Verlag des Forschungszentrums Jülich
JUEL-4097
Gerharz, Tanja
Pyruvat-Produktion durch acetatauxotrophe Escherichia-coli-Stämme
V, 124 S., 2003
Pyruvate is a central intermediate of cellular metabolism, which is also used industrially in the
synthesis of a variety of compounds, e.g. L-Dopa, L-ephedrine or L-tryptophane. The aim of
this work was to establish a biotechnological process for the production of pyruvate based on
acetate-auxotrophic Escherichia coli strains . For this purpose, E. coli YYC202 was choosen, a
strain which lacks the aceEF genes for the pyruvate dehydrogenase complex and contains
mutations in the genes encoding pyruvate oxidase (poxB), pyruvate formate lyase (pfB) and
PEP synthetase (pps). Due to this genotype, strain YYC202 is unable to convert pyruvate to
acetyl-CoA, acetate or PEP and requires acetate for growth in glucose minimal medium. In
contrast to the E. coli wild-type strain MG1655, YYC202 converted glucose and acetate
simultaneously and possessed a very high acetate resistance. The acetate auxotrophy of
YYC202 could be complemented in this work by transformation with plasmid pGS87, a
pBR322 derivative encoding the genes of the pyruvate dehydrogenase complex.
When cultivated aerobically in glucose/acetate minimal medium, E. coli YYC202
produced up to 1 .7 mol pyruvate/mol glucose, depending on the concentrations of glucose and
acetate and on the pH. This yield is significantly higher than the yield of 1 .2 mol
pyruvate/mol glucose obtained in an alternative pyruvate production process using vitamine
auxotrophic microorganisms . With resting cells of E. coli YYC202, an almost complete
conversion of glucose to pyruvate was obtained (>1 .9 mol/mol) . Formation of the by-product
lactate, which occurred at higher cell densities, was entirely prevented by disruption of the
ldhA gene encoding NAD+-dependent D-lactate dehydrogenase, leading to increased yields
and productivity.
Transriptional profiling using whole-genome DNA microarrays revealed that pyruvate
production by E. coli YYC202/pBR322 is associated with acid stress . Several genes involved
in the acid stress response, e.g. gadA and gadB encoding glutamate decarboxylases, had
increased mRNA levels in the pyruvate producer when compared with the non-producer
YYC202/pGS87. Since the external pH was 7 in these experiments, the results suggest a
cytoplasmic acidification . Besides acid stress genes, the genes for the Na+/serine symporter
SstT and for the not yet characterised transporter YbgH showed increasedmRNA levels in the
pyruvate producer. Whether these secondary transporters are involved in pyruvate import or
export remains to be shown.
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