Bettina Möckel

The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins

The complete genomic sequence of Corynebacterium glutamicum ATCC 13032, well-known in industry for the production of amino acids, e.g. of L-glutamate and L-lysine was determined. The C. glutamicum genome was found to consist of a single circular chromosome comprising 3282708 base pairs. Several DNA regions of unusual composition were identified that were potentially acquired by horizontal gene transfer, e.g. a segment of DNA from C. diphtheriae and a prophage-containing region. After automated and manual annotation, 3002 protein-coding genes have been identified, and to 2489 of these, functions were assigned by homologies to known proteins. These analyses confirm the taxonomic position of C. glutamicum as related to Mycobacteria and show a broad metabolic diversity as expected for a bacterium living in the soil. As an example for biotechnological application the complete genome sequence was used to reconstruct the metabolic flow of carbon into a number of industrially important products derived from the amino acid L-aspartate.

Biotechnological manufacture of lysine.

L-Lysine has been manufactured using Corynebacterium glutamicum for more than 40 years. Nowadays production exceeds 600,000 tons per year. Based on conventionally bred strains, further improvement of lysine productivity has been achieved by genetic engineering. Pyruvate carboxylase, aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase and the specific lysine exporter were shown to be key enzymes for lysine production and were characterized in detail. Their combined engineering led to a striking increase in lysine formation. Pathway modeling with data emerging from 13C-isotope experiments revealed a coordinated flux through pentose phosphate cycle and tricarboxylic acid cycle and intensive futile cycling between C3 compounds of glycolysis and C4 compounds of tricarboxylic acid cycle. Process economics have been optimized by developing repeated fed-batch techniques and technical continuous fermentations. In addition, on-line metabolic pathway analysis or flow cytometry may help to improve the fermentation performance. Finally, the availability of the Corynebacterium glutamicum genome sequence has a major impact on the improvement of the biotechnological manufacture of lysine. In this context, all genes of the carbon flow from sugar uptake to lysine secretion have been identified and are accessible to manipulation. The whole sequence information gives access to post genome technologies such as transcriptome analysis, investigation of the proteome and the active metabolic network. These multi-parallel working technologies will accelerate the generation of knowledge. For the first time there is a chance of understanding the overall picture of the physiological state of lysine overproduction in a technical environment.

Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: Use of a cosmid and a bacterial artificial chromosome library

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.

Identification and characterization of the last two unknown genes, dapC and dapF, in the succinylase branch of the L-lysine biosynthesis of Corynebacterium glutamicum

The inspection of the complete genome sequence of Corynebacterium glutamicum ATCC 13032 led to the identification of dapC and dapF, the last two unknown genes of the succinylase branch of the L-lysine biosynthesis. The deduced DapF protein of C. glutamicum is characterized by a two-domain structure and a conserved diaminopimelate (DAP) epimerase signature. Overexpression of dapF resulted in an 8-fold increase of the specific epimerase activity. A defined deletion in the dapF gene led to a reduced growth of C. glutamicum in a medium with excess carbon but limited ammonium availability. The predicted DapC protein of C. glutamicum shared 29% identical amino acids with DapC from Bordetella pertussis, the only enzymatically characterized N-succinyl-aminoketopimelate aminotransferase. Overexpression of the dapC gene in C. glutamicum resulted in a 9-fold increase of the specific aminotransferase activity. A C. glutamicum mutant with deleted dapC showed normal growth characteristics with excess carbon and limited ammonium. Even a mutation of the two genes dapC and ddh, interrupting both branches of the split pathway, could be established in C. glutamicum. Overexpression of the dapF or the dapC gene in an industrial C. glutamicum strain resulted in an increased L-lysine production, indicating that both genes might be relevant targets for the development of improved production strains.