ABSTRACT
Bacillus subtilis can be widely used as an important microorganism for metabolic engineering and recombinant proteins expression in industrial biotechnology and synthetic biology. However, it is difficult to make accurate regulation of exogenous gene by biological tools in B. subtilis, which limits the application of B. subtilis in synthetic biology. The purpose of this study is to develop regulatory tools for precise control of gene expression by using non-coding RNAs, by which the activation of heterologous gene could be achieved without the auxiliary protein factors. We constructed the synthetic riboswitch E and aptazyme AZ using the theophylline aptamer. Six different native promoters from B. subtilis were functionally adapted with the E and AZ to fabricate an array of novel regulatory elements activated by theophylline. Then, we determined the performance of these elements using green fluorescence protein as reporter, and then further verified using red fluorescence protein and pullulanase as cargo proteins. Results showed that the same kind of RNA elements with different promoters showed different levels of efficiency. Promoter PsigW and E combination (sigWE) had the highest induction rate in B. subtilis. Compared with the control group, it can produce the induction rate of 16.8. Promoter PrpoB and AZ combination (rpoBAZ) showed the highest induction rate of 6.2. SigWE mediated mCherry induction rate was 9.2, and P43E mediated pullulanase induction rate was 32.8, in which enzyme activity reached 81 U/mL. This study confirmed that GFP, mCherry and pullulan can all be regulated by riboswitch and aptazyme, but there were differences between different combinations of promoters with RNA regulators.
Subject(s)
Bacillus subtilis , Promoter Regions, Genetic , RNA , Recombinant Proteins , TheophyllineABSTRACT
Objective To compare the regulation effects of different activated and inhibitory riboswitches , and to facilitate the precise regulation of gene circuits .Methods A green fluorescent protein amcyan expression vector regulated by different riboswitches (addA, M6, TPP and btuB) was constructed, and the expression level of amcyan under different ligand concentrations was analyzed by RT-qPCR and relative fluorescence intensity , and then compared with the expression level of a vector without any riboswitch .The dynamic control performance was analyzed .Results Under the control of addA and M6 activated riboswitches , the expression of green fluorescent protein increased with ligand concentrations , and addA riboswitch had more dynamic regulatory effect than M 6 riboswitch.However, under the control of TPP and btuB inhibitory riboswitches , the expression of green fluorescence decreased with the increase in ligand concentrations , and the dynamic regulation of btuB riboswitch was slightly greater than that of TPP riboswitch .Conclusion The regulation efficacy of different riboswitches which have the same mechanism varies .Activated riboswitch addA and inhibitory riboswitch btuB with dynamic regulation and control advantages are more suitable for precise metabolism regulation and target gene expression in Escherichia coli.
ABSTRACT
Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous nucleotide second messenger present in a wide variety of bacteria. It regulates many important bacterial physiological functions such as biofilm formation, motility, adhesion, virulence and extracellular polysaccharide synthesis. It binds with many different proteins or RNA receptors, one of which is called riboswitch that is usually located at the 5'-untranslational region (5'-UTR) in some mRNA. Riboswitch usually comprises a specific ligand-binding (sensor) domain (named aptamer domain, AD), as well as a variable domain, termed expression platform (EP), to regulate expression of downstream coding sequences. When a specific metabolite concentration exceeds its threshold level, it will bind to its cognate riboswitch receptor to induce a conformational change of 5'-UTR, leading to modulation of downstream gene expression. Two classes of c-di-GMP-binding riboswitches (c-di-GMP-Ⅰ and c-di-GMP-Ⅱ) have been discovered that bind with this second messenger with high affinity to regulate diverse downstream genes, underscoring the importance of this unique RNA receptor in this pathway. Class Ⅰ c-di-GMP riboswitches are present in a wide variety of bacteria, and are most common in the phyla Firmicutes and Proteobacteria, while class Ⅱ c-di-GMP riboswitches typically function as allosteric ribozymes, binding to c-di-GMP to induce folding changes at atypical splicing site junctions to modulate downstream gene expression. This review introduces the discovery, classification, function, and also the affected downstream genes of c-di-GMP riboswitches.
ABSTRACT
Riboswitch is a novel type of posttranscriptional regulatory elements discovered by Breaker et al. in 2002. It can regulate gene expression by binding directly to small metabolites without the aid of protein molecules. Compared to normal protein-mediated regulation, riboswitch responds to metabolites more rapidly and sensitively. Its discovery opens a new world for RNA research. The recent advances in riboswitch researches were summarized, including crystal structure determination, mechanism and dynamics study, biosensor and antibacterial drug design. Topp et al. successfully reprogrammed E. coli to detect, follow, and precisely localize to a completely new chemical signal by using a synthetic riboswitch. This work provided new ideas for synthetic biology and artificial biology network. The advances in riboswitch 3D structure determination, reaction mechanism and dynamics provide useful information for rational drug design towards new generation of riboswitch-targeting antibacterials.