Discovery and characterisation of broad-host-range transcriptional terminators for use in solventogenic Clostridia

The bacterial butanol hyper-producer Clostridium saccharoperbutylacetonicum is of interest to industrial biotechnology and efforts are underway to engineer strains of this species and related solventogenic Clostridia species for traits such as improved tolerance and production capabilities. Recent synthetic biology engineering efforts in model organisms have been underpinned by the availability of genetic tools such as characterized biological parts. Intrinsic transcriptional terminators are crucial to the normal functioning of both natural and synthetic genetic systems but have not been thoroughly investigated in C. saccharoperbutylacetonicum. In order to study terminators in this species, two pairs of matched reporters of gene expression were explored and tested – two enzymatic (GusA and LacZ) and two fluorescent (mCherry and phiLOV2.1Opt), with the dual enzymatic system emerging as the most robust for medium-throughput use. Computational terminator prediction approaches and a terminator strength prediction model developed were extensively utilized and evaluated to construct a library of Clostridium terminators, which was combined with a library of known terminators used in other model systems. The broad-host-range dual enzymatic reporter vectors were used to assess the strength of these terminators in two model organism bacterial species - Escherichia coli and Bacillus subtilis - as well as in C. saccharoperbutylacetonicum. Terminators that function effectively in C. saccharoperbutylacetonicum were discovered; some terminators exhibited species-specific efficiency while others had broad-host-range activity. Together with the low predictive power of the strength prediction model, these observations suggest that important determinants of terminator activity are still to be determined. The dual reporter system has the potential for use in the screening of larger libraries of terminators to investigate the underlying mechanistic features for future applications in synthetic biology.