(594b) Bioproduction of Aromatic Amines Enabled By Stress-Resistant Bacteria

Pseudomonas putida is a potential chassis for bioproduction due to its high tolerance against aromatic compounds, such as p-aminocinnamic acid (pACA) – a precursor for high-performance polymers synthesis. In this work, we demonstrated that pACA can be produced in the non-model bacteria P. putida. Biosynthesis of pACA was previously challenging in E. coli due to its inherent toxicity. With optimization of enzymes of choice, production of pACA directly from glucose can be improved from a trace-level to more than 100 μM. We then fine-tuned enzyme expression number using CRISPRa/i transcriptional regulation and plasmids with varying copy-number, and therefore improved the pACA titer to 500 μM. Further investigation at different cell density suggested that conversion of pACA peaked later at the stationary stage when the other competing aromatic analogs were consumed. This finding illustrates the potential of the transcriptional CRISPR tools for tuning of gene expression level, which could provide a new methodology to optimize metabolic engineering processes in non-model bacteria.