Part:BBa_K2992036
Acetone pathway: cb_PntnH-5-UTR+RBS-ca_thl-ca_ctfAB-cp_TFdx
Botulinum toxin-predicting acetone production pathway with C. botulinum PntnH driving expression of C. acetobutylicum-derived ctfAB, thl and adc.
Usage and Biology
This parts entry represents an acetone-production pathway for plasmid-borne expression in C. sporogenes for predicting botulinum neurotoxin production. The entry comprises the thiolase gene thl BBa_K2992008 and acetoacetate decarboxylase adc gene BBa_M36585 coupled with the two units of the ctfAB complex BBa_M36581 and BBa_M36582 which are all derived from C. acetobutylicum.This operon is regulated by the promoter BBa_K2992001 and associated 5’UTR+RBS BBa_K2992015 from the non-toxic non-haemagglutinin ntnH gene of C. botulinum. Transcriptional termination for this synthetic acetone-production operon occurs through the activity of Tfdx from C. pasteurianum BBa_K2284012. In our project we used genome-scale modelling to predict the necessary genes required to produce acetone in our chosen surrogate strain C. sporogenes. We sought to link acetone production with C. botulinum neurotoxin production by the integration of the neurotoxin transcriptional regulator botR onto the chromosome of C. sporogenes and by using promoter regions from the regulon of botR to control the acetone-production operons. In doing so, we hoped to generate our surrogate host strain as a model for predicting neurotoxin production in foodstuffs following food manufacturing processes.
Characterisation
Before assessing the ability of our chosen acetone production pathways to generate acetone in our C. sporogenes reporter strains, we first assessed promoter activity using our FAST reporter BBa_K2992002. We compared promoter activities of our chosen promoters PbotR BBa_K2992012 and PntnH BBa_K2992001 alongside three constitutive clostridial promoters Pfdxc114t BBa_K2992016, Pfdxt114c BBa_ K2715011 and Pthl BBa_ K2715010 and the E. coli promoter J23106 BBa_ J23106. The plasmids were cloned upstream of the FAST reporter gene and ligated into pMTL82151 plasmids. FAST reporter assays were conducted on both E. coli and C. sporogenes lysates following transfer of genetic material thereto.
In the C. sporogenes experiments, adequate expression was detected for each of the clostridial promoters chosen for study. The two Pfdx derivatives generated the greatest level of reporter activity whilst the two C. botulinum promoters generated much lower levels of activity. Reporter activity appeared to be generally higher when analysed from the E. coli lysates as opposed to the C. sporogenes lysates. In those experiments, activity from the PbotR and Pntnh constructs were considerably greater than the no promoter control.
Having established the functionality of our chosen promoters. We next assessed the feasibility of using these to drive acetone production in our reporter strains. In our design approach, we rationalized that either C. botulinum BBa_K2992003. BBa_K2992007 BBa_K2992005 or C. acetobutylicum-derived ctfAB [BBa_M36581. BBa_M36582, encoding the A and B subunits of the butyrate-acetoacetate CoA-transferase complex, should permit acetone production. To test this, we transformed pMTL82151 plasmids encoding our acetone pathways using C. botulinum ctfAB BBa_K2992029 or C. acetobutylicum ctfAB BBa_K2992036, both under the control of PntnH, into our PbotR-botR reporter strain of C. sporogenes.
The data clearly indicates that ctfAB from C. acetobutylicum was much better suited to providing acetone production capacity to our C. sporogenes reporter strains. As a consequence, we selected the acetone production pathway cb_PntnH-5-UTR+RBS-ca_thl-ca_ctfAB-cp_TFdx BBa_K2992036 to assess acetone production in all of our promoter-botR genome integrant strains. The construct was cloned into pMTL82151 and transformed into our various promoter-botR strains and controls.
The data demonstrated appreciable acetone production of >2nM concentration when using either the native PbotR promoter and associated 5’-UTR+RBS or the RBS only construct to permit polar transcription from PpyrKDE. Considerable acetone production (4-6nM) was observed when using the constitutive clostridial promoter Pfdx. Crucially, acetone production was comparably scant when botR was absent from the genome of C. sporogenes and when no promoter was used to drive expression of the acetone production operon. These data provide experimental validation for the production of acetone in C. sporogenes as a model for Botulinum toxin prediction in foodstuffs.
Sequence and Features
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References
Heap Modular Cornillo et al 1997
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