Developing a Riboswitch-Mediated Regulatory System for Metabolic Flux Control in Thermophilic Bacillus methanolicus

Abstract

Genome-wide transcriptomic data obtained in RNA-seq experiments can serve as a reliable source for identification of novel regulatory elements such as riboswitches and promoters. Riboswitches are parts of the 5′ untranslated region of mRNA molecules that can specifically bind various metabolites and control gene expression. For that reason, they have become an attractive tool for engineering biological systems, especially for the regulation of metabolic fluxes in industrial microorganisms. Promoters in the genomes of prokaryotes are located upstream of transcription start sites and their sequences are easily identifiable based on the primary transcriptome data. Bacillus methanolicus MGA3 is a candidate for use as an industrial workhorse in methanol-based bioprocesses and its metabolism has been studied in systems biology approaches in recent years, including transcriptome characterization through RNA-seq. Here, we identify a putative lysine riboswitch in B. methanolicus, and test and characterize it. We also select and experimentally verify 10 putative B. methanolicus-derived promoters differing in their predicted strength and present their functionality in combination with the lysine riboswitch. We further explore the potential of a B. subtilis-derived purine riboswitch for regulation of gene expression in the thermophilic B. methanolicus, establishing a novel tool for inducible gene expression in this bacterium.

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol

“…Therefore, heterologous overexpression of ribR in B. methanolicus could be beneficial for increased riboflavin overproduction by this organism. Recently, Irla et al have shown for the first time that the putative lysine riboswitch detected through in silico analysis was in fact functional in B. methanolicus , indicating the potential of this type of approach (Irla et al, 2021 ). Further steps should be taken to improve the precursor supply and inhibit by‐product formation to develop strains with higher riboflavin production titres for the methanol‐based production of riboflavin.…”

Efficient cell factories for the production of N‐methylated amino acids and for methanol‐based amino acid production

“…Adaptive laboratory evolution (Hu et al ., 2016; Sandberg et al ., 2019; Hennig et al ., 2020; Wang et al ., 2020) and enforcement of production by coupling it to growth (Haupka et al ., 2020) will allow for efficient selection procedures of superior strains (Prell et al ., 2021). Moreover, development of novel genetic tools will facilitate strain engineering of methylotrophic production hosts (Irla et al., 2016; Irla et al., 2021). In addition, synthetic consortia of different microorganisms may be developed to divide labour, for example, between conversion of a substrate such as methanol to an intermediate by one microorganism and product formation from the intermediate by another (Sgobba and Wendisch, 2020).…”

The Second Class of Tetrahydrofolate (THF-II) Riboswitches Recognizes the Tetrahydrofolic Acid Ligand via Local Conformation Changes

“…Concurrently, riboswitches can be used as targets for antibacterial compounds in drug development and as strain-engineering tools in biotechnology [ 60 ]. For example, THF exists in pathogens bacteria of the order Rhizobiales , controlling critical cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens synthesis pathways.…”