Rational Genome Engineering with Genetically Encoded Biosensors at Single-Cell Scale | AIChE

Rational Genome Engineering with Genetically Encoded Biosensors at Single-Cell Scale


Marienhagen, J. - Presenter, Forschungszentrum Juelich
Binder, S., Forschungszentrum Juelich
Schendzielorz, G., Forschungszentrum Juelich
Eggeling, L., Forschungszentrum Jülich
Bott, M., Forschungszentrum Juelich

Microsoft Word - Jan Marienhagen_Abstract_Metabolic_Engineering_X

Rational Genome Engineering with Genetically Encoded Biosensors at

Single-cell Scale

Jan Marienhagen, Stephan Binder, Georg Schendzielorz, Lothar Eggeling and Michael Bott

Institute of Bio- und Geosciences, IBG-1: Biotechnology, Forschungszentrum Juelich GmbH, Juelich, Germany

The engineering of microbial strains for the production of small molecules is a time-consuming, laborious and expensive process. This can be mostly attributed to the fact that good producers cannot be readily obtained by high-throughput (HT) screening approaches since increased product formation usually does not confer a clear phenotype to producing strain variants.
Recently, advances were made in the design and construction of genetically encoded biosensors for detecting small molecules at the single-cell level [1]. In combination with fluorescent-activated cell sorting (FACS) we already demonstrated the value and potential of these new tools for microbial strain development by screening large libraries of chemically mutagenized Corynebacterium glutamicum cells for L-lysine producers with the L-lysine sensor pSenLys [2]. Whole genome sequencing of selected clones identified a single mutation leading to an amino acid substitution from glycine to glutamate at position 81 in the UDP-N-acetylmuramyl-tripeptide synthetase (MurE). This enzyme consumes the L-lysine precursor D, L-diaminopimelate and is involved cell wall synthesis, rendering MurE essential for C. glutamicum.
Motivated by the assumption that other amino acid substitutions in murE might lead to even higher L- lysine titers, we developed RecFACS for the site-directed saturation mutagenesis of microbial genomes. RecFACS combines targeted genome mutagenesis via recombineering with biosensor- guided HT screening for improved producers (Fig. 1) [3]. We successfully used RecFACS to generate and screen a site-saturation library of murE of C. glutamicum via FACS and identified 12 different amino acid substitutions causing different L-lysine production titers.
RecFACS is highly suitable to generate targeted genetic diversity in microbial genomes and screen for phenotypes in a HT-format in a single step, thus establishing a new general concept in metabolic engineering at genome-scale.

Figure 1. Genome engineering by RecFACS. After targeted genome mutagenesis of cells containing the L- lysine biosensor pSenLys via recombineering, cells were subjected to two rounds of FACS screening to first enrich and then select positive clones. Fluorescent cells were collected and cultivated in MTPs to follow growth and fluorescence of individual clones. L-lysine was quantified in culture supernatants and mutations were verified by sequencing.

[1] Schallmey M., Frunzke J., Eggeling L., Marienhagen J. (2014). Looking for the pick of the bunch: High throughput screening of producing microorganisms with biosensors. Curr. Opin. Biotechnol. 26: 148-154.

[2] Binder S., Schendzielorz G., Stäbler N., Krumbach K., Hoffmann K., Bott M. and Eggeling L. (2012). A high- throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level. Genome Biology 13: R40.

[3] Binder S., Siedler S., Marienhagen J., Bott M., Eggeling L. (2013). Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. Nucleic Acids Res. 41: 6360-6369.