Towards evolution-guided microbial engineering - tools development and applications

By enabling sustainable manufacturing of medicines, materials andchemicals, biotechnology and the use of genetically engineeredmicroorganisms promises solutions to many of todays global challenges.Key to the development of new microbial processes is the design,construction, and testing of new high-performing microorganisms.However, an insufficient understanding of biological complexity currentlyrenders strict forward engineering approaches inadequate. Our success forthe development of useful biological systems is therefore largelydetermined by our abilities to generate microbial diversity and evaluate itsphenotypic performance at large-scale.In this thesis I report the development of a number of novel tools -experimental as well as computational - that aim to accelerate theconstruction of new microbial phenotypes and functionally evaluate theirperformance in ultra high-throughput. At the very core of the thesis is thedevelopment of highly robust biosensor-based synthetic selection systemsthat enable high-throughput functional interrogation of complexphenotypic libraries. Using the model organism Escherichia coli as a host, Ideploy these systems to i) perform metagenome wide sequenceindependentidentification of novel microbial vitamin uptake systems, ii)directed protein evolution to elucidate sequence-function relationships ofbacterial small molecule transport systems, and iii) perform sequencephenotypicanalysis of combinatorially engineered metabolic pathwaysresulting in significant productivity improvements of an early-stagethiamine cell factory. Additionally, this thesis presents two advancedgenetic engineering methods that enable rapid combinatorial engineering ofboth plasmid and chromosomal DNA.Overall, the results of this thesis illustrate the benefits of advancing theconstruction and screening toolbox for microbial engineering anddemonstrates direct applications to gene discovery, protein engineering andcell factory development.