Part:BBa_K2398000

Cytochrome c for the synthesis of organosilicons

We present a codon-optimized version of the cytochrome c protein derived from Rhodotermus marinus. This protein is able to perform the conversion of organosilicons. It is a novel catalytic unit that allows utilization of silicon compounds [Arnold et al., 2016]. When in its mature form, it catalyzes a carbene insertion into silicon-hydrogen bonds.

Usage and Biology

The cytochrome c is used for the catalysis of silicon compounds. This cytochrome c variant provides an easy to use tool that is accessible to everyone in the synthetic biology community and allows the user to harness the vast potential of organosilicons. This basic part exhibits a strong tendency to form silicon-carbon bonds and is, therefore, a valuable addition to perform controlled organic chemistry in microorganisms. A triple mutant of this part has already been applied in the successful synthesis of organosilicons as a proof-of-concept. As a next step, this part can be implemented in the directed evolution approach of phage-assisted continuous evolution (PACE) or in the phage-related discontinuous evolution (PREDCEL) approach to improve organosilicon synthesis by cytochrome engineering.

Characterisation

For the characterization of this part, we conducted experiments for the production of organosilicons and therefore used a previously described triple mutant already engineered by [Arnold et al., 2016]. According to the protocol, (ref) we obtained the matured cytochrome c and performed conversion experiments analyzed via GC-MS method. The following figures show the results obtained with the previously engineered enzyme. First, we tested the enzyme on the conversion of the phenylsilane compound to the final product of

Figure 1: Used educts are depicted at the top while produced products are depicted on the bottom

Figure 2: 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 at 6.9 and 7.2, 7.4 minutes

Figure 3: 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 4: Gas chromatogram for the reaction of educt (2) and (5) to the product (4). 9.2 minutes retention time indicates product formation.

Figure 5: 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 93
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]