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Verbundprojekt: Essigsäurebakterien / Beschreibung Teilprojekt
Teilprojekt 1: Development of overexpression systems for acetic acid bacteria and the elucidation of catalytic activities of membrane-bound dehydrogenases
Laufzeit: 01.10.09-31.07.13
   Prof. Dr. Uwe Deppenmeier, Rhein. Friedrich-Wilhelms-Universität Bonn

1: Development of a novel acetic acid bacterial system to overproduce membrane proteins
2: Internal membranes as a platform for the production of membrane proteins from acetic acid bacteria
3: Analysis of product formation by uncharacterized membrane-bound dehydrogenases from acetic acid bacteria and related organisms

Membrane-bound proteins in acetic acid bacteria are very important for the process of oxidative fermentation and the application of these organisms in industrial biotransformation. Hence, acetic acid bacteria forming internal cytoplasmic membranes are thought to be ideal hosts for improved production of membrane proteins because of the increased capacity for membrane formation. We will investigate the formation of internal cytoplasmic membranes in Gluconobacter oxydans during growth on different carbon sources and at various growth stages. In parallel we will develop a novel protein expression system for acetic acid bacteria. The third aim concerns the analysis of product formation by uncharacterized membrane-bound dehydrogenases. Genes encoding PQQ or flavin-dependent enzymes will be detected from genome sequences of acetic acid and related bacteria and will be overexpressed in the aforementioned hosts and vectors. The focus of this research is the identification of membrane-bound enzyme that selectively oxidize alpha-diols to the corresponding αhydroxy ketones or hydroxy aldehydes, which are desired building blocks for chemical synthesis of biologically active compounds.

Results (status Jan 2011): The genome sequence of G. oxydans 621H is known but molecular tools are needed for the characterization of putative oxidoreductases and for the improvement of industrial strains by heterologous and homologous gene expression. To this end, promoter regions for the genes encoding G. oxydans ribosomal proteins L35 and L13 were introduced into the broad-host-range plasmid pBBR1MCS-2 to construct two new expression vectors for gene expression in Gluconobacter spp (Lit). These vectors were named pBBR1p264 and pBBR1p452, respectively, and have many advantages over current vectors for Gluconobacter spp. The uidA gene encoding β-D-glucuronidase was inserted downstream of the promoter regions and these promoter-reporter fusions were used to assess relative promoter strength. The constructs displayed distinct promoter strengths and strong (pBBR1p264), moderate (pBBR1p452) and weak (pBBR1MCS-2) promoters were identified. As proof of concept, active strep-tagged arabitol dehydrogenases (from G. oxydans and Thermotoga maritima) were purified from G. oxydans using pBBR1p264 and pBBR1p452 and protein production was similar to E. coli protein production.

The abovementioned expression system was also successfully used for the production of PQQ-dependent dehydrogenases from G. oxydans. The production strains revealed 5-fold and 2-fold increase in PQQ-dependent glucose dehydrogenase and PQQ-dependent sorbitol dehydrogenase, respectively. Furthermore, a strep-tagged unchararcterized PQQ-dehydrogenase was overproduced in G. oxydans and the protein was purified.

The presence of internal membranes in G. oxydans was verified by Dr. Hoppert (University of Göttingen). First results indicate that such ICM`s are formed under stress conditions. In agreement with these findings we could show that the activity of membrane-bound dehydrogenases is increased under stress conditions whereas the activity of “house-keeping” proteins (e.g. 6-phosphogluconate dehydrogenase) is not effected.