Engineering a highly sensitive biosensor for abscisic acid in mammalian cells

Kim, Seo Woo; Alci, Kübra; Gaever, F. Van; Driege, Yasmine; Bicalho, Keylla; Goeminne, Geert; Libert, Claude; Goossens, Alain; Beyaert, Rudi; Staal, Jens

doi:10.1002/1873-3468.14431

Cited by 2 publications

(1 citation statement)

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“…This design solely relies on the ABA-induced dimerization and does not require a functional phosphatase. In a recent study, a highly sensitive ABA biosensor for use in mammalian cells was developed based on the same design principle (Kim et al, 2022). In addition, two latest studies changed the ligand specificity of PYR/PYL receptors and developed PYR/PYL-PP2C-based biosensors for various non-native ligands, including herbicides, cannabinoids and organophosphates (Beltrán et al, 2022;Zimran et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

Sense and Screen-ability: Development of tuneable, biosensor-based screening platforms for abscisic acid

Otto

Dabirian

David

et al. 2023

Preprint

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The activities of heterologous enzymes often limit the production titers, rates and yields of cell factories. With the help of biosensors, large random mutagenesis libraries can be screened for improved enzyme variants in a high-throughput manner, even if the enzyme-of-interest is poorly characterised. We previously constructed a Saccharomyces cerevisiae cell factory for the heterologous production of abscisic acid (ABA), a high-value product with a broad range of applications in medicine, agriculture and nutrition. In the current study, we developed high-throughput screening platform strains for two rate-limiting cytochrome P450 monooxygenases, BcABA1 and BcABA2, in the ABA biosynthetic pathway. The screening platforms are designed to minimize the occurrence of false positives during screening experiments. We thoroughly characterised two plant protein-based ABA biosensor candidates. Furthermore, we designed a simple genetic switch, based on the thiamine-repressible promoter pTHI4, to regulate the expression level of enzyme variants. We demonstrated that ABA production can be fine-tuned by varying thiamine concentration in the media. In-depth analysis of the platform strains revealed that screening conditions can be optimized by varying thiamine concentration and cultivation time, making it possible to utilize the full dynamic and operational range of the biosensor. In the future, the constructed strains can be used to screen for improved BcABA1 and BcABA2 variants.

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Section: Introductionmentioning

confidence: 99%