Engineering CatM, a LysR-Type Transcriptional Regulator, to Respond Synergistically to Two Effectors

Abstract

The simultaneous response of one transcriptional regulator to different effectors remains largely unexplored. Nevertheless, such interactions can substantially impact gene expression by rapidly integrating cellular signals and by expanding the range of transcriptional responses. In this study, similarities between paralogs were exploited to engineer novel responses in CatM, a regulator that controls benzoate degradation in Acinetobacter baylyi ADP1. One goal was to improve understanding of how its paralog, BenM, activates transcription in response to two compounds (cis,cis-muconate and benzoate) at levels significantly greater than with either alone. Despite the overlapping functions of BenM and CatM, which regulate many of the same ben and cat genes, CatM normally responds only to cis,cis-muconate. Using domain swapping and site-directed amino acid replacements, CatM variants were generated and assessed for the ability to activate transcription. To create a variant that responds synergistically to both effectors required alteration of both the effector-binding region and the DNA-binding domain. These studies help define the interconnected roles of protein domains and extend understanding of LysR-type proteins, the largest family of transcriptional regulators in bacteria. Additionally, renewed interest in the modular functionality of transcription factors stems from their potential use as biosensors.

Engineering and application of a biosensor with focused ligand specificity

“…In cases where biosensor parameters, such as sensitivity or operational range do not fit the requirements of a certain application, these can be adapted by promoter engineering as it has been reported for a cis,cis-muconic acid biosensor in Saccharomyces cerevisiae 27 . In addition, the selectivity of the TR controlling reporter gene expression in response to the presence of the molecule(s) of interest can be expanded or shifted [12][13][14][15][16] . However, in this study, we showed that TRs can also be engineered toward a more focused ligand spectrum without changing other biosensor characteristics, which is of importance when a biosensor-based screening campaign yields only false-positive variants accumulating a different biosensor-detectable metabolite.…”

“…No other TR as part of a biosensor has been studied in such structural detail, even though the LTTR LysG is a member of the largest known family of prokaryotic TRs comprising >40,000 proteins plus many more functionally orthologous proteins identified in archaea and eukaryotic organisms (Interpro Entry: IPR000847) 20,31 . Since they can act either as activators or repressors of single or operonic genes and are known to accept a broad range of chemically diverse ligands, it is no surprise that many different LTTR-based biosensors have been constructed 16,27,32 . The strategy for altering ligand-binding properties of LTTRs along with a detailed understanding of the ligand-binding mode of these TRs presented in this study should spur interest in developing custom-made LTTRbased biosensors.…”

“…More recently, the repressor of the lac operon, LacI, was engineered to also accept D-fucose, lactitol, sucralose, or gentiobiose 15 . CatM of Acinetobacter baylyi ADP1, involved in the degradation of aromatic compounds, was also altered to accept benzoate 16 . However, only a few of these adapted biosensors were used for larger-scale screening campaigns, probably because biosensor properties, such as weak ligand binding or low reporter gene expression prohibited implementation of FACS-based screenings 9 .…”

Transcriptional factor engineering in microbes for industrial biotechnology

“…Tumen-Velasquez et al carried out a structure-based protein design and demonstrated that the capability of responding to multiple signals could be integrated. 42 Reconstructing the ancestral TFs Ancestral sequence reconstruction (ASR) based on relevant sequence alignment is a method initially invented in the molecular evolution research. 43 Multiple sequence alignment (MSA) is a fundamental way to gain information that could not be obtained by analyzing any individual sequence included in alignment.…”

Special Issue: Genetics of Biodegradation and Bioremediation

“…Durante-Rodríguez et al [7] documented the regulation of an anaerobic benzoate biodegradation pathway by identifying the bzdR repressor gene in different Azoarcus/Aromatoleum strains and characterising the repressor binding and repressor complex formation at the target promoter-regulatory region. The relevance of the specificity of the regulatory protein for the response to a particular inducer and how this specificity may be tuned to create variants that respond synergistically to several effectors is shown by Tumen-Velasquez et al [8] by comparing the effector specificity of two very similar LysR-type activators, the CatM and BenM paralogs in Acinetobacter baylyi ADP1. Finally, the sophisticated regulatory system consisting of three regulatory circuits that control the expression of the tetralin biodegradations genes is reviewed by Floriano et al [3].…”