L-Cystein-Bildung mit Corynebacterium glutamicum und optische Sensoren zur zellulären Metabolitanalyse

L-Cysteine is an important amino acid and is in addition to L-methionine the most abundant sulfur containing molecule inside the cell. It provides reduced sulfur for the synthesis of a variety of other sulfur containing compounds. Besides its importance for microbial growth there is also a growing interest for L-cysteine in the food, pharmaceutical and cosmetics industries and therefore a need for the production of L-cysteine by fermentation exists. Consequently, the main objective of this study was to construct a L-cysteine producer based on an efficient Corynebacterium glutamicum L-serine producing strain. Furthermore the development of metabolite sensors for intracellular detection of sulfur availability should be achieved. The following results were obtained: (1) The L-serine producer Ser4 contains the plasmid pserACB. To facilitate further genetic work concerning L-cysteine formation with this strain, the production of L-serine without a plasmid was desirable. The chromosomal integration of the gene serAfbr, coding for a feedback-resistant 3-phosphoglycerate dehydrogenase, led already to slight L-serine formation. Studies on a promoter exchange of serAfbr in combination with plasmid based expression of the genes serC and serB revealed that strong expression of serAfbr together with that of serC and serB is necessary to obtain high L-serine production. (2) In contrast to the expression of genes encoding heterologous feedback-resistant serine acetyltransferases the increased expression of native cysE from C. glutamicum led to improvement of L-cysteine formation from 0.95 ± 0.35 mM to 5.6 ± 0.8 mM in the Lserine producer. In combination with expression of cysK, coding for the O-acetylserine sulfhydrylase, 7.3 ± 0.7 mM L-cysteine accumulated in the culture supernatant. The deletion of genes encoding cysteine desulfurase AecD to prevent L-cysteine degradation and homoserine actyltransferase MetX to disable L-methionine synthesis as well as the expression of the operon fpr2-cysIXHDNYZ necessary for assimilatory sulfate reduction had a moderate effect on L-cysteine formation. (3) To construct metabolite sensors for the detection of the sulfur acceptor molecules Oacetyl- L-serine (OAS) and O-acetyl-L-homoserine (OAH) promoter regions of genes NCgl1289 and cysI including CysR-binding sites were fused to the reporter gene eyfp. Additionally, the respective sensor cassettes contained the gene of transcriptional regulator CysR, which activates the genes for the assimilatory sulfate reduction. It could be shown that intracellular elevated levels of OAS and OAH led to increased specific fluorescence at the single cell level and that cultures cultivated under limiting sulfur conditions showed increased fluorescence. (4) To analyze the impact of the transcriptional regulator CysR on L-cysteine formation the gene cysR was expressed under control of the native promoter as well as Ptac. It could be shown, that increased expression of CysR does not necessarily improve the L-cysteine formation. The results indicate the delicate regulation of sulfur metabolism in C. glutamicum, in which the three transcriptional regulators CysR, McbR and SsuR are involved.