C1 Compound Biosensors: Design, Functional Study, and Applications

Abstract

The microbial assimilation of one-carbon (C1) gases is a topic of interest, given that products developed using this pathway have the potential to act as promising substrates for the synthesis of valuable chemicals via enzymatic oxidation or C–C bonding. Despite extensive studies on C1 gas assimilation pathways, their key enzymes have yet to be subjected to high-throughput evolution studies on account of the lack of an efficient analytical tool for C1 metabolites. To address this challenging issue, we attempted to establish a fine-tuned single-cell–level biosensor system constituting a combination of transcription factors (TFs) and several C1-converting enzymes that convert target compounds to the ligand of a TF. This enzymatic conversion broadens the detection range of ligands by the genetic biosensor systems. In this study, we presented new genetic enzyme screening systems (GESSs) to detect formate, formaldehyde, and methanol from specific enzyme activities and pathways, named FA-GESS, Frm-GESS, and MeOH-GESS, respectively. All the biosensors displayed linear responses to their respective C1 molecules, namely, formate (1.0–250 mM), formaldehyde (1.0–50 μM), and methanol (5–400 mM), and they did so with high specificity. Consequently, the helper enzymes, including formaldehyde dehydrogenase and methanol dehydrogenase, were successfully combined to constitute new versatile combinations of the C1-biosensors.

Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus

“…The DNA fragment of pET/tac-Lxmdh was amplified by PCR using Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA), and was then assembled by the Blunting Kination Ligation method to construct pET/tac-Lxmdh. pFrm-GESS, a formaldehyde biosensor plasmid, was obtained from a previous study [ 19 ].…”

“…In particular, some TF-based biosensors to detect methanol (MxaYZ-TCS) have been developed for screening C1-converting enzymes and for developing biochemical-producing recombinant microorganisms [ 18 ]. Previously, we developed formaldehyde-detectable genetic enzyme screening system (Frm-GESS) that can be applied for the direct evolution of Mdh based on FACS screening systems [ 19 ]. This TF-based screening system was highly appropriate as a screening technique to engineer C1-converting enzymes such as Mdh.…”

Strategies for Improving Small-Molecule Biosensors in Bacteria

“…In this work, the authors added exogenous metabolic enzymes to Escherichia coli cells that mediated the conversion of target analytes into detectable ligands for existing biosensors, producing strains capable of sensing cocaine, nitroglycerin, chlorpropham, 2-chloro-4-nitrophenol, parathion, and hippurate [ 77 ]. Other groups have since utilized metabolic enzymes in a similar fashion, producing biosensor strains to detect a variety of compounds, including lignin [ 78 ], glycerate [ 79 ], phenylalanine [ 80 ], and methanol [ 81 ]. In addition to metabolic enzymes, biosensors have also been constructed using aminoacyl tRNA synthetase (aaRS) proteins, a class of enzyme that catalyzes ligation between an amino acid ligand and its cognate tRNA, which leads to incorporation of the amino acid into proteins.…”

Sensitive and Rapid Phenotyping of Microbes With Soluble Methane Monooxygenase Using a Droplet-Based Assay

“…Recent advances in synthetic biology have enabled the establishment of genetically engineered biosensors for highthroughput screening of biocatalysts by converting enzyme activity into a fluorescence signal upon transcriptional activation (van der Helm et al, 2018). Few biosensors have been developed for engineering methanotroph-derived enzymes (Rohlhill et al, 2017;Selvamani et al, 2017;Lee et al, 2019). However, the target ligands were cellular metabolites such as methanol and formaldehyde, which are rapidly assimilated by methanotrophs with limited extracellular transport.…”