Pseudomonas putida KT2440 has been given increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose
however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows strain growth and muconate production. In this work, we employed adaptive evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, deletion of hexR, gntZ, and gacS improved performance at similar conditions, with the resulting strain achieving 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.