The Gram-negative acetic acid bacterium Gluconobacter oxydans has superior capabilities to perform regio- and stereoselective oxidative biotransformations and therefore is used in industrial biotechnology, e.g. for vitamin C production. The aim of the GLUFACT project was to improve the biomass yield of this bacterium (WP-1), to establish a global view of the genetic organization via next-generation DNA-Seq and RNA-Seq (WP-2 and WP-3), to identify novel industrially relevant dehydrogenases (WP-4) and to establish novel bioprocesses based on the above results (WP-5). In WP-1, a series of metabolically engineered strains were constructed, in which the conversion of glucose into biomass was improved by preventing formation of gluconate and acetate, by enhancing the NADH oxidation capacity, and by restoring the incomplete citrate cycle via genomic integration of heterologous genes encoding succinate dehydrogenase, flavinylation factor and succinyl-CoA synthetase.The final strain IK003.1 has a 1.6-fold increased biomass yield on glucose. In WP-2, the transcriptional landscapes of G. oxydans were determined by establishing RNAseq, delivering the first genome-wide data set on gene expression, transcriptional organisation, and promoters. These data are highly valuable for future studies on metabolic engineering and development of expression systems for G. oxydans. In WP-3, the high-quality reference genome for G. oxydans was further improved and complex structural variants were solved by establishing a leading-edge setup for long-read nanopore sequencing using Oxford Nanopores MinION technology. The results have been made available to the public via the web-based www.gluconobacterfactory.de as a resource for future research. In WP-4, genes from different Gram-negative bacteria encoding uncharacterized PQQ-dependent dehydrogenases were cloned into expression vectors for production of periplasmic enzymes with potential novel oxidative activities of industrial relevance. An inducible expression system and modified strains were developed to improve protein production, including a strain with deletions of known membrane-bound dehydrogenases and a strain lacking the tolB gene for facilitated protein export across the cytoplasmic membrane. At least one dehydrogenase with a high application potential was identified. Furthermore, a gene encoding an endo-1,4-β-xylanase from Bacillus subtilis was expressed in G. oxydans ΔtolB enabling the utilization of the polysaccharide xylan. In WP-5, the G. oxydans wild-type strain as well as the new strains constructed in WP-1 and WP-4 were characterized. Valuable information about microbial growth, suitable pH and buffer, respiration behavior and sensitivity to osmotic pressure was gained. A fed-batch process was developed and optimized. In sum, the GLUFACT project has achieved its aims and its results provide the basis for future projects aiming at the establishment of novel bioproducts together with industrial partners.
Participating Core Groups
Prof. Dr. Michael Bott, Dr. Stephanie Bringer; Forschungszentrum Jülich, Institute of Bio- and Geosciences: Biotechnology (IBG-1, Systemic Microbiology)
Prof. Dr. Michael Bott, Dr. Tino Polen; Institute of Bio- and Geosciences: Biotechnology (IBG-1, Systemic Microbiology); Forschungszentrum Jülich
Prof. Dr. Björn Usadel; Institute of Biology I (Botany/Molecular Genetics): Plant Walls, Metabolism & Bioinformatics; Rheinisch-Westfälische Technische Hochschule Aachen
Prof. Dr. Uwe Deppenmeier; Institute of Microbiology and Biotechnology, Rheinische Friedrich-Wilhelms-Universität Bonn
Prof. Dr. Jochen Büchs; AVT – Lehrstuhl für Bioverfahrenstechnik, Rheinisch-Westfälische Technische Hochschule Aachen
1.1.2014 – 31.12.2016
The total budget of GLUFACT is € 671.148,00. GLUFACT is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Innovation, Science and Research of the German State of North Rhine-Westphalia.
Kiefler, I, Bringer, S and Bott, M (2015). Sdhe-dependent formation of a functional acetobacter pasteurianus succinate dehydrogenase in gluconobacter oxydans--a first step toward a complete tricarboxylic acid cycle. Appl Microbiol Biotechnol 99(21): 9147-9160.
Kosciow, K, Domin, C, Schweiger, P and Deppenmeier, U (2016). Extracellular targeting of an active endoxylanase by a tolb negative mutant of gluconobacter oxydans. J Ind Microbiol Biotechnol 43(7): 989-999.
Kosciow, K, Zahid, N, Schweiger, P and Deppenmeier, U (2014). Production of a periplasmic trehalase in gluconobacter oxydans and growth on trehalose. J Biotechnol 189: 27-35.
Loehrer, M, Vogel, A, Huettel, B, Reinhardt, R, Benes, V, Duplessis, S, Usadel, B and Schaffrath, U (2014). On the current status of phakopsora pachyrhizi genome sequencing. Front Plant Sci 5: 377.
Luchterhand, B, Fischoder, T, Grimm, AR, Wewetzer, S, Wunderlich, M, Schleputz, T and Buchs, J (2015). Quantifying the sensitivity of g. Oxydans atcc 621h and dsm 3504 to osmotic stress triggered by soluble buffers. J Ind Microbiol Biotechnol 42(4): 585-600.
Meyer, M, Schweiger, P and Deppenmeier, U (2015). Succinic semialdehyde reductase gox1801 from gluconobacter oxydans in comparison to other succinic semialdehyde-reducing enzymes. Appl Microbiol Biotechnol 99(9): 3929-3939.
Zahid, N and Deppenmeier, U (2016). Role of mannitol dehydrogenases in osmoprotection of gluconobacter oxydans. Appl Microbiol Biotechnol 100(23): 9967-9978.