Fluxomic, Metabolomic, and Transcriptomic Analyses\nReveal Metabolic Responses to Phenazine-1-carboxamide Synthesized\nin <i>Pseudomonas chlororaphis</i>

Phenazine-1-carboxamide\n(PCN) has been exploited as a successful\nbiopesticide due to its broad-spectrum antifungal activity. We engineered\na PCN-overproducing Pseudomonas chlororaphis strain through overexpressing shikimate pathway genes (<i>aroB</i>, <i>aroQ</i>, <i>aroE</i>, and <i>phzC</i>) and deleting negative regulatory genes (<i>relA</i>, <i>fleQ</i>, and <i>pykF</i>). The optimized strain produced\n1.92 g/L PCN with a yield of 0.11 g/g glycerol, the highest titer\never reported by using minimal media. To gain deeper insights into\nthe underlying regulatory network, the final strain and the parental\nstrain were examined using three distinct omic data sets. <sup>13</sup>C-metabolic flux analysis revealed a substantial flux reconfiguration\nin the optimized strain, including the activation of the EDEMP cycle,\nthe PP pathway, the glyoxylate shunt, and the shikimate pathway. Metabolomic\nresults indicated that central carbon was rerouted to the shikimate\npathway. Transcriptomic data identified global gene expression changes.\nThis study forms the basis for further engineering of strains to achieve\noutstanding performance.