Pyruvat-Produktion durch acetatauxotrophe - JUWEL ...

Abstract
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 .