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Abstract
Acinetobacter baylyi ADP1 degrades many aromatic compounds and has a uniquely tractable genetic system. Recent interest in aromatic compound metabolism stems from diverse biotechnology applications. My research focused on increasing the number and range of aromatic compounds metabolized by A. baylyi to enhance this bacterial host’s ability to convert a heterogenous lignin-derived mixture to a valuable product. The lignin portion of renewable lignocellulosic biomass is currently vastly underutilized. Foreign DNA segments encoding catabolic pathways were inserted in the ADP1 chromosome and amplified in tandem arrays prior to adaptive laboratory evolution. Additional research goals include understanding how metabolic functions become regulated and integrated in a new host. This approach, therefore, explores fundamental aspects of evolution, horizontal gene transfer, and metabolic regulation. In this dissertation, I describe the chromosomal integration and adaptation in A. baylyi of a meta-cleavage protocatechuate degradation pathway from Pseudoalteromonas atlantica. Similar experiments used a codon-optimized pathway for veratrate and isovanillate degradation from Pseudomonas putida. A. baylyi strains able to grow on aromatic compounds using these non-native pathways were isolated and characterized. Whole genome sequencing revealed multiple mutations, and new strains were constructed with different combinations of mutations to determine the significance of genetic changes. A single chromosomal copy of the 7-kbp meta-cleavage pathway was sufficient for growth at 25 C, whereas growth using this pathway at higher temperatures (up to 37 C) occurred only with multiple chromosomal copies. A remarkable expansion of the chromosome initially added as much as 2 Mbp of repeated DNA to the normally 3.6 Mbp chromosome. After long-term culturing, copy number decreased and mutations accrued that increase the growth temperature at which the foreign pathway is functional. Mutations unveiled the importance of a previously undescribed phosphotransferase system, several peptidyl-prolyl isomerases, and the GroE chaperone. In additional studies, I characterized aspartate metabolism and regulation in ADP1. This research was done in conjunction with teaching an undergraduate microbiology research course. All studies focused on metabolic regulation and involved the development and modification of methods that exploit the exceptional efficiency of natural transformation and allelic replacement in ADP1, an ideal model organism for synthetic biology.