Proteindesign und Stammentwicklung für die Ganzzell-Biokatalyse mittels Pyrrolochinolinchinon (PQQ)-abhängiger Oxidoreduktasen

• Antragsteller: Dr. Janosch Klebensberger
• Fachliche Zuordnung: Bioverfahrenstechnik ; Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
• Förderung: Förderung von 2014 bis 2018
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 254226746

• Erstellungsjahr: 2018

Zusammenfassung der Projektergebnisse

In this DFG research project, the two pyrroloquinoline quinone-dependent ethanol dehydrogenases (PQQ-EDHs) from Pseudomonas putida KT2440 were investigated in detail. From these studies, it was found that PedE and PedH are functionally redundant and essential for efficient growth on alcoholic volatile compounds, but differ in their cofactor dependency. In contrast to the calcium-dependent PedE, PedH was characterized as the first rare earth element (REE)-dependent PQQ-EDH from a non-methylotrophic organism. With the use of laboratory evolution, site-directed mutants, transcriptional reporter fusions, functional complementation assays, and growth experiments, this study further characterized the molecular mechanism underlying the inverse transcriptional regulation of the two PQQ-EDHs in response to REE availability and identified a two-component system that is crucial in this context. In a second part of the project, the metabolic and regulatory basis of ethylene glycol metabolism in P. putida KT2440 was elucidated by a whole-genome re-sequencing approach of suppressor mutants. A specific transcriptional regulator was found to play a central role in ethylene glycol catabolism through the repression of the glyoxylate carboligase pathway. Secondary mutations further improved growth on ethylene glycol in evolved strains, likely by balancing fluxes through the initial oxidations, thereby preventing the accumulation of toxic aldehydes. These results and the outcome of an additional collaborative work enabled the metabolic engineering of a robust strain for efficient ethylene glycol consumption, which now can serve as foundation strain for the development of biocatalytic processes based on the conversion of ethylene glycol from different waste streams. Last but not least, the project focused on the development of the two PQQ-EDHs as useful biocatalytic tools. A heterologous whole-cell assay and the use of a sequence-structure similarity database enabled us to increase the thermal and solvent stability of PedE, with only minimal impact on the overall catalytic efficiency of the enzyme. With a semi-rational design approach using small focused mutant libraries in combination with a solid-phase screening assay, we further successfully altered the activity of PedH towards chemicals, whose oxidation products represent industrially relevant precursors for polymer chemistry. In conclusion, this project resulted in i) the extensive biochemical characterization of the PQQ- EDHs PedE and PedH; ii) the successful developed of a protein engineering platform that allowed the construction of proof-of-concept protein variants of industrial relevance, and; iii) detailed understanding of the transcriptional and proteomic changes of cells in response to REE availability. Consequently, this study not only provides novel insights into the regulation and ecological function of the periplasmic oxidation system in P. putida KT2440, but will also enable the establishment of new biocatalytic routes for the production of value-added chemicals using engineered PQQ-EDHs in either cell-free or whole-cell applications.

Projektbezogene Publikationen (Auswahl)

• The PedS2/PedR2 twocomponent system is crucial for the rare earth element switch in Pseudomonas putida KT2440
  Wehrmann M., Berthelot C., Billard P., Klebensberger J.
  
• Entner–Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1. Proceedings of the National Academy of Sciences of the United States of America, 31:E4298-E4305 (2015)
  Felux A.-K., Spiteller D., Klebensberger J., and Schleheck D.
  
• Production of ω-hydroxy octanoic acid with Escherichia coli. Journal of Biotechnology 230:30-33 (2016)
  Kirtz M., Klebensberger J., Otte K.B., Richter S.M., Hauer B.
  
• SiaA/D interconnects c-di-GMP and RsmA signaling to coordinate cellular aggregation of Pseudomonas aeruginosa in response to environmental conditions. Frontiers in Microbiology, 7:179 (2016)
  Colley B., Dederer V., Carnell M., Kjelleberg S., Rice S.A., and Klebensberger J.
  
• Functional role of lanthanides in enzymatic activity and transcriptional regulation of pyrroloquinoline quinone-dependent alcohol dehydrogenases in Pseudomonas putida KT2440. mBIO, 3:e00570-17 (2017)
  Wehrmann M., Billard P., Martin-Meriadec A., Zegeye A., and Klebensberger J.
  
• Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization. Metabolic Engineering
  Franden M.A., Jayakody L., Li W-J., Wagner N.J., Cleveland N.S., Michener W.E., Hauer B., Blank L.M., Wierckx N., Klebensberger J., Beckham G.T.
  
• Engineering thermal stability and solvent tolerance of the soluble quinoprotein PedE from Pseudomonas putida KT2440 with a heterologous whole‐cell screening approach. Microbial Biotechnology, 2:399-408 (2018)
  Wehrmann M. and Klebensberger J.