This title appears in the Scientific Report :
2008
Please use the identifier:
http://hdl.handle.net/2128/3095 in citations.
Reduktive Ganzzellbiotransformation zur Gewinnung chiraler Alkohole mit Bacillus megaterium und Corynebacterium glutamicum
Reduktive Ganzzellbiotransformation zur Gewinnung chiraler Alkohole mit Bacillus megaterium und Corynebacterium glutamicum
The present work studied whether the Gram-positive bacteria Bacilus megaterium and Corynebacterium glutamicum were suitable for reductive whole-cell biotransformations. The conversion of D-fructose to D-mannitol and methyl acetoacetate (MAA) to ( R)-methyl-3-hydroxybutanoate (MHB) was investigated i...
Saved in:
Personal Name(s): | Bäumchen, Carsten (Corresponding author) |
---|---|
Contributing Institute: |
Biotechnologie 1; IBT-1 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2008
|
Physical Description: |
VII, 91 p. |
Dissertation Note: |
Düsseldorf, Univ., Diss., 2007 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
Biotechnologie |
Series Title: |
Berichte des Forschungszentrums Jülich
4265 |
Subject (ZB): | |
Link: |
OpenAccess |
Publikationsportal JuSER |
The present work studied whether the Gram-positive bacteria Bacilus megaterium and Corynebacterium glutamicum were suitable for reductive whole-cell biotransformations. The conversion of D-fructose to D-mannitol and methyl acetoacetate (MAA) to ( R)-methyl-3-hydroxybutanoate (MHB) was investigated in order to characterize the recombinant strains. Formate dehydrogenase was used to regenerate the cofactor NADH. The investigation focused on the long-term stability of the biocatalysts, which was evaluated by a quantitative analysis of the intracellular NAD(H) concentration during the biotransformation. The good cofactor retention expected for the Gram-positive organisms was confirmed. Both of the species studied displayed stable productivity over a period of up to three days and thus differed significantly from the E. coli whole-cell biotransformation, which showed an 80-90 % drop in specific productivity within 8 hours. Furthermore, the sugar transport in C. glutamicum was to be characterized in more detail for the conversion of D-fructose to D-mannitol, which had proved limiting for the biotransformation. By overexpressing the glucose/fructose facilitator gene from Zymomonas mobilis it was possible to increase the productivity of C. glutamicum 5.5-fold (final concentration 470 mM of D-mannitol after 24 hours). Genome-wide homology comparisons were used to identify two proteins (CGL0181 and CGL3058) of C. glutamicum, which had great structural similarity to GLF. Overexpression and deletion experiments showed that these transporters can transport D-fructose into the cytoplasm in a non-phosphorylated form, which had not previously been shown for C. glutamicum. The D-mannitol productivity of C. glutamicum was doubled with the aid of these transporters and, in addition to B. megaterium, C. glutamicum proved to be a stable microorganism for the biotransformation. With C. glutamicum and B. megaterium, the conversion of MAA to MHB proceeded significantly more slowly and within 24 hours yielded low final concentrations (5-10 mM, 10 % conversion). A substrate turnover of 50-80 % was achieved by increasing the solvent fraction for biotransformations with substrate-coupled cofactor regeneration. Both species displayed a high solvent tolerance, which provides a starting point for more extensive work with solvent-dependent biotransformation systems. |