Selection of biocatalysts for chemical synthesis

Abstract

To determine whether microbial chemosensors can be used to find new or better biocatalysts, we constructed Escherichia coli hosts that recognize the product of a biocatalytic conversion through the transcriptional activator NahR and respond by expression of a lacZ or tetA reporter gene. Equipped with a benzaldehyde dehydrogenase (XylC from Pseudomonas putida), the lacZ-based host responded to the oxidation of benzaldehyde and 2-hydroxybenzaldehyde to the corresponding benzoic acids by forming blue colonies, whereas XylC- cells did not. Similarly, the tetA-based host was able to grow under selective conditions only when equipped with XylC, enabling selection of biocatalytically active cells in inactive populations at frequencies as low as 10(-6).

Fig. 1.

Overview of the biocatalyst selection system.

Fig. 2.

Reporter vector pVSF2-lacZ and selector vector pVSF2-tet^r carry lacZα and tetA fused to the salicylate promoter. The divergently and constitutively expressed NahR mutant regulates expression of the reporter and selector genes.

Fig. 3.

Effect of various concentrations of benzoate (a) and 2-hydroxybenzoate (b) on the growth of DH10B-pVSF2-tet^r. Under nonselective conditions, DH10B-pVSF2-tet^r is able to grow in the absence of an inducer (○), whereas, under selective conditions, an inducer is required for growth (♦). Error bars indicate SDs. Note that the tetracycline concentrations for the benzoate and 2-hydroxybenzoate assays were 22.5 and 25 μg/ml, respectively.

Fig. 4.

Effect of various concentrations of benzaldehyde (a) and 2-hydroxybenzaldehyde (b) on the growth of cells containing an active enzyme. Under nonselective conditions, cells with (○) and without (•) active enzyme are able to grow. Growth under selective conditions requires an active enzyme and an aldehyde substrate. Cells containing an active enzyme are able to grow upon addition of benzaldehyde or 2-hydroxybenzaldehyde (□), whereas cells lacking an active enzyme fail to grow in the presence of benzaldehyde or 2-hydroxybenzaldehyde (♦). Error bars indicate SDs. Note that the tetracycline concentrations for the benzaldehyde and 2-hydroxybenzaldehyde assays were 22.5 and 25 μg/ml, respectively

Fig. 5.

Selection of biocatalytically active cells from a background of inactive cells. Biocatalytically active cells that contain the active enzyme XylC were mixed with inactive cells in a ratio of 1:10⁵. Approximately 10⁷ cells were plated and incubated on nonselective (Upper) and selective (Lower) plates. Ten randomly chosen colonies from the selective plate were tested for XylC; all contained the plasmid.