Untersuchungen zur L-Serin-Bildung mit Corynebacterium glutamicum
L-serine is a very important precursor of many cellular metabolites with a central position in the metabolism of corynebacterium glutamicum. This is especially because of the reaction catalyzed by the serine hydroxymethyltransferase (SHMT) in which serine is degraded to glycine and a C1 unit. It was an aim of this work to analyze the formation of the pharmaceutical important L-serine with metabolic engineering. Another aim was to analyze the regulation of the glyA-gene, coding for the SHMT.1. The wildtype of c. glutamicum does not accumulate L-serine and the overexpression of the serine pathway genes serA(fbr)CB only leads to a small increase in serine production to 0,05 mM. If the L-serine dehydratase is deleted, up to 3 mM L-serine is accumulated. With an insertion of the tac-promoter into the chromosome it was possible, to decrease the specific-activity of the essential SHMT from 30 to 1.3 nmol/min*mg protein and a very high production of 100 mM L-serine was achieved. Another further increase to 140 mM was gained by deleting the gene coding for the pyruvate kinase.2. The strain with glyA under control of the tac-promoter was instable, which was due to a mutation A13T in the LacIq-repressor protein. Because of this instability the genes for the folate-biosynthesis pabABC were deleted, because folate is needed for the synthesis of the SHMT-cofactor tetrahydrofolate. Strains with deleted pabABC-genes need 1 mM folate or p-aminobenzoate for growth. Folate-deficiency, overexpression of serA(fbr)CB and deletion of the L-serine dehydratase leaded to a very strong increase in L-serine production to 600 mM in a 20 l fermenter.3. The -10- and the -35-region of the glyA-gene could be determined by primer-extension-analysis. The transcriptional start site is 64 bp before the glyA-translational start site. Transcriptional fusions of the glyA-promoter with the chloramphenicol-acetyltransferase gene showed, that the glyA-promoter is – with 2 U/mg protein – a very strong promoter. With affinity chromatography two regulatory proteins were isolated which bind specifically to the promoter of the glyA-gene: ArsR and RamB. The ArsR-protein binds specifically to the glyA-gene in presence of 200 µM nickel(II) and enhances the glyA-expression by 20-25% in the stationary phase.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, L-Serin, Aminosäuren, SHMT, Produktion, L-serine, amino acids, SHMT, production
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