Team:Heidelberg/Organosilicons
Organosilicons
Synthesis of organosilicons and cytochrome engineering
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 pharmacyRecent 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
As a proof-of-principle, we wanted to show and harness the potential of organosilicon-forming proteins. Therefore, we used a previously engineered cytochrome c enzyme and coupled organosilicon-production directly to a reporter expression. Thereby, we were focusing on a small molecule-sensing riboswitch as proposed underlying mechanism
Figure 1:
Our workflow for organosilicon production and cytochrome engineering.
Figure 2:
3D structure of the cytochrome c derived from Rhodotermus marinus (PDB) that is used as the catalytic unit in the production of organosilicon. Depicted as yellow sticks is the heme prosthetic group of the electron carrier protein. The protein part of the cytochrome c is illustrated as orange ribbon structure.
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 compound 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 compound 4-(dimethylsilyl)aniline (1). The corresponding riboswitch was designed accordingly using the MAWS 2.0 software developed by the iGEM Team Heidelberg 2015. To obtain the riboswitch sequence, the chemical structure of the desired product was in silico aligned to randomly generated RNA sequences which were scored according to their ability to form hydrogen bonds with the product.The most favorable sequence (BBa_K2398555) was ordered as oligos which were annealed in a single-cycle Touch-Down PCR, diminishing the temperature by 0.1°C x sec-1 95°C to 10°C. The sequence was ordered as oligos and not as gBlock to ensure overhangs of a specific length and sequence at the 5’ and 3’ ends. The vector and reporter were amplified via PCR and purified by gel extraction (Qiagen). The final plasmid was assembled by using an equimolar concentration of the vector, reporter, and the riboswitch in a golden gate reaction. The plasmid was amplified after transformation in DH10beta cells and purified via plasmid purification (Qiagen). To make organosilicon production more approachable 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 desired best basic part (BBa_K2398000). For cloning purposes, we ordered the optimized version as gBlock containing a biobrick prefix and suffix for restriction cloning. We were using this part as a triple mutant, created by F. Arnold
Riboswitch binding assay
Figure 3:
The riboswitch binding assay in a 96-well plate immediately after substrate addition. Rows show biological triplicates (n = 3) and riboswitch activators are compound (3) ethyl 2-(dimethyl(phenyl)silyl)propanoate and compound (1) dimethyl(phenyl)silane.
The assay reaction 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 OD600nm of 0.6 per well. Following, the riboswitch activity was detected in the wells that additionally contained either the educt (2) as the initial substrate or the product (3) to which it was specifically designed.
Therefore, the reaction efficiency was tested for the compounds (2) and (3) using either 5mM or 15mM concentrations, respectively. The reaction mixtures were incubated at RT for 10min without the Nano-Glo substrate. After the incubation, the Nano-Glo substrate was added fresh to each well (125µl) to ensure its maximum reactivity. The light absorbance was immediately measured using the Tecan Infinite M200 Pro plate reader.
Results
Synthesis of the organosilicon compounds
Following the successful synthesis of the two compounds (3) and (4), they were subjected to gas chromatography-mass spectrometry (GC-MS) for the analysis of validation and calculation of the conversion rate.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 no complete conversion, possibly due to enzyme inactivity or an unfavorable reaction. Indeed, the conversion rate is 47.5%, which differs from the literature value of 70%
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. The successful product synthesis was further confirmed by mass spectrometry. Fig. 5 shows the mass spectrum of the breakdown of the product and its correct mass of 251 Dalton.
Figure 4:
Gas chromatogram for the reaction of educt (1) and (5) to the product (3). 11.7 minutes retention time, indicates product formation. Unconverted educts converge 6.9 and 7.2, 7.4 minutes
Figure 5:
Mass chromatogram shows the breakdown of the product (3) ethyl 2-((4-aminophenyl)dimethylsilyl)propanoate. The product itself corresponds to a mass of 251 dalton
Figure 6:
Gas chromatogram for the reaction of educt (2) and (5) to the product (4). 9.2 minutes retention time indicates product formation.
Figure 7:
Mass chromatogram shows the breakdown of the product (4) ethyl 2-(dimethyl(phenyl)silyl)propanoate. The product itself corresponds to a mass of 236 dalton
The reaction without the enzyme present served as negative control and is depicted in Fig. 8. As expected, only the two educts emerged after their respective retention time and no product was obtained.
Conclusively, satisfactory concentrations of the products could be achieved and, in case of the organosilicon (3), it could be purified to >98% chemical purity.
The emission rates detected at 460nm were plotted as indicated in Fig. 9. The conditions of each reaction are specified below their respective bars. All measurements are already subtracted for the background emission. Interestingly, the addition of 15 mM of the compound (3) resulted in a higher emission rate compared to the sample containing the 5 mM concentration. As the compound (3) was specifically designed for the riboswitch it was striking to detect a significantly higher emission rate compared to the original precursor compound (1), indicating a higher binding affinity. Notably, these results were very reproducible along biological triplicates, as suggested by the small error bars.
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.
This is demonstrated in Fig. 10 that shows the 1.5 fold enzyme activity when using the newly synthesized substrate.
Figure 8:
Gas chromatogram for the control reaction where the enzyme has been substituted.
Figure 9:
Light emission detection of the NanoLuc reaction for different riboswitch activators and concentrations. Addition of compound (3) to the reaction resulted in increased enzyme activity as indicated by the two bars on the left-hand side compared to the compound (1) reaction.
Figure 10:
Fold enzyme activity upon addition of compound (3) compared to compound (1) at either 5 mM concentration (left bar) or 15 mM concentration (right bar). At both concentrations, compound (3) exceeded the reaction activity of compound (1) by 1.5 fold.
Outlook
Figure 11:
The selection of any small molecule synthesis is implemented into the product formation drive PREDCEL approach. The selection pressure is applied by the most efficient riboswitch binding and subsequent gene expression activation.
A pool of preselected enzyme isoforms generated by the software tool AiGEM manufactures a wide variety of products. In the case of the positive selection, the desired product binds to the riboswitch that was specifically designed to be activated by it. The subsequent conformational change then facilitates phage propagation via the enhanced expression of the gene III. Thus, the enzyme that most efficiently forms the product of interest is enriched over time in a positive selection process. To ensure that enzymes that synthesize undesired products are not propagated, negative selection is conducted. Thereby, different riboswitches mediate the negative gene III expression upon product binding. The negative gene III inhibits the further phage propagation and thus, decreases the abundance of all enzymes not specifically forming the desired product
