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 use a previously engineered cytochrome c enzyme and couple organosilicon-production directly to a reporter expression. Thereby, we are focusing on the “small-molecule binding riboswitch” as proposed underlying mechanism. (citation) 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 luciferaseuntil today and is able to show us a significant output despite using only a small amount of substrate .

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.

Results

The PACE experiments we carried out ranged from 3 to 7 days with a turbidostat volume of about 1.2 l. We observed E. coli generation times of 30 to 40 min.

Results

The PACE experiments we carried out ranged from 3 to 7 days with a turbidostat volume of about 1.2 l. We observed E. coli generation times of 30 to 40 min.

Since the goal was to improve the method reproducibility was prioritized over minimizing the medium consumption and we always used lagoon volumes of 100 ml. The turbidostat volume was then adjustet to the number of lagoons.