Dokument 2.pdf - OPUS-Datenbank - Universität Hohenheim

2 Summary
The main focus of this work was a quantitative proteome analysis of a variety of Pseudomonas
strains with respect to the biotechnological synthesis of the base chemicals
glyoxylic acid, butanol and vanillin. In addition, effects of the terpene citronellol on
the proteome of P. aeruginosa were investigated.
In a first set of experiments, the effects of glyoxylic acid and ethylene glycol, the educt of
a possible biotechnological route to glyoxylic acid, on the proteome of P. putida KT2440
and JM37 were analysed. Using a 2D-DIGE and a label-free MS-based quantification
approach it could be demonstrated that P. putida JM37 metabolizes glyoxylic acid via
three different catabolic routes, whereas P. putida KT2440 appeared to be restricted to
a single pathway. Furthermore, it was shown that the alcohol dehydrogenases PedE and
PedH as well as the aldehyde dehydrogenase PedI play a crucial role in the conversion of
ethylene glycol to glyoxylic acid in both P. putida KT2440 and JM37. All three enzymes
exhibit a high degree of homology to the respective proteins in P. aeruginosa and P.
putida U. Moreover, the increased expression of the regulatory proteins PedR1 and
PedS1 indicates a role of the conserved PedR1-regulon in the degradation of ethylene
glycol in P. putida KT2440 and JM37.
Samples for the proteome analysis of butanol metabolism were either obtained from
shaking-flask cultures or from cultivation in a bioreactor. In both samples, an increased
expression of the alcohol and aldehyde dehydrogenases (PedE, PedH, PedI) as well as
an induction of several regulatory proteins of the PedR1-Regulon was observed. This
result is consistent with the findings from the ethylene glycol experiment and suggests
a role of this regulon in the degradation of both substances. In addition, an induction
of the β-oxidation pathway was detected upon treatment with butanol. This finding is
in line with the biodegradation pathway of butanol that was suggested by Rühl et al.
(Rühl et al. (2009) Appl. Environ. Microbiol.) based on a 13 C-flux analysis. Moreover,
an induction of several membrane transport systems like the ABC-Transporter PedA,
B, C and the porin PP_2662 was observed. However, an increased biogenesis of fatty
acids as shown by Rühl et al. (Rühl et al. (2012), Microb. Biotechnol.) could not be
confirmed since no up-regulation of corresponding proteins was detected.
After growth on vanillin, P. putida KT2440 showed an increased expression of almost
all enzymes involved in the multistep conversion of protocatechuate to succinyl-
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