Development of Artificial Riboswitches for Monitoring of Naringenin In Vivo

Abstract

Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing. However, productivity and titer of current recombinant strains still do not meet the requirements of industrial processes. Genetically encoded biosensors have been applied for high-throughput screening or dynamic regulation of biosynthetic pathways for enhancing the performance of microbial strains that produce valuable chemicals. Currently, few protein sensor-regulators for flavonoids exist. Unlike the protein-based trans-regulating controllers, riboswitches can respond to their ligands faster and eliminate off-target effects. Here, we developed artificial riboswitches that activate gene expression in response to naringenin, an important flavonoid. RNA aptamers for naringenin were developed using SELEX and cloned upstream of a dual selectable marker gene to construct a riboswitch library. Two in vivo selection routes were applied separately to the library by supplementing naringenin at two different concentrations during enrichments to modulate the operational ranges of the riboswitches. The selected riboswitches were responsive to naringenin and activated gene expression up to 2.91-fold. Operational ranges of the riboswitches were distinguished on the basis of their selection route. Additionally, the specificity of the riboswitches was assessed, and their applicability as dynamic regulators was confirmed. Collectively, the naringenin riboswitches reported in this work will be valuable tools in metabolic engineering of microorganisms for the production of flavonoids.

RNA-based dynamic genetic controllers: development strategies and applications

“…In vitro and in vivo selections can then be performed to empirically validate each design in a high-throughput manner in an environment where the dynamic RNA controllers should work. The potential of empirical selection should be further explored because the quantitative properties of RNA controllers can be modulated using well-designed in vivo selection schemes [43 ]. Emerging approaches in the development of dynamic RNA controllers will expand the repertoire of genetic parts for applications in metabolic engineering and synthetic biology.…”

“…In vitro selection recently published work utilizing a coupled in vitro-in vivo selection strategy [43 ]. In this study, an aptamer library that binds to a flavonoid, naringenin, was cloned upstream of a dual selectable marker gene.…”

Toehold Switch-Based Genetic Modulator for Tuning of Riboswitch Sensor

“…Previous studies showed that modifying binding kinetics and affinity by directed mutagenesis of aptamer domains of riboswitches shifted the dose-response curves [18][19][20]. Another approach focused on modifying an expression platform, which was also effective for tuning of riboswitches [21,22].…”

Dynamic metabolic control: towards precision engineering of metabolism

“…Common biosensors in dynamic control are transcriptional riboswitches [7] and transcription factors [75]. Progress in RNA engineering has led to a growing number of riboswitches that respond to specific metabolites [66,26]. Studies have shown that RNA sequences shape the sensitivity and threshold of riboswitch dose-response curves [52,5], yet the precise tuning of riboswitch function remains a significant challenge.…”