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Establishment of a Biosensor-based High-Throughput Screening Platform for Tryptophan Overproduction
Yongfei Liu1,2,3, Huiling Yuan1,2,3, Dongqin Ding1,2,3
1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
ACS Synthetic Biology
|June 3, 2021
Summary
Researchers developed a high-throughput screening platform using RNA aptamer biosensors and droplet sorting to enhance tryptophan production in *Escherichia coli*. A mutant strain showed a 165.9% increase in tryptophan titer, attributed to CRP(T29K) improving stationary phase tolerance.
Area of Science:
- Synthetic Biology
- Metabolic Engineering
- Biotechnology
Background:
- RNA's structural versatility enables its function as a cellular sensor for environmental changes.
- Efficiently screening for microbial strains with enhanced metabolic output is crucial for industrial applications.
- Developing robust biosensors is key to identifying high-producing microbial strains.
Purpose of the Study:
- To establish a high-throughput screening platform for identifying high-producing tryptophan (*Trp*) strains.
- To isolate and characterize mutant strains with significantly improved *Trp* production.
- To investigate the genetic basis for enhanced *Trp* production in selected mutants.
Main Methods:
- Development of a screening platform integrating a *Trp*-specific aptamer-based biosensor with fluorescence-activated droplet sorting (FADS).
- Random mutagenesis of *Escherichia coli* to generate a diverse library of candidate strains.
- High-throughput screening of the mutant library using the established platform.
- Genomic sequencing of high-producing mutants to identify genetic modifications.
Main Results:
- A high-throughput screening platform combining aptamer biosensors and FADS was successfully established.
- One mutant strain exhibited a 165.9% increase in *Trp* titer compared to the parental strain.
- Genomic analysis identified 10 single-nucleotide polymorphisms (SNPs) in the mutant, with CRP(T29K) identified as a key contributor to increased *Trp* production by enhancing stationary phase tolerance.
Conclusions:
- The developed aptamer-based biosensor and FADS platform is effective for high-throughput screening of microbial strains for enhanced amino acid production.
- The CRP(T29K) mutation significantly improves *Trp* production in *E. coli* by enhancing tolerance during the stationary phase of fermentation.
- This strategy holds promise for improving the production of other amino acids using *E. coli* as a host.
