Yannik Brack

• This dissertation comprises a systematic process for exploring and analysing aromatic ammonia lyases (AALs) via phylogenetic tree building. AALs can be used in medical as well as in industrial applications, for example to produce p-coumaric acid (p-CA). In general, there are two pathways to obtain p-CA. One is the phenylalanine ammonia lyase (PAL)-cinnamate 4-hydroxylase (C4H) pathway, encompassing two steps from L-phenylalanine. Due to the great challenges in the expression of plant-sourced P450 C4H in E. coli, the main focus was on the second pathway named as tyrosine ammonia lyase (TAL) pathway, which entails the one-step deamination of L-tyrosine. However, one of the main drawbacks of the TAL pathway is the generally high pH optimum of TALs, posing challenges for the use in a synthetic microbial consortium (SMC). Thus, with the aim to discover better performing TALs or phenylalanine/tyrosine ammonia lyases (PTALs), the phylogenetic tree building approach was used in Article I. After a BLAST search of the UniParc database, 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases were identified. Through further investigation of 15 enzymes, eight novel enzymes (TALs and PTALs) were characterized. Two of these enzymes showed high specific activity towards L-tyrosine, and one of these enzymes showed an unusually low optimum pH of 8.5. Articles II and III systematically analysed the sequences of AALs, focusing on their substrate switch motifs and MIO moieties, respectively. Article II identified new residue combinations at the substrate switch motif which lead to a substrate change from L-tyrosine to L-phenylalanine in TAL. Article III uncovered numerous enzymes with divergent MIO-forming residues as well as structurally similar enzymes without a MIO moiety. New clusters with unclassical or no MIO forming residues were discovered and the characterization of six novel enzymes (two histidine ammonia lyases, one phenylalanine ammonia mutase, and three ergothionases) was performed. Overall, the efficient TALs from Article I were applied to optimize p-CA production in an E. coli strain, which has been utilized in a SMC for flavonoid production. Articles II and III provide novel insights into critical positions of AALs, paving the way for future protein engineering strategies.