Organosilicons or compounds containing bonds between silicon and carbon and provide completely new structural moieties with altered properties and metabolism. By utilizing a well-known and previously engineered Cytochrome cKan.2016 as a catalyst, it is possible to synthesize carbon-silicon compounds suitable for medical and agricultural applications e.g. in Alzheimer’s disease or as insecticides. In our project, we are focusing on the application of novel organosilicon-forming organisms by evolving enhanced cytochrome c variants. This is implemented by the use of a phage-assisted continuous evolution (PACE) approach. In a stepwise proof of principle design, we can show 1) the production of two different organosilicons analyzed via the GC-MS method and 2) the viability of a riboswitch-coupled reporter system detecting one of the most valuable compounds derived from Organosilicon formation. This proof of principle will lead us to biocatalysts which are environmentally friendly and will greatly contribute to the production of novel carbon-silicon bonds as they are highly efficient.

Introduction

Organosilicons are organometallic compounds that consist of carbon-silicon bonds. They are comparable to their corresponding organic analogs but differ in their intrinsic properties. These differences, especially the chemical properties of silicon and the bond formation tendencies, have a significant impact on their bioavailability and their application in medicine. Recent publications cluster their unique features into three categories: The first category comprises the chemical properties of silicon bonds. Typically, silicon forms longer bonds at different angles, which leads to 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. They are more likely to overcome the membrane barrier of cells as they are more lipophilic compared to their respective carbon counterparts. The third - and most important - category deals with the medical application of these 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 to carbon-based molecules. Additionally, the more electropositive nature of silicon facilitates hydrogen bond formation and conveniently increases the acidity of the compounds. As a result, organosilicons address the major issue in the synthesis of bioactive pharmaceuticals, the design of pro-drugs, as well as a safe medicine with a genuine biomedical benefit. Thus, their main advantage is to operate as pro-drugs due to their thermodynamic stability, but aqueous and acidic instability. On top, as we know so far, silicon is nonhazardous by itself, which makes it a valuable source for further biomedical research.

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-binding riboswitch as proposed underlying mechanismHenkin.2008. This riboswitch was designed in silico using the MAWS software, that was provided by the iGEM Team Heidelberg 2015. In a step-by-step approach, we wanted to produce an organosilicon which could, in the end, be tested with the designed riboswitch to express the NanoLuc reporter (Promega). NanoLuc is the most sensitive luciferase currently today and is able to show us a significant output despite using only a small amount of substrate.

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 (x) and (x) or were custom synthesized by Fabian Ebner (Greb group, ACI Heidelberg, Germany). The corresponding riboswitch was designed accordingly using the MAWS 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 view part was ordered as oligos which were annealed in a single-cycle Touch-Down PCR, decreasing 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).

Riboswitch binding assay

The purified construct was retransformed into DH10beta and incubated at 37°C overnight. To carry out the assay in biological triplicates, three clones were picked and inoculated separately overnight in 5ml LB including chloramphenicol. The assay reaction was performed in a transparent 96-well plate (Greiner) with a final volume of 250µl per well. The light absorbance was measured at 460nm. The background absorbance was determined by measuring 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. Following, the riboswitch activity was detected in the wells that additionally contained either the educt (compound x) as the initial substrate or to the product (x) to which it was specifically designed. Therefore, the reaction efficiency was tested for the compounds (x) and (y) 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

Outlook

By establishing this proof of principle, we aim to further extend the use of organosilicon-producing proteins especially in combination with our PREDCEL approach and to bring silicon to life one big step closer.