Publikationsserver der RWTH Aachen University
Molekulargenetische und reaktionskinetische Untersuchungen nativer und rekombinanter saccharolytischer Clostridien zur Optimierung der biologischen Wasserstoffproduktion
Abstract
dc:descriptionSaccharolytic 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.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2009
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Klein, Mathias Carsten
- Contributors dc:contributor
-
- Hartmeier, Winfried
Subjects
dc:subject × 13Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger