Background
What are biosensors and allosteric transcription factors？
Biosensors use biological recognition elements (proteins, enzymes, etc.) to interact with specific analytes, and transform physical or chemical signals into easily collected quantitative data through a series of reactions. As a new type of analytical instrument, biosensors are capable of performing residue screening and offer the potential for rapid, quantitative and high-throughput analysi [1].
Allosteric transcription factors (aTFs) are important molecules that control gene expression. They can bind to specific DNA elements, and regulate downstream DNA transcription into mRNAs, leading to target gene expression at specific intensity in designated time points and locations. aTF-based biosensors with innately high ligand specificity are widely used in molecule detection, enzyme-directed evolution, dynamic control of metabolic pathways, and adaptive laboratory evolution[2]. Despite promising applications in synthetic biology, the number of compounds that can be recognized by natural TFs is limited, so it is of significance to adjust ligand specificity of existing aTFs and create desired biosensors through directed evolution.
The biosynthetic dilemma of aromatic compounds
Aromatic chemicals refer to compounds containing benzene ring structures, and represent an important class of microbial fermentation chemicals. They are important raw materials, and widely used in medicine, agriculture, spices and other fields, with excellent economic values. Aromatic compounds typically have a long and complicated synthetic route in microbial cells and very low natural levels. To obtain high-yielding strains that satisfy the needs of fermentation, it is necessary to create tools that allow in cellulo detection of the levels of target aromatic compounds and intermediate metabolites.
PobR: targets for mutagenesis and evolution
PobR, a member of the iclR superfamily, is an aTF responsive to
4-hydroxybenzoic
acid (4HB). PobR consists of 271 amino acids, and its N- and C-terminal
structural domains are responsible for DNA binding and ligand recognition,
respectively. In the Acinetobacter strain ADP1, when PobR activates the pobA
promoter (PpobA) involved in 4HB metabolism
[3],
low
4HB levels trigger the
pobA gene expression
[4].
The selection of novel PobR mutants was driven by three major reasons:
1. Biosynthesis of aromatic compounds shows promising applications but lacks
specific biosensors.
2. PobR-based biosensors have demonstrated excellent responsiveness.
3. PobR has the potential to be evolved to become retrofitted TFs responsive
to other aromatic compounds.
Strategy
The purpose of this study is to develop a selection system that drives directed evolution and discovers ideally effective mutants in an established mutant library. Distinct from most conventional biosensor screening methods, a dual selection system created by us can efficiently alter the ligand specificity of transcriptional regulators. The system is composed of various genes for a transcriptional regulator, a red fluorescent protein, a cytosine deaminase, and antibiotic resistance. As a result, we generated a PobR mutant library based on random mutagenesis, and obtain PobR mutants through the dual selection system. For more information, please visit the Design.
