Engineering and application of a biosensor with focused ligand specificity

Abstract

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

Fig. 1. Functional principle of the biosensor pSenLys and structure of its TR-component LysG.

a Schematic representation of the pSenLys biosensor for the intracellular detection of basic amino acids in C. glutamicum and phase-contrast/fluorescence microscopy images of C. glutamicum cells carrying pSenLys. In the presence of elevated intracellular concentrations of any of the three basic amino acids l-lysine, l-histidine, or l-arginine, the transcriptional activator LysG binds the respective inducer amino acid and activates expression of the reporter gene eyfp. As a result, cells of C. glutamicum show fluorescence. b Cartoon representation of the LysG homodimer in complex with the effector l-arginine. The DNA-binding domains (DBD) of the compact (light blue) and extended (blue) protomers dimerize to form a winged helix-turn-helix motif responsible for DNA binding. l-arginine bound in the regulatory domain (RD) of the extended protomer is shown as pink stick model. c Coordination of l-arginine in the ligand-binding site of the RD. The RD is shown in cartoon representation; bound l-arginine and selected residues of the binding pocket later targeted for pairwise mutation are shown in stick representation.

Fig. 2. FACS-based positive/negative screening to identify an l-lysine-insensitive biosensor.

a FACS plots obtained during the five-step screening/counterscreening campaign for identifying l-lysine-insensitive l-histidine biosensor variants. During this procedure, fluorescent cells responding to l-His-l-Ala dipeptides were collected during positive screening, alternating with a collection of nonfluorescent cells during negative screening in the presence of l-Lys-l-Ala dipeptides. During the last positive screening, 96 individual clones were sorted for further characterization. b Initial characterization of the identified L-lysine-insensitive biosensor variant. Fluorescence response of the biosensors pSenLys (top) and pSenHis (bottom) to the presence of various dipeptides at different concentrations during microtiter plate cultivations. In each case, the specific fluorescence as ratio of the fluorescence determined after 22 h over the culture backscatter (as measure for cell density) is shown. All data represent mean values from three biological replicates including standard deviations.

Fig. 3. Microscale cultivations of C. glutamicum ΔlysEG carrying pSenLys or pSenHis.

At left: growth of cells in microfluidic chambers in the presence of l-His-l-Ala dipeptides, l-Lys-l-Ala dipeptides, or no dipeptides. At right: distribution of the corresponding fluorescence as measure of the biosensor response at the single-cell level across microcolonies.

Fig. 4. Distribution of open and closed conformations of LysG and LysG-A219L binding domains.

a The 2 µs simulation of LysG and 2 µs simulation of LysG-A219L. Both samples have an open (blue) and closed (tan) conformation of the ligand-binding domain, where openness is defined by distance >10 Å between atoms 96 Cα and 219 Cα. It is proposed that the open conformation corresponds to an eyfp fluorescence promoting sensor. b Three predominant hydrogen bridges between l-lysine and the L219 linker stabilize the closed conformation in LysG-A219L. c Wedge function of LysG that forces the open conformation of the regulator to remain stable. d Distributions of open (blue) and closed (tan) conformations as sampled in simulations for TRs LysG (two times 1 µs—black (from crystal conformation) and magenta (from open start conformation) dots) and LysG-A219L (two times 1 µs—magenta and black dots), and for complexes of both sensors with ligands l-lysine and l-histidine in the respective binding site (four times 250 ns). For l-arginine, joint distance distributions from two starting configurations are shown (four times 250 ns—black (from crystal conformation) and magenta (from rotated ligand conformation) dots).

Fig. 5. Amino acid substitutions in the ATP-phosphoribosyltransferase (HisG).

Location of the amino acid substitutions in the allosteric l-histidine binding site in HisG of C. glutamicum. The structure model for HisG of C. glutamicum was built based on the HisG crystal structure of the closely related Mycobacterium tuberculosis (PDB code: 1NH8) using SWISS-MODEL.