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Recombinant RNA Polymerase from Geobacillus sp. GHH01 as tool for rapid generation of metagenomic RNAs using in vitro technologies
Author(s) -
Kinfu Birhanu M.,
Jahnke Maike,
Janus Mareike,
Besirlioglu Volkan,
Roggenbuck Michael,
Meurer Richard,
Vojcic Ljubica,
Borchert Martin,
Schwaneberg Ulrich,
Chow Jennifer,
Streit Wolfgang R.
Publication year - 2017
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26436
Subject(s) - rna polymerase , biology , transcription (linguistics) , t7 rna polymerase , metagenomics , polymerase , recombinant dna , rna , computational biology , dna , gene , microbiology and biotechnology , biochemistry , escherichia coli , bacteriophage , linguistics , philosophy
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 His 6 ‐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.