Targeting riboswitches with synthetic small RNAs for metabolic engineering

Highlights

• •Design and implementation of a new RNA tool to target bacterial riboswitches (rtRNAs).

• •The synthetic rtRNA activates gene expression at the transcription level.

• •The rtRNA is suitable for use in cell-free and cellular systems.

• •Synthetic rtRNAs can be combined into arrays for multiplex riboswitch-targeting.

• •The rtRNA is an easy-to-construct and versatile tool.

Abstract

Our growing knowledge of the diversity of non-coding RNAs in natural systems and our deepening knowledge of RNA folding and function have fomented the rational design of RNA regulators. Based on that knowledge, we designed and implemented a small RNA tool to target bacterial riboswitches and activate gene expression (rtRNA). The synthetic rtRNA is suitable for regulation of gene expression both in cell-free and in cellular systems. It targets riboswitches to promote the antitermination folding regardless the cognate metabolite concentration. Therefore, it prevents transcription termination increasing gene expression up to 103-fold. We successfully used small RNA arrays for multiplex targeting of riboswitches. Finally, we used the synthetic rtRNAs to engineer an improved riboflavin producer strain. The easiness to design and construct, and the fact that the rtRNA works as a single genome copy, make it an attractive tool for engineering industrial metabolite-producing strains.

Introduction

RNAs are important regulatory tools in the synthetic biology toolbox for controlling gene expression and constructing synthetic gene networks (Chappell et al., 2015b; Leistra et al., 2019). Among all regulatory RNAs, trans-acting antisense small RNAs (sRNAs) are particularly attractive to engineer due to their simplicity, fast response to external signals, and usefulness for fine-tuning of gene expression (Shimoni et al., 2007). The simplicity of the RNA sequence and the predictability of RNA folding has powered the development of sRNA rational design to tune gene expression. Moreover, the specificity of base-pairing and easiness to construct make sRNA an ideal tool for circuit design and multiplex control (Bao et al., 2021; Kelly et al., 2018; Noh et al., 2017). Compared to protein-based regulation, sRNAs offer multiple options for the design of orthogonal control with minimal or no metabolic burden associated. Therefore, synthetic sRNAs are simple and versatile tools to regulate gene expression and engineer metabolic pathways.

Synthetic sRNAs have been mostly used as antisense RNAs to block the access of the ribosome to the mRNA and prevent translation in E. coli. sRNA-mediated knockdown has been successfully used to fine-tune gene expression and develop E. coli superproducers of industrially relevant compounds such as tyrosine, proline, cadaverine, putrescine, 4-hydroxycoumarin, resveratrol, and naringenin (Na et al., 2013; Noh et al., 2017; Yang et al., 2015). The strategy has been once used in B. subtilis to engineer a N-acetylglucosamine producer strain (Liu et al., 2014). Now, we expand the functionality of sRNAs to target riboswitches and activate gene expression at the transcription level.

Riboswitches play an important role in the regulation of gene expression in bacteria. In B. subtilis there are 41 identified riboswitches that regulate ∼2% of all genes, many of them related to the biosynthesis of industrially relevant compounds (Kalvari et al., 2021; Mandal et al., 2003). Bacterial riboswitches ultimately regulate the levels of metabolites in the cell through the control of biosynthesis and/or transport processes (Mandal et al., 2003; Mars et al., 2016). Although tempting, deletion of riboswitches aiming to create constitutive expression leads to severe decrease in gene expression (Boumezbeur et al., 2020; Shi et al., 2014). Alternatively, we propose the use of riboswitch-targeting sRNAs (rtRNA) for dynamic control of gene expression.

Riboswitches that bind organic small molecules, such as purines, vitamins, and amino acids, keep the cell homeostasis preventing unneeded accumulation of cellular compounds. In B. subtilis and other bacteria the control exerted by riboswitches is most pronounced during the exponential growth phase when the intense cell metabolism favors biosynthetic processes (Lins et al., 2021; Pedrolli et al, 2012, 2015). The developed rtRNAs act at that time point, interfering with the riboswitch aptamer folding, preventing transcription termination, which leads to metabolite accumulation.