Dokument: NADPH-related studies performed with a SoxR-based biosensor in Escherichia coli
| Titel: | NADPH-related studies performed with a SoxR-based biosensor in Escherichia coli | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=52384 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20200324-101925-9 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Spielmann, Alina [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Bott, Michael [Gutachter] Prof. Dr. Martina Pohl [Gutachter] | |||||||
| Stichwörter: | NADPH biosensor, RseC, RsxABCDGE complex, transhydrogenases PntAB and SthA, enzyme optimization, fluorescence-activated cell sorting, Lactobacillus brevis, NADPH biosensor, NADPH-dependent alcohol dehydrogenase, random mutagenesis | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The SoxRS regulatory system of Escherichia coli responds to NADPH, presumably due to the NADPH-dependent reduction of the transcriptional regulator SoxR, switching it to the inactive state. In a previous study, this NADPH-responsiveness was used to construct the genetically encoded NADPH biosensor pSenSox, in which the SoxR-activated soxS promoter controls expression of the reporter gene eyfp, allowing detection of SoxR activation at the single cell level via eYFP fluorescence. The biosensor was reported to sense intracellular NADPH availability, because increased cellular NADPH demands during the biotransformation of methyl acetoacetate (MAA) to R-methyl 3-hydroxybutyrate (MHB) by the strictly NADPH-dependent alcohol dehydrogenase of Lactobacillus brevis (LbADH) led to increased eyfp expression. Most importantly, the specific eYFP fluorescence of E. coli cells catalyzing MAA reduction to MHB correlated not only with the amount of MAA reduced by the cells, and consequently with the NADPH demand, but also with the specific LbADH activity when a fixed MAA concentration was provided. The latter property enables high-throughput screening of large NADPH-dependent enzyme libraries for variants with improved activity using fluorescence-activated cell sorting (FACS). Based on the correlation of the specific fluorescence of the biosensor with the cellular NADPH demand and the activity of NADPH-dependent enzymes, the pSenSox biosensor was used in this thesis to (1) study NADPH-related processes in E. coli and to (2) identify variants of the NADPH-dependent LbADH with improved catalytic properties.
(1) NADPH plays a crucial role in cellular metabolism for biosynthesis and oxidative stress responses. Here, pSenSox was used to study the influence of various NADPH-related parameters on the soxRS response in E. coli. Specifically, the influence of different growth media, of the redox-cycling drugs paraquat and menadione, of the SoxR-reducing system RsxABCDGE and RseC, and of transhydrogenases SthA and PntAB on the pSenSox signal was examined. Redox-cycling drugs activated the NADPH biosensor. The absence of RsxABCDGE and/or RseC caused an enhanced biosensor response, in agreement with their function as a SoxR-reducing system. The absence of the membrane-bound transhydrogenase PntAB caused an increased biosensor response, whereas the lack of the soluble transhydrogenase SthA or of SthA and PntAB was associated with a strongly decreased response. These data support the opposing functions of PntAB in NADP+ reduction and of SthA in NADPH oxidation. In conclusion, the biosensor pSenSox was shown to be a useful tool for analyzing environmental conditions and genes with respect to their influence on the NADPH availability in the cell. (2) The NADPH-dependent LbADH is widely used in industrial biotechnology for the biocatalytic production of chiral alcohols. To find an optimized LbADH variant for the substrate 2,5-hexanedione, the biosensor pSenSox was used for FACS-based high-throughput screening of an LbADH library to isolate clones showing increased fluorescence during the biotransformation of 2,5-hexanedione. Using this approach, the improved variant LbADHK71E was identified in which lysine-71 was replaced by glutamate, causing a charge reversal at the surface of the protein. Kinetic measurements with purified enzymes revealed that LbADHK71E has a 16% higher affinity (KM= 4.3 ± 0.5 mM) and a 17% higher activity (Vmax= 173.3 ± 11.1 μmol min-1mg-1) compared to the wild-type enzyme (KM= 5.1 ± 0.6 mM; Vmax= 148.5 ± 12.3 μmol min-1mg-1) with 2,5-hexanedione as substrate. Moreover, the LbADHK71E enzyme also showed higher activity for the alternative substrates acetophenone, acetylpyridine, 2-hexanone, 4-hydroxy-2-butanone, and MAA. The isolation of the optimized variant LbADHK71E demonstrates that the application of the biosensor combined with high-throughput screening is a powerful workflow for the identification of unexpected beneficial mutations of NADPH-dependent ADHs. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie | |||||||
| Dokument erstellt am: | 24.03.2020 | |||||||
| Dateien geändert am: | 24.03.2020 | |||||||
| Promotionsantrag am: | 15.05.2019 | |||||||
| Datum der Promotion: | 01.07.2019 |
