Biosensor-Based Evolution and Elucidation of a Biosynthetic Pathway in Escherichia coli

Abstract

The successful evolution of metabolite-producing microbes requires a high-throughput screening method to obtain the desired properties within a short time. In this study, we developed a transcription-factor-driven device that combines a metabolite-responsive element and a selection module. This device was able to specifically sense intracellular l-phenylalanine (l-Phe) and convert this signal into an observable phenotype. Applying this device, we successfully improved l-Phe production by screening hyperproducing phenotypes from a ribonucleotide binding site library and a random mutagenesis library. In addition, several site mutations introduced by random mutagenesis were identified and elucidated to facilitate the improvement of l-Phe production. Our results present a paradigm for screening of compounds that are not easily observable to raise the yield of targeted compounds from a large candidate library. This approach may guide further applications in rewiring metabolic circuits and facilitate the directed evolution of recombinant strains.

Fine‐tuning the (2S)‐naringenin synthetic pathway using an iterative high‐throughput balancing strategy

“…Perhaps the greatest challenge in preforming IHTB is establishing a universally used high‐throughput screening procedure. Inspired by previous biosensor studies (Liu et al, ; Qian & Cirino, ; Wang, Tang, & Yang, ; Xiong et al, ), combining the advantages of transcriptomic and biosensor technology will allow many inducible promoters to be screened and novel biosensors constructed that respond to different concentrations of target chemicals for the high‐throughput screening of high‐titer strains.…”

Transcription Factor‐Based Biosensors in High‐Throughput Screening: Advances and Applications

“…Microorganisms can produce a number of valuable metabolites, such as amino acids, alcohols, flavonoids, theophylline, however, the titers achieved using naturally‐occurring microbes are often inadequate for large‐scale applications. To identify microorganisms capable of achieving higher intracellular product concentrations, TFs have been integrated into diverse synthetic regulatory circuits for optimization of metabolic pathways, thereby enabling the intracellular semi‐quantification of these metabolites (Figure ).…”

Strategies for directed and adapted evolution as part of microbial strain engineering

“…However, for the case of short regions of DNA, such as promoter and RBS elements, the ssDNA library has a great advantage in its ability to establish the required set of variants. For example, Liu et al . used synthetic ssDNA library to randomly mutate, generate, and evaluate a RBS library with a size of 8192.…”

“…When these sites were altered via synthetic ssDNA libraries, 49 T 50 15 values for the enzyme were substantially improved by up to 4.3 ∘ C. However, for the case of short regions of DNA, such as promoter and RBS elements, the ssDNA library has a great advantage in its ability to establish the required set of variants. For example, Liu et al 50 used synthetic ssDNA library to randomly mutate, generate, and evaluate a RBS library with a size of 8192. Thus, when compared with Ep-PCR and DNA shuffling, synthetic ssDNA libraries have the advantage of a more focused mutation rate and region which makes the approach more compatible with rational design strategies that are based on protein 3D-structure or other types of modeling (Table 1).…”