Sungyeon Jang

Development of Artificial Riboswitches for Monitoring of Naringenin In Vivo

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

Dynamic regulation of gene expression in response to various molecules is crucial for both basic science and practical applications. RNA is considered an attractive material for creating dynamic genetic controllers because of its specific binding to ligands, structural flexibility, programmability, and small size. Here, we review recent advances in strategies for developing RNA-based dynamic controllers and applications. First, we describe studies that re-engineered natural riboswitches to generate new dynamic controllers. Next, we summarize RNA-based regulatory mechanisms that have been exploited to build novel artificial dynamic controllers. We also discuss computational methods and high-throughput selection approaches for de novo design of dynamic RNA controllers. Finally, we explain applications of dynamic RNA controllers for metabolic engineering and synthetic biology.

Design and optimization of genetically encoded biosensors for high-throughput screening of chemicals

Evolutionary engineering of microbes for the production of metabolites requires efficient screening methods to test vast mutant libraries. Genetically encoded biosensors are regarded as promising screening devices owing to their wide range of detectable ligands and great applicability to high-throughput screening and selection. Here, we reviewed the current progress in design and optimization of biosensors for high-throughput screening of chemicals. First, we summarized genetic parts of biosensors and strategies for their discovery and development. Next, we explained the properties of biosensors that are relevant to high-throughput screening. Finally, we described various methods for tuning biosensors to fulfill requirements of an efficient screening.

Molecular parts and genetic circuits for metabolic engineering of microorganisms

Microbial conversion of biomass into value-added biochemicals is a highly sustainable process compared to petroleum-based production. In this regard, microorganisms have been engineered via simple overexpression or deletion of metabolic genes to facilitate the production. However, the producer microorganisms require complex regulatory circuits to maximize productivity and performance. To address this issue, diverse genetic circuits have been developed that allow cells to minimize their metabolic burden, overcome metabolic imbalances and respond to a dynamically changing environment. In this review, we briefly explain the basic strategy for constructing genetic circuits by assembling molecular parts such as input, operation and output modules. Next, we describe recent applications of the circuits in the metabolic engineering of microorganisms to improve biochemical production. Beyond those achievements, genetic circuits will facilitate more innovative approaches to future strain development through mining and engineering new genetic elements and improving the complexity of genetic circuit design.

Toward tunable dynamic repression using CRISPRi

CRISPR interference (CRISPRi) is widely utilized for regulation of target gene expression by repressing transcription. Simple design rules for the single guide RNA (sgRNA) and multiplexity won this method immense popularity. However, quantitative control of the expression levels at varying degrees in a dynamic manner using CRISPRi has been regarded difficult. To deal with this limitation, Fontana et al. modulated the expression levels of the components of CRISPRi, the deactivated Cas9 (dCas9), and the sgRNAs, using various constitutive or inducible promoters (Fontana et al., Biotechnol. J. 2018, 13, 1800069). They found that the expression level of sgRNA is the key to controlling CRISPRi. Modulation of sgRNA expression levels enabled quantitative tuning of the CRISPRi-regulated gene expression level. This approach is expected to be easily applied to diverse applications owing to its simplicity compared to the conventional approaches that modified target sequence or changed the expression level of dCas9.

Novel Hybrid Input Part Using Riboswitch and Transcriptional Repressor for Signal Inverting Amplifier

Genetic circuits are composed of input, logic, and output parts. Construction of complex circuits for practical applications requires numerous tunable genetic parts. However, the limited diversity and complicated tuning methods used for the input parts hinders the scalability of genetic circuits. Therefore, a new type of input part is required that responds to diverse signals and enables easy tuning. Here, we developed RNA-protein hybrid input parts that combine a riboswitch and orthogonal transcriptional repressors. The hybrid inputs successfully regulated the transcription of an output in response to the input signal detected by the riboswitch and resulted in signal inversion because of the expression of transcriptional repressors. Dose-response parameters including fold-change and half-maximal effective concentration were easily modulated and amplified simply by changing the promoter strength. Furthermore, the hybrid input detected both exogenous and endogenous signals, indicating potential applications in metabolite sensing. This hybrid input part could be highly extensible considering the rich variety of components.