Recombinant RNA Polymerase from Geobacillus sp. GHH01 as tool for rapid generation of metagenomic RNAs using in vitro technologies

Abstract

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 -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.

Thermostablein vitrotranscription-translation for enzyme screening in microdroplets

“…For instance, the RNA polymerase the thermophilic Geobacillus sp. GHH01 has been described to be stable and active at elevated temperatures up to 55 °C, recognizing DNA template sequences from a wide variety of organisms, including bacteria and archaea (119), which would contribute to the applicability of thermostable IVTT extracts for recognition of diverse native promoters in the activity-based screening of metagenomic libraries.…”

Thermostable in vitro transcription-translation compatible with microfluidic droplets

“…An example of the latter would be the RNA polymerase of Geobacillus sp. GHH01, which is stable and active up to 55 °C and recognizes DNA template sequences from a wide variety of organisms [ 55 ], which is an asset in the activity-based screening of metagenomic libraries. Also, the addition of helper molecules such as T. thermophilus chaperones DnaK/ClpB and homologs of GroEL [ 56 , 57 ], compatible solutes [ 58 – 60 ] or stabilizers, such as trehalose, may increase the stability of the RNA polymerase.…”

Approaches to Unmask Functioning of the Uncultured Microbial Majority From Extreme Habitats on the Seafloor

“…Although the use of large insert metagenomic libraries has the potential to counteract some of these challenges, problems with heterologous gene expression in the surrogate host, e.g., failed gene expression and incorrect post-transcriptional processing, remain one of the major limitations of functional metagenomic approaches ( Perner et al, 2011b ; Johnson et al, 2017 ). The use of custom expression strains, alternative vector systems, ionic liquids, or even in vitro recombinant transcription systems are promising techniques to mitigate these shortcomings ( Lam et al, 2015 ; Kinfu et al, 2017 ; Mital et al, 2021 ).…”