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@PHDTHESIS{Klein:50485,
author = {Klein, Mathias Carsten},
othercontributors = {Hartmeier, Winfried},
title = {{M}olekulargenetische und reaktionskinetische
{U}ntersuchungen nativer und rekombinanter saccharolytischer
{C}lostridien zur {O}ptimierung der biologischen
{W}asserstoffproduktion},
address = {Aachen},
publisher = {Publikationsserver der RWTH Aachen University},
reportid = {RWTH-CONV-113028},
pages = {VI, 132 S. : Ill., graph. Darst.},
year = {2009},
note = {Aachen, Techn. Hochsch., Diss., 2009},
abstract = {Saccharolytic clostridia produce a wide range of
industrially relevant substances which explains their
intensive exploration concerning biotechnological
applications. Besides butyric and acetic acid and the
organic solvents butanol and acetone molecular hydrogen is
one of the major fermentation products of these obligate
anaerobic organisms. Many clostridia applied for biological
hydrogen production are still not characterised genetically.
The profitable biotechnological production of hydrogen,
especially as an alternative energy carrier still affords
the enhancement of product yields as well as volumetric
productivities. Besides technical process optimisations
genetically engineered production strains may be a promising
option. An organism often applied for biological hydrogen
production is C. butyricum. To further characterise this
organism several genes coding for enzymes of the energy
metabolism were cloned by genome walk. The activity of the
[FeFe]-hydrogenase could be demonstrated by its coexpression
with the corresponding maturation proteins in E. coli
BL21(DE3). Furthermore Thiolase was purified by affinity
chromatography and its biochemical and kinetic properties
were determined. The physiological condensation of
acetyl-CoA was measured applying a coupled enzymatic assay
where beta-hydroxybutyryl-CoA dehydrogenase from the same
organism was used as the second enzyme. As all biosynthetic
thiolases the native enzyme from C. butyricum forms
homotetramers in solution and its kinetic characteristics
were comparable to the ones from closely related enzymes.
Sequence analysis of the cryptic plasmid pCB101 from C.
butyricum revealed the gene coding for the bacteriocin
butyricin 7423. It was expressed as a fusion protein in E.
coli and its activity was confirmed by plate diffusion
assays employing the purified protein. The bacteriocin
exhibited activity only on clostridia and several Bacillus
species. Gelfiltration revealed that the protein forms large
soluble aggregates and for further biochemical
characterisation its isoelectric point was determined. To
optimise the biological hydrogen production of C.
acetobutylicum genetically modified strains were developed.
The genes coding for the [FeFe]-hydrogenases from C.
butyricum and C. acetobutylicum were cloned into a suitable
shuttle-vector and C. acetobutylicum was transformed with
the constructed vectors by electroporation. The recombinant
strains were subjected to fermentations and plasmids as well
as the corresponding transcripts could be detected in
withdrawn cell samples. The overexpression of the plasmid
encoded endogenous hydrogenase was verified by western blot
analysis and determination of the specific enzyme activities
whereas the expression of the C. butyricum hydrogenase was
rather low. Fermentation patterns of the recombinant strains
did not show any alteration at several applied conditions.
The hydrogen yields and productivities were comparable to
those of the control strains indicating that the cells’
hydrogenase concentrations are not limiting for the
biotechnological hydrogen production. Another strategy
involved the construction of shuttle-vectors for the
expression of antisense-RNAs (asRNAs) targeting the
transcript of the C. acetobutylicum biosynthetic thiolase to
alter the organic acid ratios and therefore influence the
produced hydrogen yields. Again no alteration of the
fermentation pattern could be observed although the
corresponding transcripts could be detected specifically in
cells derived from the conducted fermentations. In all cases
the cellular specific thiolase activities were reduced
drastically compared to those of the control strains.
Therefore the reduction of the intracellular thiolase
concentration does not directly influence the organism’s
metabolism. In summary no optimisation of the biological
hydrogen production of C. acetobutylicum could be achieved
by means of metabolic engineering.},
keywords = {Clostridium butyricum (SWD) / Clostridium (SWD) /
Gentechnologie (SWD) / Wasserstoff (SWD)},
cin = {162610 / 160000},
ddc = {570},
cid = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$},
typ = {PUB:(DE-HGF)11},
urn = {urn:nbn:de:hbz:82-opus-27088},
url = {https://publications.rwth-aachen.de/record/50485},
}
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