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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.

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Figure 1: Workflow for the optimization of a whole-cell biocatalyst performing the conversion of cellobiose into vitamin B2.
Figure 2: B2 quantification in nLRs by genetically encoded sensors.
Figure 3: Screening for high B2-producing B. subtilis strain variants.

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Acknowledgements

We thank the Swiss National Foundation Research Equipment and the Swiss Commission of Technology and Innovation for their generous support, B. Chevreux (DSM) for the bioinformatics work on the sequence assembly and detection of mutations, T. Roberts (Department of Biosystems Science and Engineering (BSSE)) for carefully reading this manuscript and D. Gerngross (BSSE) for his support in generating the figures.

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Authors and Affiliations

Authors

Contributions

A.M. developed the sensor strains and the aptamers. R.P. developed the incubation and COPAS protocols. K.B., A.M. and R.P. performed the screening. S.Po. generated the B. subtilis library and characterized the isolated strains. All the authors discussed the results and commented on the manuscript. All the authors assisted in co-writing the paper.

Corresponding author

Correspondence to Martin Held.

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Competing interests

H.P.H. and S.Po. are with DSM NP, which co-sponsored the development. A.M., R.P., S.Pa. and M.H. are affilliates of FGen GmbH, which develops similar protocols.

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Meyer, A., Pellaux, R., Potot, S. et al. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors. Nature Chem 7, 673–678 (2015). https://doi.org/10.1038/nchem.2301

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