Development of a Metabolite Sensor for High-Throughput Detection of Aldehydes in Escherichia Coli

Abstract

We have developed a fluorescence-based metabolite sensor enabling in vivo detection of various aldehydes of biotechnological interest in Escherichia coli. YqhC is a transcriptional regulator that is known to be involved in the upregulation of the yqhD-dgkA operon in the presence of aldehydes. We took advantage of this property by constructing a bi-modular biosensor, in which a sensing module constitutively expresses yqhC while a reporter module drives the expression of the syfp2 reporter gene that is put under control of the yqhD promoter. The sensitivity of the sensor has been optimized by engineering the 5′-UTRs of both the sensing and the reporter modules resulting in a 70-fold gain of fluorescence in response to the model compound glycolaldehyde at 5 mM. The optimized sensor further responded to other aldehydes when supplemented to the cultivation medium at concentrations of 1–10 mM. We furthermore showed that this metabolite sensor was functional in vivo as it responded to the presence of glycoladehyde that is specifically produced upon induction of a synthetic xylulose-1-phosphate pathway expressed in E. coli. This bi-modular sensor can therefore be employed as an exquisite tool for FACS-based ultra-high-throughput screening of aldehyde (over) producing enzymes.

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

“…As observed on the EMSA assays, Van2 responds to vanillin and was still responsive to Other genetically encoded biosensors for aromatic aldehydes have been reported [66][67][68][69][70][71]. Among transcriptional based biosensors, a system based on the BldR aTF of archea Sulfolobus solfataricus was developed in E. coli for the detection of benzaldehyde [66].…”

“…A more recent study generated a biosensor in E. coli based on the native YqhC aTF for the detection of aliphatic and aromatic aldehydes including glycoaldehyde and vanillin [71]. The…”

Construction of a synthetic pathway for the production of 1,3-propanediol from glucose

“…Generation and screening of larger mutant libraries appears to be required for obtaining OHB decarboxylase as well as DHB dehydrogenase enzymes with adequate activities to solve this issue and favor the DHB-PDO production pathway. In this regard, our recently developed transcription-based aldehyde sensor 36 , which also responded to the PDO pathway intermediate 3-hydroxypropanal, could be an exquisite tool for in vivo screening of more efficient OHB decarboxylases as well as enzyme(s) mutant libraries generated of upstream reaction steps in the pathway.…”

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

“…Other genetically encoded biosensors for aromatic aldehydes have been reported [69–74]. Among transcription factor-based biosensors, a system based on the BldR aTF of archea Sulfolobus solfataricus was developed in E. coli for the detection of benzaldehyde [69].…”