Genetically encoded biosensors + FACS + Recombineering: versatile tools for strain and enzyme development

Michael Bott, Georg Schendzielorz, Stephan Binder, Solvej Siedler, Stephanie Bringer, Jan

Marienhagen, Julia Frunzke and Lothar Eggeling

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

Transcriptional regulators usually function as sensors and regulators, as their activity is controlled by specific stimuli, such as metabolites, redox status, toxic compounds, or inorganic ions. We recently have exploited this property for the construction of biosensors, which are able to detect the concentrations of amino acids such as L-lysine or L-methionine in single bacterial cells and convert this information into a fluorescence output by using EYFP or other autofluorescent proteins as reporter. The response was linear in a certain concentration range (Binder et al. 2012; Mustafi et al.
2012). The lysine sensor was used to isolate single producer cells from a library of randomly mutagenized cells of Corynebacterium glutamicum by fluorescence-activated cell sorting (FACS). Characterization of these clones led to the identification of known, but also of novel mutations triggering lysine overproduction (Binder et al. 2012).
As random mutagenesis often leads to hundreds of mutations whose relevance for production is not obvious, a novel methodology termed RecFACS was developed, which combines biosensors, FACS, and recombineering. It allows to rapidly identify those point mutations that cause a â??productiveâ? phenotype, such as lysine overproduction, and can be used to screen hundreds of mutations, including those in essential genes, such as murE (Binder et al. 2013). Another highly useful application of the biosensors was demonstrated for altering the allosteric properties of key enzymes in biosynthetic pathways. Using plasmid-encoded mutant libraries of aspartate kinase (LysC), N- acetylglutamate kinase (ArgB), and ATP-phosphoribosyltransferase and cells carrying the lysine sensor, which also detects arginine and histidine, it was possible to rapidly isolate feedback-resistant variants of the corresponding enzymes, whose presence was sufficient to cause overproduction of lysine, arginine, and histidine (Schendzielorz et al. 2014). Besides sensors for amino acids, also sensors for the cysteine and methionine precursors O-acetyl serine and O-acetyl homoserine were developed based on the transcriptional regulator CysR of C. glutamicum. They allowed monitoring of intracellular sulfur availability (Hoffmann et al. 2013).
More recently, we also developed a biosensor for the NADPH/NADP+ ratio based on the transcriptional regulator SoxR of Escherichia coli. SoxR is a homodimer with each subunit containing a [2Fe-2S] cluster. Only when oxidized to [2Feâ??2S]2+ they confer transcriptional activity to SoxR, which
in turn results in expression of soxS. SoxS then activates expression of the SoxRS regulon, which mediates the cellular response to superoxide, to diverse redox-cycling drugs like paraquat, or to nitric oxide and includes e.g. the genes for superoxide dismutase (sodA), glucose 6-phosphate dehydrogenase (zwf), or fumarase C (fumC). Inactivation of SoxR involves its NADPH-dependent reduction catalyzed by the rsxABCDGE and rseC products. The observation that soxS expression was strongly increased during the NADPH-dependent reductive biotransformation of methyl acetoacetate to (R)-methyl 3-hydroxybutyrate by an alcohol dehydrogenase (Siedler et al. 2014a) suggested that SoxR is responsive to the NADPH/NADP+ ratio, which was already indicated in previous studies (Liochev and Fridovich 1992; Krapp et al. 2011). By placing the eyfp gene under the control of the

soxS promoter, correlations of the specific fluorescence intensity with the period of high NADPH demand and with alcohol dehydrogenase activity were found. In a proof-of-principle study, the pSenSox sensor was used to isolate via FACS an alcohol dehydrogenase variant with improved

activity for the substrate 4-methyl-2-pentanone out of mutant library. Thus, this sensor is suitable for
HT-screening of NADPH-dependent enzymes (Siedler et al. 2014b).
Binder S, Schendzielorz G, Stäbler N, Krumbach K, Hoffmann K, Bott M, Eggeling L (2012) A high- throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level. Genome Biol 13
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
Hoffmann K, Grünberger A, Lausberg F, Bott M, Eggeling L (2013) Visualization of imbalances in sulfur assimilation and synthesis of sulfur-containing amino acids at the single-cell level. Appl Environ Microbiol 79:6730-6736
Krapp AR, Humbert MV, Carrillo N (2011) The soxRS response of Escherichia coli can be induced in the absence of oxidative stress and oxygen by modulation of NADPH content. Microbiology
157:957-965
Liochev SI, Fridovich I (1992) Fumarase C, the stable fumarase of Escherichia coli, is controlled by the

soxRS regulon. Proc Natl Acad Sci USA 89:5892-5896

Mustafi N, Grünberger A, Kohlheyer D, Bott M, Frunzke J (2012) The development and application of a single-cell biosensor for the detection of L-methionine and branched-chain amino acids. Metab Eng 14:449-457
Schendzielorz G, Dippong M, Grünberger A, Kohlheyer D, Yoshida A, Binder S, Nishiyama C,
Nishiyama M, Bott M, Eggeling L (2014) Taking control over control: Use of product sensing in single eells to remove flux control at key enzymes in biosynthesis pathways. ACS Synth Biol
3:21-29
Siedler S, Bringer S, Polen T, Bott M (2014a) NADPH-dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: influence on global gene expression and role of oxygen supply. In revision
Siedler S, Schendzielorz G, Binder S, Eggeling L, Bringer S, Bott M (2014b) SoxR as a single-cell
biosensor for NADPH-consuming enzymes in Escherichia coli. ACS Synth Biol 3:41-47