Engineering Pseudomonas putida for efficient aromatic conversion to bioproduct using high throughput screening in a bioreactor

Pseudomonas putida KT2440 is an emerging biomanufacturing host amenable for use with renewable carbon streams including aromatics such as para-coumarate. We used a pooled transposon library disrupting nearly all (4,778) non-essential genes to characterize this microbe under common stirred-tank bioreactor parameters quantitative fitness assays. Assessing differential values by monitoring changes in mutant strain abundance identified 33 gene mutants improved across multiple formats. Twenty-one deletion strains from subset were reconstructed, GacA, regulator, TtgB, ABC transporter, and PP_0063, lipid A acyltransferase. Thirteen roles varying cellular functions evaluated conversion of para-coumarate, heterologous bioproduct, indigoidine. Several mutants, the ΔgacA 35 fold showed 8-fold improvement indigoidine production (4.5 g/L, 0.29 g/g, 23% maximum theoretical yield) para-coumarate source.