Final Activity Report Summary - EVOCOMBINE (Evolution of New modules for Combinatorial Biosynthesis)
The goal of this project was to make new enzymes in order to be able to produce new biologically active peptides.
The non-ribosomal peptide synthetases (NRPSs) are enzymes that produce biologically active peptides, like antibiotics and immunosuppressants. They work in a modular fashion, like an assembly line where each module corresponds to one amino acid in the final peptide. One way of producing new peptides with new activities is by changing the building blocks of the peptide. Since it is not known which peptides will be active several combinations of non-natural amino acids should be tried, through a process which is called combinatorial biosynthesis. Changing the peptide structure by exchanging modules has been tried, but did not result in high yields of the new peptide.
Therefore, we chose another method, namely directed evolution. By mimicking evolution in a test tube, we could change the substrate specificity of a module. This evolution process consisted of introducing random mutations and could be directed towards the desired goal either by applying the right selection pressure, i.e. by survival of the fittest, or by screening all available variants.
In this project selection was not possible, so we had to develop a high throughput screening method. The technique had to be high throughput, since we generated at least a million different variants during each evolution step. Firstly, we tried to optimise a continuous spectrophotometric assay. Unfortunately, this assay did not appear to be sensitive enough for our purposes. In a second attempt we tried to optimise the radioactive adenosine triphosphate / pyrophosphate (ATP / PPi) exchange assay for high throughput screening. This assay was much more sensitive and the use of radioactivity was limited by the small volumes. Furthermore, by doing several discontinuous reactions in one plate kinetic parameters of the enzyme could be determined in a very reliable manner. As a result, this assay not only served our goal of developing a sensitive and reliable assay, but could also be used for other scientific purposes.
Since I didn't finish the project I couldn't test a mutant library of an NRPS module; nevertheless, the assay was very well suitable for quick determination of new activities and could be easily used by other people in the field.
The non-ribosomal peptide synthetases (NRPSs) are enzymes that produce biologically active peptides, like antibiotics and immunosuppressants. They work in a modular fashion, like an assembly line where each module corresponds to one amino acid in the final peptide. One way of producing new peptides with new activities is by changing the building blocks of the peptide. Since it is not known which peptides will be active several combinations of non-natural amino acids should be tried, through a process which is called combinatorial biosynthesis. Changing the peptide structure by exchanging modules has been tried, but did not result in high yields of the new peptide.
Therefore, we chose another method, namely directed evolution. By mimicking evolution in a test tube, we could change the substrate specificity of a module. This evolution process consisted of introducing random mutations and could be directed towards the desired goal either by applying the right selection pressure, i.e. by survival of the fittest, or by screening all available variants.
In this project selection was not possible, so we had to develop a high throughput screening method. The technique had to be high throughput, since we generated at least a million different variants during each evolution step. Firstly, we tried to optimise a continuous spectrophotometric assay. Unfortunately, this assay did not appear to be sensitive enough for our purposes. In a second attempt we tried to optimise the radioactive adenosine triphosphate / pyrophosphate (ATP / PPi) exchange assay for high throughput screening. This assay was much more sensitive and the use of radioactivity was limited by the small volumes. Furthermore, by doing several discontinuous reactions in one plate kinetic parameters of the enzyme could be determined in a very reliable manner. As a result, this assay not only served our goal of developing a sensitive and reliable assay, but could also be used for other scientific purposes.
Since I didn't finish the project I couldn't test a mutant library of an NRPS module; nevertheless, the assay was very well suitable for quick determination of new activities and could be easily used by other people in the field.