RESUMO
The exciting promises of functional metagenomics for the efficient discovery of novel biomolecules from nature are often hindered by factors associated with expression hosts. Aiming to shift functional metagenomics to a host independent innovative system, we here report on the cloning, heterologous expression, and reconstitution of an RNA polymerase (RNAP) from the thermophilic Geobacillus sp. GHH01 and in vitro transcription thereafter. The five genes coding for RNAP subunits, a house keeping sigma factor and two transcription elongation factors were cloned and over expressed as His6 -tagged and/ or tag-free proteins. Purified subunits were reconstituted into a functional polymerase through either the classical method of denaturation and subsequent renaturation or through a new resource and time efficient thermo-reconstitution method which takes advantage of the subunits' temperature stability. Additionally, all subunits were cloned into a single vector system for a co-expression and in vivo reconstitution to the RNAP core enzyme. Both the core and holoenzyme form of the RNAP exhibited a robust transcription activity and were stable up to a temperature of 55°C close to their fullest activity. The Geobacillus RNAP showed a remarkable in vitro transcription profile recognizing DNA template sequences of diverse bacteria and archaea as well as metagenomic samples. Coupled with a subsequent in vitro translation step, this recombinant transcription system could allow a new, clone-free, and functional metagenomic screening approach.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Melhoramento Genético/métodos , Geobacillus/genética , Metagenoma/genética , RNA/biossíntese , Proteínas Recombinantes/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , RNA/genética , Proteínas Recombinantes/metabolismoRESUMO
Foliar application of micronutrients (e.g. Fe3+ ) onto plants over an extended time is challenging and often not possible due to insufficient rainfastness. Smart delivery systems which enable micronutrient release over several weeks would offer innovative and sustainable options to improve plant health and food production. Herein, we report a novel foliar fertilizer delivery system based on functional pH-responsive biohybrid microgels that have orthogonal functionality as carriers of micronutrients and employ peptides (termed anchor peptides) as foliar adhesion promoters. The anchor peptides bind to hydrophobic surfaces and the waxy "islands" of plant leaves. Our system requires no auxiliaries and is loadable, storable, and applicable from aqueous dispersion. We report the synthesis and functionalization of microgels, their loading with Fe3+ ions, and a proof of concept for the biofunctional microgel-based fertilizer system is demonstrated for iron-deficient cucumber plants.