Development of a Synthetic 3-Dehydroshikimate Biosensor in Escherichia coli for Metabolite Monitoring and Genetic Screening

Abstract

Biosensors for target metabolites provide powerful high-throughput screening tools to obtain high-performing strains. However, well-characterized metabolite-sensing modules are often unavailable and limit rapid access to the robust biosensors with successful applications. In this study, we developed a strategy of transcriptome-assisted metabolite-sensing (TAMES) to identify the target metabolite-sensing module based on selectively comparative transcriptome analysis between the target metabolite producing and nonproducing strains and a subsequent quantative reverse transcription (RT-qPCR) evaluation. The strategy was applied to identify the sensing module cusR that responds positively to the metabolite 3-dehydroshikimate (DHS) and proved it was effective to narrow down the candidates. We further constructed the cusR-based synthetic biosensor and established the DHS biosensor-based high-throughput screening (HTS) platform to screen higher DHS-producing strains and successfully increased DHS production by more than 90%. This study demonstrated that the TAMES strategy was effective at exploiting the metabolite-sensing transcriptional regulator, and this could likely be extended to develop the biosensor-based HTS platforms for other molecules.

Mining and fine-tuning sugar uptake system for titer improvement of milbemycins in Streptomyces bingchenggensis

“…More importantly, we provided a simple workflow to discover useful transporters by establishing the relationship between time-course transcription levels of all putative sugar transporters and titers of desired products. In comparison to previous work depending on structural analogues of specific compounds [ 44 ] or omics data [ 45 , 46 ], our approach is less information required and more feasible to obtain unidentified candidates.…”

Recent trends in biocatalysis

“…129 Lastly, the physicochemical stability of DNA can be used to detect natural products such as biotin, vitamin D, and folate at nanomolar levels by strand displacement reactionbased biosensors, which have been shown to exhibit increased sensitivity, low interference, and high controllability. 130,131 The application of (small-molecule) biosensors and the development and engineering of new sensory devices is certainly of interest for different industries to meet performance criteria through the directed evolution of enzymes, 132,133 for the optimisation of microbial cell factories, [134][135][136] and the real-time monitoring of the production of target molecules 137 including (aromatic) alcohols, aldehydes, and acids, 112,113,[138][139][140][141] precursors for the synthesis fatty acids and their derivatives, 84,92,93,[142][143][144][145][146][147][148] isoprene and terpenoids, 149,150 steroids, as well as flavonoids. Biosensor systems for the last two will be highlighted in the following.…”

An engineered lactate responding promoter system operating in glucose-rich and anaerobic environments

“…Another approach for lactate detection is to use whole-cell biosensors. These sensors based on living cells, often bacteria, generally use a specific transcription factor responding to a signal of interest and its target promoter to regulate the expression of a reporter gene 18,19 . This strategy has produced a wide range of biosensors responding to a variety of metabolites including heavy-metals, butanol, alkanes, acyl-or malonyl-CoA [20][21][22][23][24][25][26][27] .…”