title: Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors.
abstract: Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.
authors.affiliation: 1] Department of Biosystems Science and Engineering, ETH Zurich, Basel 4058, Switzerland [2] FGen GmbH, Basel 4057, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Meyer
authors.affiliation: 1] Department of Biosystems Science and Engineering, ETH Zurich, Basel 4058, Switzerland [2] FGen GmbH, Basel 4057, Switzerland.
authors.forename: René
authors.initials: R
authors.lastname: Pellaux
authors.affiliation: DSM Nutritional Products, Kaiseraugst 4303, Switzerland.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Potot
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel 4058, Switzerland.
authors.forename: Katja
authors.initials: K
authors.lastname: Becker
authors.affiliation: DSM Nutritional Products, Kaiseraugst 4303, Switzerland.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Hohmann
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel 4058, Switzerland.
authors.forename: Sven
authors.initials: S
authors.lastname: Panke
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Basel 4058, Switzerland.
authors.forename: Martin
authors.identifier: 0000000259525659
authors.initials: M
authors.lastname: Held
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D004798:Enzymes; D004926:Escherichia coli / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D049329:Nanostructures / Q000737:chemistry
chemical_list: D004798:Enzymes; D005486:Flavin Mononucleotide