Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast

Abstract

In cell factory development, screening procedures, often relying on low-throughput analytical methods, are lagging far behind diversity generation methods. This renders the identification and selection of the best cell factory designs tiresome and costly, conclusively hindering the manufacturing process. In the yeast Saccharomyces cerevisiae, implementation of allosterically regulated transcription factors from prokaryotes as metabolite biosensors has proven a valuable strategy to alleviate this screening bottleneck. Here, we present a protocol to select and incorporate prokaryotic transcriptional activators as metabolite biosensors in S. cerevisiae. As an example, we outline the engineering and characterization of the LysR-type transcriptional regulator (LTTR) family member BenM from Acetinobacter sp. ADP1 for monitoring accumulation of cis,cis-muconic acid, a bioplast precursor, in yeast by means of flow cytometry.

Evolution-guided engineering of small-molecule biosensors

“…Stable S. cerevisiae strains were routinely cultivated at 30 °C on YPD (1% (w/v) yeast extract, 2% (w/v) peptone 2% (w/v) dextrose, solidified with 2% (w/v) agar) medium, whereas plasmid-containing strains or libraries were cultivated in synthetic complete medium lacking leucine (SC-Leu; 6.7 g/L yeast nitrogen base without amino acids, 1.62 g/L yeast synthetic drop-out medium supplement without leucine (Y1376, Sigma-Aldrich), 2% (w/v) dextrose, pH 5.6, 2% (w/v) agar in case of plates). Mineral medium supplemented with tryptophan (7.5 g/L (NH 4 ) 2 SO 4 , 14.4 g/L KH 2 PO 4 , 0.5 g/L MgSO 4 •7H 2 O, 2 g/L dextrose, trace metals, vitamins, 0.02 g/L tryptophan, pH 4.5) was freshly prepared as described previously 58 , and medium was handled as outlined by Ambri et al 59 . Diacids, cis, cis -muconic acid (Sigma, CAS Number 1119-72-8, Product Number: 15992) or adipic acid (TCI, CAS Number:124-04-9 Product Number: A0161), were dissolved freshly to the medium on the day of handling, after which the pH was adjusted to 4.5 and the solution filter-sterilized as previously described 59 .…”

“…Mineral medium supplemented with tryptophan (7.5 g/L (NH 4 ) 2 SO 4 , 14.4 g/L KH 2 PO 4 , 0.5 g/L MgSO 4 •7H 2 O, 2 g/L dextrose, trace metals, vitamins, 0.02 g/L tryptophan, pH 4.5) was freshly prepared as described previously 58 , and medium was handled as outlined by Ambri et al 59 . Diacids, cis, cis -muconic acid (Sigma, CAS Number 1119-72-8, Product Number: 15992) or adipic acid (TCI, CAS Number:124-04-9 Product Number: A0161), were dissolved freshly to the medium on the day of handling, after which the pH was adjusted to 4.5 and the solution filter-sterilized as previously described 59 .…”

Yeast-Based Biosensors: Current Applications and New Developments

“…The design of such biosensors relies on systematic engineering and can use small-molecule binding transcriptional activators of various origins including prokaryotic. This was notably demonstrated by Skjoedt et al, who showed that several activators from different bacterial species are indeed able to function as allosterically regulated transcription factors in S. cerevisiae, thus creating a biosensor resource useful for future biotechnological [80,81]. Following this demonstration, they further developed a synthetic selection system that couples the concentration of muconic acid (a plastic precursor) to cellular fitness using the prokaryotic transcriptional regulator BenM upstream of the Kan r antibiotic resistance gene [82].…”

Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: current state and perspectives

“…Recent development in metabolite-responsive transcriptional factor (MRTF) based biosensors have expanded our ability to reprogram gene expression or control metabolic activity [19][20][21][22]. Most of these biosensors are developed in bacterial system.…”