Foundations for genetic engineering in the shikimate pathway of Bacillus methanolicus MGA3

Numerous widely-used chemicals, such as adipic acid, are produced from hydrocarbons that come from petroleum. Consumption of petroleum contributes to global climate change and ocean acidification, which has created a great need for the development of petroleum independent chemical synthesis methods. Recently, considerable interest has turned toward using methanol as a feedstock for chemical synthesis because of its abundance, ease of purification, and lack of ties to the food industry. Adipic acid precursor cis,cis-muconic acid has been biosynthesized in a variety of production hosts, but it has not been biosynthesized using a methanol feedstock. In this work, groundwork was laid for genetically engineering the thermophilic, methylotrophic bacterium Bacillus methanolicus MGA3 for cis,cis-muconic acid production from methanol via the shikimate pathway. The first enzyme in the shikimate pathway is 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase. This enzyme has been shown to play an important role in the regulation of carbon flow into the pathway. B. methanolicus MGA3 DAHP synthase isozymes AroG1 and AroG2 were characterized. Both isozymes were found to be Type Iβ DAHP synthases, with AroG1 being unregulated at the protein level and AroG2 being allosterically regulated by the aromatic amino acid precursors chorismate and prephenate. Additionally, methods for storing and screening B. methanolicus shikimate pathway mutants were developed. The established storage method allows B. methanolicus to be mutagenized in large batches and subsequently stored as spores. Agar plate growth medium recipes were developed for germinating B. methanolicus spores and phenotypic screening of mutants.