Dokument 2.pdf - OPUS-Datenbank - Universität Hohenheim

and acetyl-CoA (β-ketoadipate pathway). An exclusive role of vanillin dehydrogenase
(Vdh) in the first step of the vanillin degradation pathway, the conversion of vanillin to
vanillic acid, was not supported by the proteomics data as Vdh was not up-regulated
in vanillin grown cells. Other aldehyde dehydrogenases that were significantly induced
in the presence of vanillin, like conifer aldehyde dehydrogenase (PP_5120), a potential
benzaldehyde dehydrogenase (PP_1948), and the aldehyde dehydrogenases PP_2680
and PP_0545, might also catalyze this reaction. Besides the induction of the solvent extrusion
systems TtgABC and MexE, MexF, OprN, an induction of transporters specific
for vanillin and its degradation intermediates was observed. Among these transporters
were the proteins PcaK/PcaP (protocatechuate and 4-hydroxy benzoate transport) and
PP_3739/PP_3740 (putative vanillic acid transport-system). The potential of proteomics
to contribute to biotechnological strain development was demonstrated by the
accumulation of vanillin in P. putida KT2440 mutants, which were generated by Nadja
Graf (AG Altenbuchner, Institut für Industrielle Genetik, University of Stuttgart) on
the basis of these proteomics data.
Since P. putida lacks several essential genes for the degradation of terpenes, the analysis
of the terpene metabolism was carried out in P. aeruginosa. In this analysis, the
proteome response of cells grown in the presence of citronellol was compared to the
proteome of cells grown in the presence of octanoic acid or glucose. The obtained data
indicate that the degradation of citronellol is catalyzed by enzymes encoded by the liuand
atu-gene clusters and by enzymes involved in the β-oxidation of fatty acids. Only
one enzyme postulated to be part of the terpene degradation pathway, the “putative
very-long chain acyl-CoA synthetase” (PA2893), eluded identification in this study.
Instead, several acetyl- and acyl-CoA dehydrogenases could be identified, which were
up-regulated in cells grown in the presence of citronellol and might be able to catalyse
this reaction. Differences in the regulation pattern of enzymes involved in β-oxidation
between cells grown on terpenes and the ones grown on the straight chain fatty acid
octanoic acid suggest a preference of several of these enzymes for terpenes.
A second key aspect of this work involved the establishment of proteomics methods
for the analysis of complex samples, especially for the analysis of membrane proteins.
Using carbonate extraction followed by label-free MS-based quantification allowed the
identification and quantification of a significant number of hydrophobic proteins which
were not covered by the 2D-DIGE approach. In addition, the GeLCMSMS workflow
was found to be a simple and efficient method for the analysis of total bacterial lysates.
Using this method, about 30% of all proteins encoded by the P. putida KT2440 genome
could be identified and quantified. In conclusion, this work demonstrated that different
proteomics methods can substantially contribute to biotechnological strain development
and the understanding of cellular networks.
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