RESUMO
As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks.
Assuntos
Arabidopsis/genética , Biocombustíveis , Ácidos Graxos/genética , Microbiologia Industrial/métodos , Óleos Industriais , Microalgas/genética , Saccharomyces cerevisiae/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Biocombustíveis/análise , Biocombustíveis/microbiologia , Diacilglicerol Colinofosfotransferase/genética , Diacilglicerol Colinofosfotransferase/metabolismo , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Graxos/metabolismo , Óleos Industriais/análise , Óleos Industriais/microbiologia , Engenharia Metabólica/métodos , Microalgas/enzimologia , Microalgas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismoRESUMO
CDP--choline:1,2-diglyceride choline phosphotransferase (CPT; EC 2.7.8.2) undergoes a marked surge in activity in human amniotic fluid when assayed from 30 weeks of gestation to term. The activity of this enxyme, plotted against gestational age, follows a highly significant regression correlation from which an equation can be obtained for the prediction of gestational age.
Assuntos
Líquido Amniótico/enzimologia , Diacilglicerol Colinofosfotransferase/metabolismo , Fosfotransferases/metabolismo , Feminino , Feto/fisiologia , Crescimento , Humanos , Pulmão/embriologia , Gravidez , Terceiro Trimestre da GravidezRESUMO
To achieve an isolated, ventilated, perfused newborn rabbit lung preparation, newborn rabbits are anesthetized and ventilated. The pulmonary vasculature is perfused with a Krebs-Ringer bicarbonate solution containing glucose and plasma expanders, and the lungs are isolated in a 37 degrees C chamber at 100% humidity. The success of a perfusion is evaluated on three levels. 1) Visual. 2)mechanical: a, drop in flow of greater than 20% indicates failure; b, an increase of more than 25% of the initial lung weight indicates fluid accumulation and failure. 3) Biochemical: a, the endogenous ATP concentration after perfusion; b, the level of the enzyme choline phosphotransferase (CPT) after perfusion. Isolated, perfused, ventilated newborn rabbit lungs maintained for 4 h with no changes in the monitored physical and mechanical parameters have an endogenous ATP level of 1.06 +/- 0.06 (2 SD) mumol ATP/mg wet weight and a CPT level of 1.34 +/-0.15 (2 SD nmol [14C]CDP-choline per milligram protein in 30 min. These values are stable at a level lower than observed in intact, hypoxic, newborn rabbit's lungs. In constrast, if perfusion is maintained after any of the monitored criteria indicate failure, the endogenous ATP concentration and CPT activity are significantly depressed.