Group Leader: Dr. Lothar Eggeling
Worldwide more then 1.7 × 106 tonnes of amino acids are produced annually, mostly used as feed and food additives. L-glutamate and L-lysine, which represent the major production volume, are produced exclusively with the apathogenic Corynebacterium glutamicum. Our work is focused on the quantification and improvement of the cellular fluxes necessary for overproduction of metabolites. This is done by molecular and physiological means and carried out in close connection with industrial and international collaborations. Current research activities concentrate on L-lysine, L-isoleucine, and L-serine oversynthesis by C. glutamicum, as well as the characterization of lipid and arabinogalactan synthesis in C. glutamicum whose cell wall resembles in many respects that of Mycobacterium tuberculosis. The current research group consists of technicians, postdocs, PhD students and undergraduates. Guests are welcome.
Sensors for the quantification of metabolites in single cells
Bacteria must be improved to be useful in industrial processes. To expedite metabolic engineering, we have developed a new general tool to visualize the cytosolic concentration of small molecules at the single cell level of bacteria such as E. coli or C. glutamicum. The system is based on transcriptional regulators sensing the molecule of interest and which, in response to this signal, drive transcription of an autofluorescence protein. We have constructed individual sensors to monitor the concentration of L-lysine, O-acetyl-serine, or L-serine in C. glutamicum, and in E. coli the concentration of L-arginine. In one example, we screened a population of C. glutamicum carrying the L-lysine sensor for mutants exhibiting increased L-lysine formation. Within a few minutes, 6.5 x 106 cells were screened by fluorescence-activated cell sorting (FACS) and 270 cells were selected, of which 225 accumulated 3-38 mM L-lysine. From 10 mutants with no mutation in known targets, the entire genome was sequenced using Illumina HiSeq 2000 technology. As a new target, a murE mutation was identified, which improved the L-lysine titers significantly when introduced into existing L-lysine producers. In another example, we used our technology together with a microfluidic chip device to follow arginine synthesis in single cells by time-lapse microscopy.
Engineering an L-serine producer
Overproduction of L-serine was a big challenge, since this amino acid is rather a central metabolite than the endproduct of a pathway. Using 13-C-NMR studies we found that L-serine is not only degraded to glycine to generate the C-1 compound 5,10-methylenetetrahydrofolate but also to pyruvate. A serine dehydratase was identified and deleted. Moreover, a no longer inhibitable 3-phosphoglycerate dehydrogenase was engineered. A key step in engineering was to place the glyA encoded essential serine hydroxymethyltransferase in the genome under control of an adjustable promoter to reduce expression. Thus deletion, together with down regulation of glyA and overexpression of the three L-serine biosynthesis genes led to a producer strain with highest specific productivities and titers not previously listed.
Cell Wall and lipid synthesis
C. glutamicum grows fast, is easy to handle, and apathogenic but shares with the pathogenic Mycobacterium tuberculosis many features of cell wall biosynthesis and structure. A number of genes apparently related to cell wall synthesis are syntenic. We found that two carboxylase β-subunits (DtsR2, DtsR3) are necessary for mycolic acid synthesis and that these together with the biotin-carrying α-subunit (AccBC) form a complex. The complex is involved in carboxylation of one acyl-chain (the merochain) to result in mature mycolic acid. The mycolic acids are α-branched β-hydroxylated fatty acids which are either bound to arabinogalactan or to trehalose to form an outer lipid bilayer as typically known for Gram-negative bacteria. This outer lipid bilayer is a permeability barrier, and therefore both of relevance for substrate import and naturally of course also for product export. Current efforts focus on arabinogalactan synthesis.