Screening of an Escherichia coli promoter library for a phenylalanine biosensor

Abstract

In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors.

Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli

“…Among them, promoter engineering has been proposed as one of the most efficient ways of fine-tuning transcriptional control in Escherichia coli , Corynebacterium glutamicum , Bacillus subtilis , and yeasts [ 3 – 10 ]. For example, the E. coli strain with engineered l -phenylalanine-responsive promoter could produce fourfold higher titer of phenylalanine than wild-type promoter [ 11 ], and the engineered tac promoter library could decrease leakage of antibody fragment expression in E. coli [ 12 ].…”

Metabolic engineering for the production of l-phenylalanine in Escherichia coli

“…As synthetic biology develops, more and more tools can be applied to the generation of large diversified libraries and high-throughput screening processes. In 2016, a mtr promoter-based biosensor was constructed and employed in FACS high-throughput screening of an E. coli MG1655 mutant library (Mahr et al 2016). The best mutant could produce 4.3-fold l-phenylalanine levels compared with the wild-type strain.…”

RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes

“…However, traditional chromatography-reliant metabolite detection modalities such as gas chromatography-mass spectrometry (GC-MS) and high pressure liquid chromatography–mass spectrometry (HPLC-MS) are too slow without brute-force parallelization and associated high costs. Alternative approaches, such as protein-based biosensors, can link target metabolite concentration to fluorescence or growth-selectable traits and thus enable more rapid and efficient screening 21 – 24 . However, biosensor development can be laborious for each new target and often requires host-cell modifications or DNA rewiring.…”