Iterative genome engineering of E. coli for fed-batch production of L-tyrosine.

In this project we aim to construct a tyrosine-producing E. coli strain through iterative steps of genome engineering. High PEP availability through knockout of the PTS was combined with the precise, in-place genomic integration of several engineering interventions, known to increase L-tyrosine production yields, to create a tyrosine-overproducing E. coli strain that can function as a platform for further engineering and optimization. Utilizing a design-build-test-learn (DBTL) cycle, an evolved pts-knockout E. coli strain was equipped with optimizations of the aroG, aroB and tyrA genes and cultivated under batch and fed-batch conditions. Subsequently, metabolomics, transcriptomics and proteomics samples from the fed-batch experiments were analyzed to inform the design of new genomic interventions. Overall design: An E. coli strain engineered to produce high titers of tyrosine (SDT387) and a reference E. coli strain (SDT400), which only bears a PTS knockout, were grown under fedbatch conditions, after a short batch phase. Each of the strains was grown in two separate reactors and samples for RNA-seq were taken at four timepoints. The first sample represents the batch phase, whilst the three subsequent samples were taken early, mid, and late in the fed-batch phase of the experiment.