Strains of Escherichia coli were constructed and evaluated that synthesized cis,cis ‐muconic acid from d ‐glucose under fed‐batch fermentor conditions. Chemical hydrogenation of the cis , cis ‐muconic acid in the resulting fermentation broth has also been examined. Biocatalytic synthesis of adipic acid from glucose eliminates two environmental concerns characteristic of industrial adipic acid manufacture: use of carcinogenic benzene and benzene‐derived chemicals as feedstocks and generation of nitrous oxide as a byproduct of a nitric acid catalyzed oxidation. While alternative catalytic syntheses that eliminate the use of nitric acid have been developed, most continue to rely on petroleum‐derived benzene as the ultimate feedstock. In this study, E. coli WN1/pWN2.248 was developed that synthesized 36.8 g/L of cis , cis ‐muconic acid in 22% (mol/mol) yield from glucose after 48 h of culturing under fed‐batch fermentor conditions. Optimization of microbial cis,cis ‐muconic acid synthesis required expression of three enzymes not typically found in E. coli . Two copies of the Klebsiella pneumoniae aroZ gene encoding DHS dehydratase were inserted into the E. coli chromosome, while the K. pneumoniae aroY gene encoding PCA decarboxylase and the Acinetobacter calcoaceticus catA gene encoding catechol 1,2‐dioxygenase were expressed from an extrachromosomal plasmid. After fed‐batch culturing of WN1/pWN2.248 was complete, the cells were removed from the broth, which was treated with activated charcoal and subsequently filtered to remove soluble protein. Hydrogenation of the resulting solution with 10% Pt on carbon (5% mol/mol) at 3400 kPa of H ₂ pressure for 2.5 h at ambient temperature afforded a 97% (mol/mol) conversion of cis , cis ‐muconic acid into adipic acid.

Summary: Genome‐scale reconstructions of metabolism are computational species‐specific knowledge bases able to compute systemic metabolic properties. We present a comprehensive and validated reconstruction of the biotechnologically relevant bacterium Pseudomonas putida KT2440 that greatly expands computable predictions of its metabolic states. The reconstruction represents a significant reactome expansion over available reconstructed bacterial metabolic networks. Specifically, i JN1462 (i) incorporates several hundred additional genes and associated reactions resulting in new predictive capabilities, including new nutrients supporting growth; (ii) was validated by in vivo growth screens that included previously untested carbon (48) and nitrogen (41) sources; (iii) yielded gene essentiality predictions showing large accuracy when compared with a knock‐out library and Bar‐seq data; and (iv) allowed mapping of its network to 82 P . putida sequenced strains revealing functional core that reflect the large metabolic versatility of this species, including aromatic compounds derived from lignin. Thus, this study provides a thoroughly updated metabolic reconstruction and new computable phenotypes for P . putida , which can be leveraged as a first step toward understanding the pan metabolic capabilities of Pseudomonas .