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Theophylline Riboswitch - GeneIII for the Evolution of Enzymes

BBa_K2398019: Theophylline Riboswitch - GeneIII for the Evolution of Enzymes

One of our main goals was to facilitate for the in vivo directed evolution of small molecule binding enzymes. The concept behind this idea is the use of riboswitches for the activation of geneIII. To prove our idea, we made use of a theophylline binding riboswitch in relation with the Enzyme CYP1A2, which catalyzes the reaction of caffeine to theophylline. As we could show that the principle of enzyme evolution with the help of riboswitches works, our best composite part is the theophylline riboswitch, followed by geneIII and flanked ba homology regions, which make it compatible with our cloning standard.


Figure 1: Selection process for improved CYP1A2 variants in directed evolution experiments
The evolutionary circle starts by M13 phages injecting their genome (SP) into bacterial cells, already containing two additional plasmids, AP and MP. The SP encodes CYP1A2 among genes (except geneIII) that are crucial for phage propagation. If through MP activation mutations in the CYP1A2 gene lead to improved CYP1A2 variants the intracellular level of theophylline increases. Theophylline molecules activate the theophylline riboswitch on the AP and thereby enhance geneIII expression. The assembled phages containing the improved CYP1A2 variant can leave the cell and propagate by infecting new cells.
Our evolution circuit for cytochrome engineering works as follows: Bacteriophages infect bacterial cells by introducing their genome. The genome encodes a Selection Plasmid (SP) that contains the human CYP1A2 variant and all necessary components for virus propagation except geneIII. The Accessory Plasmid (AP) codes for geneIII driven by a Psp-tet promoter and contains a riboswitch, located between the promoter and geneIII, which regulates the expression rate of geneIII. The riboswitch on the AP is only active if theophylline reaches a certain concentration within the bacterium. If CYP1A2 is active, caffeine is converted to theophylline. Gradually the theophylline concentration increases and acts upon the riboswitch, which enables expression of geneIII. Additionally, the AP encodes for the chaperone HDJ-1, which is essential to receive the functional CYP1A2 enzyme (Fig. 1).
In a first step, we wanted to validate our AP. Therefore, we added theophylline with a concentration of 100 µM to our inoculated culture and performed two rounds of PREDCEL. Afterwards, we determined the phage titers by plaque assays. Our theophylline treated culture displayed approximately two times higher phage titers than the non-treated control culture. Using the same experimental conditions, but replacing the theophylline treatment by a 300 µM caffeine treatment, we verified the functionality of CYP1A2 and thus of our SP. If caffeine is added to the culture, CYP1A2 catalyzes the reaction from caffeine to theophylline. The resulting increase of the theophylline concentration further activates the riboswitch on the AP and phage propagation is stimulated (Fig. 2). For the evolution of proteins via PREDCEL the addition of a Mutagenesis Plasmid (MP) is essential. For our cytochrome engineering approach we have chosen MP4, which induces a medium mutation rate badran2015development. After six iterations of our optimized PREDCEL workflow, we performed plaque assays and sequenced single plaques. The sequenced plaques showed five recurrent mutations demonstrating that we are able to induce mutations with our experimental setup and that we are able to evolve enzymes (Fig. 7).
Figure 2:
Plaque assays performed after two passages of CYP1A2 PREDCEL with either adding 100 µM theophylline or 300 µM demonstrating the functionality of the Accessory Plasmid and the Selection Plasmid, respectively. Adding theophylline increases the geneIII expression 2-fold. Adding caffeine enhances the conversion of caffeine to theophylline and thus increases the geneIII expression as well.
Figure 3:
Sequencing results of eight plaques after 6 iterations of the PREDCEL workflow with MP4 illustrating recurring mutations of the CYP1A2 gene. Recurrent mutations with amino acid exchange are indicated in red, without amino acid exchange in orange. Single mutations with amino acid changes are shown in yellow, and without amino acid changes in blue.
Figure 4:
Cell lysates were used for high-performance liquid chromatography to distinguish theophylline (left peak) from caffeine (right peak). This assay allows a quantification of the educt and the product of CYP1A2 and thus a conclusion about the conversion efficiency can be made.