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Figure 5:

The optimized PREDCEL procedure starts by the initial inoculation with phages carrying CYP1A2. After three hours of incubation the phage supernatant is transferred and inoculated with a fresh PREDCEL culture for three hours. While phage variants with beneficial mutations develop over several transfers, the phage concentration in the flask decreases gradually. To prevent phage washout after only a few rounds of selection and mutation, the last phage supernatant of the day is used to inoculate an over night culture for phage enrichment. The next PREDCEL transfer can then be started with a sufficient phage titer of phage variants carrying the best mutations at this point. By following this protocol, more PREDCEL rounds can be conducted consecutively enabling further optimization of the CYP1A2.

       
        
Results
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. 6).
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 6:

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 7:

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 8:

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.

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