Fig. 2: Sensing protocatechuate in a bacterial co-culture.

A P. putida SENS was grown in DBM minimal medium with varying concentrations (conc.) of protocatechuate (PCA). The msfGFP fluorescence was continuously monitored during growth and is plotted in semi-logarithmic scale. Abbreviation: AU, arbitrary units. B Correlation between final fluorescence signals and supplemented PCA. The inset shows the regression at low PCA concerns. (50 μM to 1 mM, R² = 0.97). C Principle of the co-culture for detection of PCA produced by cell factories. A P. putida strain engineered for PCA biosynthesis (P. putida PCA) consumes glucose and produces PCA, while the sensor strain is unable to utilize sugars, but grows on the PCA secreted by the producer strain. P. putida PCA has several competing pathways knocked out (ppc, pyk, pykA, pcaG, and pcaH), rate-limiting enzymes are overproduced (TkTA, AroQ, and QuiC), and the aroF-I gene was modified to remove feedback inhibition. Abbreviations: 6PG, 6-phosphogluconate; PEP, phosphoenolpyruvate; Pyr, pyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; CMA, β-carboxy-cis,cis-muconate; EDP, Entner-Doudoroff pathway; PPP, pentose phosphate pathway; TCA cycle, tricarboxylic acid cycle. D msfGFP fluorescence in co-cultures of the producer strain (P. putida PCA) and the sensor strain (P. putida SENS) in DBM medium with 20 mM glucose. In control experiments, the producer strain and wild-type P. putida KT2440 were individually grown in the same conditions. Wild-type P. putida KT2440 was also co-cultured with the SENS strain, which could not grow under these conditions. In all cases, results represent average values ± standard deviations from three independent experiments.