Fig. 3: Testing a library of E. coli strain engineered for D-lactate production with a P. putida sensor strain.

A Screening high-titer D-lactate producers with a whole-cell biosensor. E. coli engineered for D-lactate production can grow on glucose, which cannot be utilized by the sensor strain (P. putida SENS·L). Assimilation of acetate by strain SENS·L, a major by-product of E. coli, was prevented by deleting aceA. D-Lactate is used by the sensor strain as the only growth substrate, coupling the D-lactate concentration (concn.) secreted by engineered E. coli to the fluorescent (msfGFP) output by P. putida SENS·L. Abbreviations are as indicated in the caption of Fig. 1. B Calibrating the growth and fluorescence output of the sensor SENS·L strain with respect to the D-lactate concn. The sensor strain was incubated in DBM medium supplemented with D-lactate at different concns.; the msfGFP signal was normalized to the cell density and plotted in semi-logarithmic scale over time. C Relationship between maximum msfGFP fluorescence by P. putida SENS·L and the D-lactate concn. The lowest range of analyte concns. ( < 1 mM) is zoomed-in in the inset; the maximum D-lactate concn. quantifiable within the linearity range is highlighted in orange. D Engineering E. coli for D-lactate production from glucose. Product degradation was prevented by deleting dld, and a combinatorial library of constitutive promoters and ribosome binding sites (RBS), regulate the expression of ldhA_PP, a D-lactate dehydrogenase gene from P. putida, was introduced in the E. coli Δdld strain. E Exploiting P. putida SENS·L to screen for high-titer D-lactate producers. E. coli Δdld, carrying the combinatorial ldhA_PP library, was grown on M9 minimal medium with 20 mM glucose; individual culture supernatants were transferred to a new microtiter plate, inoculated with P. putida SENS·L and incubated for 24 h. Normalized fluorescence signals by strain SENS·L are shown for selected promoter and RBS combinations. The culture supernatant of E. coli ∆dld carrying a plasmid with constitutive expression of a mutant version of the ldhA from Bos taurus was used as a positive control (+); the negative control (–) consisted of the same E. coli strain carrying an empty plasmid. In all cases, results represent average values ± standard deviations from three independent experiments. The inset shows the relationship between D-lactate quantification via msfGFP fluorescence with the sensor strain and HPLC for all experimental samples.