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Abstract
Microbial aromatic compound metabolism has biotechnology applications, including the valorization of lignin, a renewable feedstock. Lignin is underutilized because it is chemically complex and recalcitrant to degradation. No individual microbe consumes all the aromatic components of lignin. In this dissertation, I use synthetic biology tools to integrate foreign DNA and alter metabolic pathways in the soil bacterium Acinetobacter baylyi ADP1. A. baylyi is an ideal chassis because of its genetic malleability. The goal involves developing methods for mixing, matching, and optimizing genetic modules to create novel pathways. Aromatic catabolic pathways can be divided into three general modules: (1) conversion of diverse aromatic compounds into ring-cleavage substrates, (2) ring-opening, and (3) routing ring-cleavage products to central metabolism. For this approach, I use native and nonnative chromosomal genes to enable conversion of aromatic substrates such as 4-hydroxybenzoate, anthranilate, benzoate, and guaiacol to catechol or protocatechuate, substrates for ring-cleavage (module 1). Ring-cleavage is catalyzed by native or nonnative ortho-cleavage dioxygenases or by nonnative meta-cleavage dioxygenases (module 2). Finally, cleavage products are routed to central metabolism using the native catechol branch of the β-ketoadipate pathway or a nonnative Pca 2,3-cleavage pathway which degrades meta-cleavage products (module 3). Modular pathways enable A. baylyi mutants to consume 4-hydroxybenzoate, benzoate, anthranilate, and guaiacol as sole carbon sources via nonnative and novel routes. Mutants were obtained after integrating foreign DNA into different chromosomal regions. In some cases, targeted gene amplification was used to increase expression. Laboratory evolution and selection for faster growth yielded isolates that were characterized by whole-genome sequencing. An important challenge in laboratory evolution is sorting beneficial mutations from neutral or deleterious changes. I demonstrate a new method, Combinatorial Evaluation of Mutations Examined by Natural Transformation (CEMENT), that harnesses the transformability of A. baylyi to determine the necessity and sufficiency of mutations. Significant mutations are discussed here and emphasize the importance of balanced regulation across different modules. Together, this research offers insights into metabolic engineering and evolution of synthetic pathway hosts. Groundwork is laid for building pathways to accomplish transformations that have not been demonstrated in nature by mixing and matching modules from functional pathways.