In Vivo Evolution of Enzymes and Metabolic Pathways for the Production of Chemicals

The development of efficient microorgansims for the production of chemicals by fermentation relies most of the time on the construction of genetically optimized microorganisms based on a rational metabolic engineering approach. However, improved enzymes or new enzyme activities are frequently needed and it is time and money consuming to obtain them either by rational protein engineering or by the now classical evolutionary methods like error prone PCR or DNA shuffling.

We have developed and patented an efficient in vivo molecular evolution method  to rapidly evolve enzymes or metabolic pathways. The simple principle of this method is to rationally design a microorganism to link the activity of the enzyme(s) or metabolic pathway  to be evolved to the growth of the host organism.  When such a strain is constructed, it is then easy to evolve the enzyme(s) (thanks to the “natural mutation frequency” of living organisms) by selecting for growth improvement in a continuous culture. To highlight efficiency of this approach, we will present five examples where in less than a month improved or new enzymes were successfully obtained. In the context of developing new bioprocesses involving fermentations or bioconversions, we will demonstrate how this method can really speed up the time to market of new “biochemicals”.