Screening and selection: Applications for vitamin biosynthesis in Escherichia coli

The chemicals industry provides the foundation of our modern lifestyle, but it has far reaching consequences for the planet. It is no longer insider knowledge that industrial biotechnology can offer more sustainable solutions to production via fermentative biosynthesis. Nevertheless, acceptance of fermentation processes is contingent on a single factor: cost-effectiveness. Therein lies the challenge of bacterial strain engineering: to generate a cell factory capable of producing the target molecule more efficiently than a highly optimised chemical process.
Rerouting and redesign of bacterial metabolism has become a complex skill, yet rational strain engineering is insufficient to garner the required concentrations of target molecule. Random and permutatory mutagenesis can be utilised to find mutations that will increase target titers but come with a new challenge: testing thousands of mutated strains for production capacity.
In this thesis, we construct systems that can offer solutions to the “problem of screening” for biotin biosynthesis in Escherichia coli. After a brief introduction to our target molecule biotin with particular emphasis on papers published in the last two years, we review the relevant literature on screening and selection. We then develop a fluorescent output in E. coli cells based on biotin availability, and adapt it for co-encapsulation in alginate beads. Further, we reroute biotin metabolism in such a way that strain can only survive if high concentrations of the target molecule are available and use this to identify high production variants. Finally, we address the problem of false positives with a targeted construct to sieve out evolutionary cheaters.