This title appears in the Scientific Report :
2006
Please use the identifier:
http://hdl.handle.net/2128/2467 in citations.
Untersuchungen zur 5-keto-D-Gluconat Bildung mit Gluconobacter oxydans
Untersuchungen zur 5-keto-D-Gluconat Bildung mit Gluconobacter oxydans
The aim of this work was a rational strain development of the acetic acid bacterium Gluconobacter oxydans DSM 2343 for whole-cell-biotranformation of glucose to 5-keto- D-gluconic acid (5-KGA), a precursor of L-(+)-tartaric acid. G. oxydans exhibits two separately localised enzyme systems for the ox...
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Personal Name(s): | Merfort, Marcel (Corresponding author) |
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Contributing Institute: |
Biotechnologie 1; IBT-1 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2006
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Physical Description: |
V, 102 S. |
Dissertation Note: |
Düsseldorf, Univ., Diss., 2006 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
Biotechnologie |
Series Title: |
Berichte des Forschungszentrums Jülich
4222 |
Subject (ZB): | |
Link: |
OpenAccess |
Publikationsportal JuSER |
The aim of this work was a rational strain development of the acetic acid bacterium Gluconobacter oxydans DSM 2343 for whole-cell-biotranformation of glucose to 5-keto- D-gluconic acid (5-KGA), a precursor of L-(+)-tartaric acid. G. oxydans exhibits two separately localised enzyme systems for the oxidation of glucose to 5-KGA. On the one hand, there is a system of pyrrolochinoline-quinonedependent (PQQ) dehydrogenases which are orientated to the periplasmatic space. On the other hand, there are cytosolic, NADP$^{+}$-dependent oxidoreductases which perform the oxidation of glucose to 5-KGA, too. Besides the favoured oxidation product 5-KGA, G. oxydans was also able to accumulate the undesired structural isomer, 2-keto-Dgluconic acid (2-KGA). By inserting a gene-sequence of a kanamycine-resistance protein into the chromosomal part of the gluconate-2-dehydrogenase (mGA2DH), the formation of 2-KGA was completely prevented. This action led to an increase in the total 5-KGA-formation rate and final concentration, respectively. By homologues overexpression of the cga5dh-gene, which encodes for a cytosolic NADP$^{+}$-dependent gluconate-5-dehydrogenase (cGA5DH), the 5-KGA concentration could be enhanced by 20%, as compared to the wild-type. To ensure the availability of the necessary cofactor NADP$^{+}$, the gene for a transhydrogenase (UDHA) of E. coli was heterologously coexpressed thus guaranteeing the reoxidation of NADPH via NAD and the respiratory chain. By inactivating the mga5dh-gene it could be demonstrated that the membrane-bound gluconate-5-dehydrogenase (mGA5DH) was the enzyme mainly responsible for 5-KGA formation. The resulting mutant strain showed a basal 5-KGA formation of 20% as compared to the wild-type strain. Exchanging the native promoter of the mga5dh-gene against the stronger tufB and mgdh-promoter led to an increase of the 5-KGA formation activity and the final 5-KGA concentration. Finally, a 5-KGA production strain could be developed, which showed an enhanced final 5-KGA concentration (300 mM), which was 20-fold higher than that of the wild-type strain. |