico , Lesão Pulmonar/tratamento farmacológico , Pancreatite/complicações , Animais , Aquaporinas/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Enteropatias/etiologia , Enteropatias/genética , Enteropatias/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/lesões , Lesão Pulmonar/etiologia , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Mevalonate, an important intermediate product of the mevalonate pathway, has a broad spectrum of applications. With the rapid growth of metabolic engineering and synthetic biology, mevalonate biosynthesis by microorganisms is feasible and holds great promise in the future. In this review, we summarize the applications of mevalonate and its derivatives and describe the biosynthesis pathways of mevalonate. The current status of mevalonate biosynthesis is also detailed with an emphasis on metabolic engineering strategies to enhance mevalonate production in typical industrial organisms, including Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida, suggesting new insights for the efficient production of biosynthesized mevalonate.