Introduction

Organosilicons are organometallic compounds that contain carbon-silicon bonds. In comparison to their respective organic analogs, they display different intrinsic properties due to the distinct chemical properties of silicon. The bond formation tendencies of silicon have a significant impact on their bioavailability and their applicability in pharmacyFranz.2013.
Recent publications cluster their unique features into three categories: The first category comprises the chemical properties of silicon bonds. Typically, silicon forms longer bonds with variable angles, which allows diverse ring conformations and thus alterations in reactivity. Furthermore, its preference to form single bonds leads to chemical compounds that have a higher intrinsic stability than their carbon analogs.
The second category represents the bioavailability of organosilicons. As they are more lipophilic compared to their respective carbon counterparts, they are able to easily overcome the membrane barrier of cells. The third - and most important - category addresses the potential pharmaceutical application of carbon-silicon compounds. Due to their aforementioned tendency to form single rather than double or triple bonds, they display a viable source for stable pharmaceuticals, which are inaccessible as carbon-based molecules. Additionally, the more electropositive characteristic of silicon facilitates hydrogen bond formation and conveniently increases the acidity of compounds. Considering these facts, organosilicons offer major opportunities in the synthesis of bioactive pharmaceuticals, the design of pro-drugs, as well as a safe medicine with a genuine biomedical benefit.

Recently, a cytochrome c variant was described, which is able to catalyze the formation of C-Si bonds (Referenz) and therefore offers the opportunity to further develop C-Si bond catalyzing enzymes.

Our Idea

According to our idea to evolve proteins by PACE and PREDCEL, we also envisioned to further develop the previously engineered cytochrome c. Therefore, we linked organosilicon-production directly to a reporter expression via a small molecule-sensing riboswitchHenkin.2008. This riboswitch was designed in silico using the MAWS 2.0 software, that was provided by the iGEM Team Heidelberg 2015. To obtain the riboswitch sequence, the chemical structure of the desired product was in silico aligned to randomly generate RNA sequences which were scored according to their ability to form hydrogen bonds with the product. The expression was evaluated using the NanoLuc reporter provided by Promega.

Experimental procedures

Design and cloning of the riboswitch and cytochrome c constructs

The educts for the organosilicon synthesis were commercially available in the case of dimethyl(phenyl)silane (2) and ethyl 2-diazopropanoate (5) or were custom synthesized by Fabian Ebner (Greb group, ACI Heidelberg, Germany) in the case of 4-(dimethylsilyl)aniline (1). The corresponding riboswitch was designed accordingly using the MAWS 2.0 software developed by the iGEM Team Heidelberg 2015. The most favorable sequence (BBa_K2398555) was ordered as oligos, which were annealed in a single-cycle Touch-Down PCR, decreasing the temperature by 0.1°C x sec^-1 from 95°C to 10°C. The sequence was ordered as oligos and not as gBlock to ensure overhangs of a specific length at the 5’ and 3’ ends. Vector and reporter were amplified via PCR and purified by gel extraction (Qiagen). The final plasmid was assembled by using equimolar concentrations of vector, reporter, and the riboswitch in a golden gate reaction. The plasmid was transformed into DH10beta cells and purified by plasmid purification (Qiagen).
To make organosilicon production more accessible for other iGEM Teams, we codon optimized the wild-type cytochrome c derived from Rhodotermus marinus and cloned it into the pSB1C3 vector. We are proud to present you this part as our best basic part (BBa_K2398000). For our purpose, we used a triple mutant created by F. ArnoldKan.2016, that showed a higher activity towards our substrates.

Riboswitch binding assay

The purified construct was re-transformed into DH10beta and incubated at 37°C overnight. Three clones were picked for biological triplicated and inoculated separately overnight in 5ml LB including chloramphenicol. The assay was performed in a transparent 96-well plate (Greiner) with a final volume of 250µl per well (fig. 3). The light absorbance was measured at 460nm. The background absorbance was determined by measuring separately medium only, medium including cells, and medium including cells and the Nano-Glo substrate. The cell concentration was adjusted to a final OD_600nm of 0.6 per well. The riboswitch activity was detected in wells that additionally contained 5mM or 15mM of either the educt (2) as the initial substrate or the intended product (3), respectively. The reaction was incubated at room temperature for 10min without the Nano-Glo substrate. Afterwards, the Nano-Glo substrate was added to each well (125µl) to ensure its maximum reactivity. The light absorbance was measured immediately using the Tecan Infinite M200 Pro plate reader.

Results

Synthesis of the organosilicon compounds

The compounds ethyl 2-((4-aminophenyl)dimethylsilyl) propanoate (3) and ethyl 2-(dimethyl(phenyl)silyl) propanoate (4) were successfully synthesized, validated by gas chromatography-mass spectrometry (GC-MS) and the conversion rate was determined (Fig. 5-8). Figure 4 shows the ethyl 2-((4-aminophenyl)dimethylsilyl)propanoate product with a retention time corresponding to 11.7 minutes. The silicon educt 4-(dimethylsilyl)aniline that emerges at 6.9 minutes implies that there is an incomplete conversion due to enzyme inactivity or an unfavorable reaction. The value of the conversion rate is 47.5%, which differs from the literature value of 70%Kan.2016. Nonetheless, the amount of the product formed was sufficient for all further experiments. The unconverted diazo educt ethyl 2-diazo propanoate corresponds to the retention time peaks at 7.2 and 7.4 minutes. The small peak at 10.8 minutes is likely a side product of the reaction. Figure 6 shows the respective mass spectrum of the breakdown of the product and its correct mass of 251 Dalton.
The GC-MS analysis of the second organosilicon ethyl 2-(dimethyl(phenyl)silyl)propanoate is demonstrated in the Figure 6 and 7. The educt dimethyl(phenyl)silane was completely converted, and the product emerges at a retention time of 9.2 minutes (Fig. 8). The mass spectrometry analysis verified the product with a mass of 236 Dalton (Fig. 9). The reaction without the enzyme represents our negative control and is depicted in Figure 9. As expected, only the two educts emerged after their respective retention time.

In summary, sufficient concentrations of the organosilicon products were synthesized, and in case of the organosilicon (3) also purified to >98% chemical purity.

Validation of the Riboswitch

We further tested our synthesized compounds by using the NanoLuc Reporter linked to our riboswitch. As expected, the addition of 15 mM of the compound (3) resulted in a higher emission rate compared to the sample containing only 5 mM. Remarkably, the specifically designed compound (3) for the riboswitch showed significantly higher activity compared to the original precursor compound (1), indicating a higher binding affinity (Fig. 10). The increase in enzyme activity upon addition of the specific riboswitch activator was determined in relation to the enzyme activity in presence of the initial substrate. The result, demonstrated in Figure 11, shows a 1.5 fold enzyme activity when using the newly synthesized substrate.

Outlook

We were able to synthesize two organosilicons and investigated their purity using GC-MS. Employing the (MAWS) 2.0 software, we designed to our knowledge the first organosilicon dependent riboswitch, and validated its specificity using a luciferase reporter assay. Together with our improved directed evolution method (PREDCEL) and our Cytochrome Engineering approach, our organosilicon part paves the way towards an in vivo directed evolution of enzymes, and brings us closer to the application of organosilicons in pharmacy. In addition, we provide a general circuit that allows an easy design for the evolution of in principle any enzyme based on riboswitches mediated by positive and negative selection (Fig. 12). A pool of pre-selected enzyme isoforms are provided by the software tool AiGEM. In the case of a positive selection, the desired product binds to the riboswitch that was specifically designed to be activated by it. The subsequent conformational change then enhances phage propagation via the increased expression of the geneIII. Consequently, the enzyme that most efficiently forms the product of interest is enriched over time in a positive selection process. In contrast, to ensure that enzymes that synthesize undesired products are not propagated, negative selection is conducted. Therefore, riboswitches mediate a negative geneIII expression upon product binding. The negative geneIII prevents the further phage propagation and thus, decreases the abundance of all enzymes not specifically forming the desired productCarlson.2014. In any case, directed evolution is driven towards the enzyme of interest.