Shikimic acid (SA) and 3-dehydroshikimate (DHS) are intermediates for synthesizing fine and commodity chemicals including oseltamivir, adipic acid, vanillin, and catechol. Current methods for producing these molecules rely on non-renewable petrochemicals, low-yielding plant extractions, or market-saturated agricultural feedstocks. Data presented in this report encompass multiple efforts to develop alternate routes toward chemicals, including both in vivo and in vitro methods. The first method entails building an economy from agricultural residues utilizing mixed sugars derived from farm waste for SA biosynthesis. The second method builds on an economy based on methanol by metabolizing methanol for DHS biosynthesis.Escherichia coli RB791 strains expressing three different glucose transport systems – one phosphotransferase system (PTS), and two non-PTS – were characterized in a fed-batch fermenter for their abilities to co-utilize glucose and xylose for SA overproduction. Strain YJ1.144/pSC6.090B, expressing transport enzymes GlF and GlK, out-performed a strain expressing its native glucose PTS and a strain evolved to take up glucose via one of its non-native transport systems. YJ1.144/pSC6.090B achieved a high SA titer of 94.9 g L-1 when grown on a 70:30 (w/w) mixture of glucose/xylose. YJ1.144/pSC6.090B achieves lower SA titer (55.6 g L-1) and higher quinic acid titer when grown on sugars derived from a ball milling process, which also contained acetate. The exact mechanism of this inhibition was not characterized. CO2 emission data collected from the fed-batch fermentations motivated a comparative LCA/TEA study. Results of LCA depended on assumptions of allocation. As a waste product, stover contributed 29% more than grain to global warming, 86% less to eutrophication, 96% less to water usage, and 69% less to land usage. With allocation based on economic value of the feedstocks, stover contributed 36% more than grain to global warming and had eutrophication, water usage, and land usage impacts that were over 2-fold higher than that of corn grain. TEA indicated both biorefinery models were profitable. SA sourced from corn grain is more profitable than SA sourced from corn stover. Deriving commodity chemicals from methanol suffered from a lack of a genetic toolset for modifying the genome of the host strain Bacillus methanolicus MGA3. A method was developed for mutagenesis and mutant screening of shikimate pathway auxotrophs in B. methanolicus MGA3. Two strategies were also employed to knockdown shikimate dehydrogenase in MGA3 to accumulate DHS: RNAi (RNA interference) and CRISPRi (CRISPR interference). Quantifiable amounts of DHS have yet to be assayed during the knockout and both knockdown strategies. Results from the knockdown suggest non-mutants are enriched during the mutagenesis procedure. More MGA3 mutants need to be isolated, and/or a more effective knockdown strategy must be developed for accumulating quantifiable amounts of DHS.
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