Engineering Pseudomonas putida for improved utilization of syringyl aromatics

Lignin is a largely untapped source for the bioproduction of value-added chemicals. Pseudomonas putida KT2440 has emerged as a strong candidate for bioprocessing of lignin feedstocks due to its resistance to several industrial solvents, broad metabolic capabilities, and genetic amenability. Here we demonstrate the engineering of P. putida for the ability to metabolize syringic acid, one of the major products that comes from the breakdown of the syringyl component of lignin. The rational design was first applied for the construction of strain Sy-1 by overexpressing a native vanillate demethylase. Subsequent adaptive laboratory evolution (ALE) led to the generation of mutations that achieved robust growth on syringic acid as a sole carbon source. The best mutant showed a 30% increase in the growth rate over the original engineered strain. Genomic sequencing revealed multiple mutations repeated in separate evolved replicates. Reverse engineering of mutations identified in agmR, gbdR, fleQ, and the intergenic region of gstB and yadG into the parental strain recaptured the improved growth of the evolved strains to varied extent. These findings thus reveal the ability of P. putida to utilize lignin more fully as a feedstock and make it a more economically viable chassis for chemical production.