Exploring the Potential of Networked Directed Evolution Based on Novel LacI/effector Pairs

Understanding of the evolution of functional proteins remains a daunting challenge, despite intense research efforts in basic and applied research. Nature's ability to create functional proteins is still unrivalled, raising the question in which respect we can can improve biomimetic efforts in directed evolution to make laboratory evolution more powerful. One key difference is the context in which evolution occurs: laboratory evolution usually deals with enzymes as single species, but natural enzymes are typically embedded in regulated networks and interdependent pathways. This project attempts to build simple networks in which expression of a coupled reporter marks E. coli cells that successfully express a new, functional protein and renders them selectable. This approach could expand the reaction classes that are accessible for directed evolutino, as current laboratory evolution is primarily targeted at functions that are assayed directly, e.g. by generating a fluorescent product. We hope to develop a method that will make directed evolution more versatile and efficient, but also provide insight into how network regulation might have contributed to natural evolution.

A novel approach to directed evolution will be set-up in which small networks that involve novel LacI/effector pairs act as mediators in reporting the success of selections identifying hits. This principle will be used for the identification and evolution of LacI/effector pairs. These pairs are then used to carry out enzyme selections, using a generic GFP-reporter system that will expand the scope of directed evolution beyond selections that generate fluorescent prodct directly. The enhancement afforded by a feedback loop is hoped to make this system suitable for the evolution of very weak activities that currently fall below the detection limit of conventional evolution systems such as colony screening.

The skills, methods and results generated in this project will first be important for companies and research institutions that engage in protein or metabolic engineering ranging from small biotech start-ups to large pharmaceutical companies. These include, for example, Novozymes, MedImmune, Novacta, Vernalis, Danisco and many others. Such companies may directly use the novel approaches developed in this project, but they will also be looking for skilled staff for introducing these methods. Thus the postdoctoral workers to be employed in this project will receive training that will give them an excellent position to join smaller biotech start-ups or larger pharmaceutical companies. Several members of the Hollfelder group are already working in biotech and pharma companies.
Although this is fundamental, basic research this project has long-term use for the general public and businesses. Currently many industrial, fine chemical, therapeutic and molecular biology processes could be made more efficinet and 'greener' by use of tailor-made enzymes, but only a limited number of transformation can be assayed at sufficiently high-throughput. Our approach could change this situation and provide a more general evolution system, that makes many more enzymes accessible for evolutionary improvements.