RESUMO
Feedback control allows cells to dynamically sense and respond to environmental changes. However, synthetic controller designs can be challenging because of implementation issues, such as determining optimal expression levels for circuit components within a feedback loop. Here, we addressed this by coupling rational design with selection to engineer a synthetic feedback circuit to optimize tolerance of Escherichia coli to the biojet fuel pinene. E. coli can be engineered to produce pinene, but it is toxic to cells. Efflux pumps, such as the AcrAB-TolC pump, can improve tolerance, but pump expression impacts growth. To address this, we used feedback to dynamically regulate pump expression in response to stress. We developed a library with thousands of synthetic circuit variants and subjected it to three types of pinene treatment (none, constant, and varying pinene). We were able to select for strains that were biofuel tolerant without a significant growth cost in the absence of biofuel. Using next-generation sequencing, we found common characteristics in the designs and identified controllers that dramatically improved biofuel tolerance.
Assuntos
Biocombustíveis/toxicidade , Escherichia coli/metabolismo , Retroalimentação Fisiológica , Engenharia Metabólica , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Liases Intramoleculares/análise , Liases Intramoleculares/biossíntese , Liases Intramoleculares/toxicidade , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Espectrometria de FluorescênciaRESUMO
As a precursor to a large variety of compounds, myo-inositol is a central molecule required for cell metabolism and plant growth. The de novo synthesis of myo-inositol requires the activity of the enzyme D-myo-inositol-3-phosphate synthase (MIPS). MIPS cDNAs encoding one or more isoforms have been cloned from a number of species, nevertheless, little is known about the regulation of MIPS expression in developing seed. Seed-specific expression of a soybean isoform (GmMIPS-1) has been demonstrated, but tissue-specific localization during embryo development has not been reported. Using immunolocalization techniques, a specialized area of GmMIPS-1 expression was identified in the outer integumentary layer during early soybean seed development. In addition, localization data provided evidence that MIPS was associated with oxalate crystal idioblasts.
Assuntos
Glycine max/enzimologia , Liases Intramoleculares/análise , Proteínas de Plantas/análise , Isoformas de Proteínas/análise , Sementes/enzimologia , Oxalato de Cálcio/metabolismo , Liases Intramoleculares/metabolismo , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/metabolismo , Sementes/crescimento & desenvolvimento , Glycine max/embriologiaRESUMO
A cDNA encoding chalcone isomerase (CHI) was isolated from the petals of Nicotiana tabacum and the effect of its suppression on flavonoid biosynthesis was analyzed in transgenic tobacco plants. CHI-suppression by RNA interference (RNAi) showed reduced pigmentation and change of flavonoid components in flower petals. The plants also accumulated high levels of chalcone in pollen, showing a yellow coloration. Our results first demonstrated that suppression of CHI by genetic transformation is possible in higher plants. This suggests that CHI plays a major part in the cyclization reaction from chalcone to flavanone, and that spontaneous reactions are few, if any, in tobacco plants.
Assuntos
Flavonoides/metabolismo , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Nicotiana/enzimologia , Pigmentação , Chalcona/metabolismo , Clonagem Molecular , DNA Complementar/genética , Flavanonas/biossíntese , Flores/enzimologia , Flores/genética , Genes de Plantas/genética , Liases Intramoleculares/análise , Fenótipo , Pigmentação/genética , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , Supressão Genética , Nicotiana/genética , Transformação GenéticaRESUMO
The enzyme isopentenyl pyrophosphate (IPP) isomerase catalyzes the reversible isomerization of IPP to produce dimethylallyl pyrophosphate, the initial substrate leading to the biosynthesis of carotenoids and many other long-chain isoprenoids. Expression of IPP isomerase, and of two enzymes specific to the carotenoid pathway (lycopene beta-cyclase and beta-carotene-C-4-oxygenase), was followed in the green unicellular alga Haematococcus pluvialis after exposure to high illumination. This alga uniquely accumulates carotenoids in the cytoplasm and in late developmental stages turns deep-red in color because of accumulation of ketocarotenoids in the cytosol. The carotenoid/chlorophyll ratio increased 3-fold in wild type and 6-fold in a precocious carotenoid-accumulating mutant (Car-3) within 24 h after increasing the illumination from 20 to 150 micromol photon m-2.s-1. Two cDNAs encoding IPP isomerase in Haematococcus, ipiHp1 and ipiHp2, were identified. Although otherwise highly similar (95% identity overall), the predicted sequence of ipiHp1 contained a 12-aa region not found in that of ipiHp2. This was reflected by a size difference between two polypeptides of 34 and 32.5 kDa, both of which reacted with an antibody to the product of ipiHp1. We suggest that the 32.5-kDa form is involved with the carotenoid accumulation in the cytoplasm, since the 32.5-kDa polypeptide was preferentially up-regulated by high light preceding the carotenoid increase and only this form was detected in red cysts.
