Pyruvat-Produktion durch acetatauxotrophe Escherichia coli-Stämme

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 $\textit{Escherichia coli}$ strains. For this purpose, $\textit{E. coli}$ YYC202 was choosen, a strain which lacks the $\textit{aceEF}$ genes for the pyruvate dehydrogenase complex and contains mutations in the genes encoding pyruvate oxidase ($\textit{poxB}$), pyruvate formate lyase ($\textit{pflB}$) and PEP synthetase ($\textit{pps}$). 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 $\textit{E.coli}$ 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, $\textit{E. coli}$ 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 $\textit{E. coli}$ 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 $\textit{ldhA}$ 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 $\textit{E. coli}$ YYC202/pBR322 is associated with acid stress. Several genes involved in the acid stress response, e.g. $\textit{gadA}$ and $\textit{gadB}$ 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 increased mRNA levels in the pyruvate producer. Whether these secondary transporters are involved in pyruvate import or export remains to be shown