Verlag des Forschungszentrums Jülich
JUEL-4259
A recombinant strain of E. coli for obtaining D-mannitol from D-fructose was the reference
system for studying whether the mutations in the NAD metabolism influenced the long-term
stability of whole-cell transformations. Here, D-fructose was reduced to D-mannitol by a
mannitol dehydrogenase from Leuconostoc pseudomesenteroides. The cofactor NADH was
regenerated by a formate dehydrogenase from Mycobacterium vaccae N10. The uptake of Dfructose
was made possible by a glucose facilitator from Zymomonas mobilis. In the present
study, it was found that during the biotransformation the cells lost NAD(H) via a
permeabilized cell membrane. The cause of cell permeabilization was found to be an
accumulation of D-mannitol in the cell, which led to bursting of the cell due to increased
osmotic pressure. By cloning a putative mannitol transport gene from L.
pseudomesenteroides, it was possible to increase the long-term stability of the recombinant E.
coli, so that after three days of biotransformation the mannitol yield was increased by 20 %.
Heuser, Florian
Einfluss des NAD(H) Pools auf die Produktivität bei Ganzzellbiotransformationen
110 S., 2008
The aim of this work was to limit the intracellular NAD turnover and to increase the
intracellular NAD concentration in Escherichia coli in order to construct a strain
characterized by higher productivity and improved long-term stability in the reductive wholecell
biotransformation. To this end, first the genes yrfE and yjaD were deleted, which code for
the NADH-pyrophosphatases that probably play a part in intracellular NAD degradation.
After deletion of these genes, the specific activity of the NADH-pyrophosphatase in the raw
extract was reduced by 70 %. By overexpressing the genes pncB and nadE from the NAD
biosynthesis it was possible to double the intracellular NAD concentration. The joint
overexpression of the two genes increased the NAD pool by a factor of seven.
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