title: Metabolic engineering of Gluconobacter oxydans for improved growth rate and growth yield on glucose by elimination of gluconate formation.
abstract: Gluconobacter oxydans N44-1, an obligatory aerobic acetic acid bacterium, oxidizes glucose primarily in the periplasm to the end products 2-ketogluconate and 2,5-diketogluconate, with intermediate formation of gluconate. Only a minor part of the glucose (less than 10%) is metabolized in the cytoplasm after conversion to gluconate or after phosphorylation to glucose-6-phosphate via the only functional catabolic routes, the pentose phosphate pathway and the Entner-Doudoroff pathway. This unusual method of glucose metabolism results in a low growth yield. In order to improve it, we constructed mutants of strain N44-1 in which the gene encoding the membrane-bound glucose dehydrogenase was inactivated either alone or together with the gene encoding the cytoplasmic glucose dehydrogenase. The growth and product formation from glucose of the resulting strains, N44-1 mgdH::kan and N44-1 DeltamgdH sgdH::kan, were analyzed. Both mutant strains completely consumed the glucose but produced neither gluconate nor the secondary products 2-ketogluconate and 2,5-diketogluconate. Instead, carbon dioxide formation of the mutants increased by a factor of 4 (N44-1 mgdH::kan) or 5.5 (N44-1 DeltamgdH sgdH::kan), and significant amounts of acetate were produced, presumably by the activities of pyruvate decarboxylase and acetaldehyde dehydrogenase. Most importantly, the growth yields of the two mutants increased by 110% (N44-1 mgdH::kan) and 271% (N44-1 DeltamgdH sgdH::kan). In addition, the growth rates improved by 39% (N44-1 mgdH::kan) and 78% (N44-1 DeltamgdH sgdH::kan), respectively, compared to the parental strain. These results show that the conversion of glucose to gluconate and ketogluconates has a strong negative impact on the growth of G. oxydans.
authors.affiliation: Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Krajewski
authors.forename: Petra
authors.initials: P
authors.lastname: Simic
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Mouncey
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bringer
authors.forename: Hermann
authors.initials: H
authors.lastname: Sahm
authors.forename: Michael
authors.initials: M
authors.lastname: Bott
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002245:Carbon Dioxide / Q000378:metabolism; D003470:Culture Media; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D005942:Gluconates / Q000378:metabolism*; D020600:Gluconobacter oxydans / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D042843:Glucose 1-Dehydrogenase / Q000235:genetics*; D009154:Mutation
chemical_list: D003470:Culture Media; D005942:Gluconates; D002245:Carbon Dioxide; D042843:Glucose 1-Dehydrogenase; D005947:Glucose
references.citation: Biochim Biophys Acta. 1959 Nov;36:294-6
references.pmid: 13797599
references.citation: Arch Microbiol. 2001 Apr;175(4):282-94
references.pmid: 11382224
references.citation: J Biol Chem. 1954 Jun;208(2):821-31
references.pmid: 13174591
references.citation: Biochem J. 1966 Mar;98(3):795-803
references.pmid: 4287842
references.citation: J Biol Chem. 1970 Nov 25;245(22):5974-8
references.pmid: 4320794
references.citation: Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648-52
references.pmid: 377280
references.citation: Appl Microbiol Biotechnol. 2006 Nov;73(2):443-51
references.pmid: 16820953
references.citation: Adv Microb Physiol. 1994;36:247-301
references.pmid: 7942316
references.citation: J Microbiol Methods. 2006 Mar;64(3):391-7
references.pmid: 15987659
references.citation: J Bacteriol. 1997 Oct;179(20):6228-37
references.pmid: 9335267
references.citation: J Bacteriol. 1969 Apr;98(1):232-7
references.pmid: 4239577
references.citation: Biotechniques. 1994 May;16(5):800-2
references.pmid: 8068328
references.citation: Appl Microbiol Biotechnol. 2002 Nov;60(3):233-42
references.pmid: 12436304
references.citation: Plasmid. 1985 May;13(3):200-4
references.pmid: 2987998
references.citation: J Mol Biol. 1969 May 14;41(3):459-72
references.pmid: 4896022
references.citation: J Mol Microbiol Biotechnol. 2001 Jul;3(3):445-56
references.pmid: 11361077
references.citation: Nat Biotechnol. 2005 Feb;23(2):195-200
references.pmid: 15665824
references.citation: J Mol Microbiol Biotechnol. 2007;13(1-3):147-55
references.pmid: 17693722
references.citation: Appl Microbiol Biotechnol. 2004 Mar;64(1):86-90
references.pmid: 14564486
references.citation: J Mol Biol. 1983 Jun 5;166(4):557-80
references.pmid: 6345791
references.citation: J Gen Microbiol. 1962 Dec;29:589-601
references.pmid: 14025877
references.citation: Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110-4
references.pmid: 4559594
references.citation: Arch Microbiol. 2009 May;191(5):477-83
references.pmid: 19340412
references.citation: Gene. 1995 Dec 1;166(1):175-6
references.pmid: 8529885
references.citation: Biosci Biotechnol Biochem. 2009 Jan;73(1):241-4
references.pmid: 19129636
references.citation: J Bacteriol. 1995 May;177(10):2637-43
references.pmid: 7751271
references.citation: Gene. 1994 Jul 22;145(1):69-73
references.pmid: 8045426