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.

Directed Evolution Methods for Enzyme Engineering

“…This in vivo transcription factor then provides a fluorescent signal proportional to the amount of the analyte present by transcribing a fluorescent protein and can be used to monitor the activity of a biosynthetic enzyme which produces the analyte of interest. This concept has been recently demonstrated by Della Corte et al [ 55 ], among others. Meister et al [ 56 ] use a variation which involves the in vivo expression of a fluorescent protein fused to an aggregation inducing peptide separated by a protease site.…”

Engineering of Synthetic Transcriptional Switches in Yeast

“…Besides, Umeno et al argued that TFs are intrinsically evolvable; therefore, mutants that are responsive to non-native compounds can be reached within a few directed evolution cycles (89). Indeed, dozens of new or evolved genetic switches (biosensors) based on bacterial TFs have been reported within the last 2 years (1,90,91,92,93,94,95,96,97,98). Library creation guided by machine-learning technology combined with deep mutagenesis and extensive sequencing technology will further accelerate the discovery of novel TF mutants (98).…”

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

“…These techniques have, inter alia, enabled the construction of genome reduced strains such as C. glutamicum C1*, CR099, and their derivatives, which have emerged as a valuable chassis for basic research and industrial development [49,50]. Other interesting developments have involved the use of biosensors to translate intracellular product levels into optical outputs to screen for superior phenotypes [51][52][53]. Evolutionary approaches have provided strains with elevated tolerance to industrial processing environments, including high temperatures [54] and oxidative stress [55], which are important from a production standpoint.…”