BioVT-2010-16 [BibTeX]
Thomas Palmen, Jens Nieveler, Bettina Frölich, Wiltrud Treffenfeldt, Martina Pohl, Jochen Büchs:
Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli
Microbial Cell Factories, 2010, 9:76
Abstract:
Background:
The benzoylformate decarboxylase (BFD) from Pseudomonas putida is a biotechnologically interesting
biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for
stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to
improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino
acid composition of a protein does not significantly influence the level of expression or media requirements. To
determine, which effects these modifications might have on cultivation and product formation, six different BFDvariants
with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009
pKK233-2. The oxygen transfer rate (OTR) as parameter for growth and metabolic activity of the different E. coli
clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity
MOnitoring System (RAMOS).
Results:
Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity
surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished.
Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro) had typical OTR curves, the second
type (clones expressing the wild type BFD, Ser181Thr or His281Ala) showed an early drop of OTR in LB and TB
medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone
expressing Leu476Gln) behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the
first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the
concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor
binding strength of the different BFD-variants correlated with the differences in metabolic activity of their
respective host strain.
Conclusions:
The BFD-variants with high cofactor binding affinity (wild type, His281Ala, Ser181Thr) obviously
extract thiamine from the medium and bind it tightly to the enzyme. This might explain the hampered growth of
these clones. In contrast, growth of clones expressing variants with low cofactor binding affinity (Leu476His,
Leu476Pro, Leu476Pro-Ser181Thr) is not impaired. Leu476Gln has an intermediate cofactor binding strength, thus,
growth of its host strain depends on the specific cultivation conditions. This paper shows that slight differences of
the amino acid composition can affect protein expression and cultivation and might require an adaptation of
media components. Effects such as the observed are hardly foreseeable and difficult to detect in conventional
screening processes. Via small scale experiments with on-line measurements in shake flasks such effects influencing
the cultivation and product formation can be detected and avoided.
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