Development of a cell-free strategy for the directed evolution of enzymes for high-value natural products

Cell-free protein synthesis (CFPS) provides a robust method for protein production in a system in which all the contained energy can be channelled toward protein synthesis by removing the life goals of the cell. Herein, we sought to produce the haem dependant cytochrome P45enzyme BM3 using a cell-free system as a proof of concept that complex cytochrome P450 enzymes can be studied using this technique and to provide a foundation for further study into directed evolution. To facilitate the production of BM3 in a cell-free reaction, we optimised a CFPS methodology by varying the reaction composition and expressed and characterised the BM3 protein using in vivo methods to act as a positive control. We observed several conditions that affect CFPS activity including, Mg2+, L-glutamate, maltodextrin, and D-ribose. However, while E. coli in vivo production of BM3 was strong, in contrast, the optimised E. coli CFPS system was unable to synthesise BM3. This could be due to several factors, including low promoter strength or low extract activity. In parallel, we also sought to produce a cell-free system for making haem the prosthetic group required for BM3 activity. Therefore, we expressed and characterised the enzymes for cell-free haem biosynthesis. Intriguingly, in an attempt to make haem b, instead, intermediates such as coproporphyrinogen III were formed.

We suggest that either one of the enzymes was limiting (HemH - ferrochelatase), or a co-factor was absent in the reaction. In this research we were unable to synthesise BM3 using CFPS, this is most likely due to the low activity of the extract, by further refining the crude extract production method and the composition of the cell-free reaction it may be possible to produce BM3.