PartC: catalysis optimisation

For this part of the project, Phil and Corinna will try to increase the efficiency of the DCMation project. In order to be able to test for the presence of dcmA, we will optimise a coupled enzymatic assay of two reactions:

(1) DCM + H2O -> formaldehyde + 2HCl

(2) formaldehyde + NAD+ -> formate + NADH + 2H+

where (1) is catalysed by dcmA, and (2) is catalysed by formaldehyde dehydrogenase. The change in the redox state of the NAD cofactor is easily observed in a spectrophotometer, and thus allows us to indirectly measure the catalytic activity of dcmA.

We will also attempt to improve the catalytic efficiency of dcmA by directed evolution. This is achieved using hypermutagenic PCR to introduce random point mutations. Some of these can lead to a higher turnover and thus increase the efficiency of dcmA.

Finally, we will introduce microcompartments into both E. coli and P. putida, and target the enzymes required for DCM metabolism into these microcompartments. DCM metabolism has previously been reported to be toxic to E. coli and P. putida, thus making these otherwise lab-friendly strains unsuitable for DCM bioremediation. We are hoping to improve this by the use of microcompartments. We have therefore designed several plasmids, including a plasmid that contains dcmA fused to the sfGFP-gene as a control and one that has a microcompartment-tag fused to the N-terminus of dcmA:

We will also insert a gene that encodes formaldehyde-dehydrogenase, fdhA, into one of these plasmids. This will ensure that the product of DCM-dehalogenation, formaldehyde, is converted into formate, which is much less harmful for the cell.

Equivalent constructs of the ones described for E. coli have been made for P. putida. However, as the microcompartment is not reported to have been expressed in Pseudomonas previously, we have an additional construct for P. putida which allows us to express microcompartment-tagged GFP. This will be used in conjunction with the microcompartment vector to ensure that the microcompartment is successfully expressed in P. putida.