The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins
Introduction
In the mid-1950s, Kinoshita and co-workers in Japan isolated a bacterium, which was shown to excrete large quantities of l-glutamic acid into the culture medium (Kinoshita et al., 1957). This bacterium, Corynebacterium glutamicum, was described as a short, aerobic, gram-positive rod capable of growing on a variety of sugars or organic acids. Under optimal conditions, this organism converted glucose into high yields of l-glutamic acid within a few days. Currently about 1×106 tons of this amino acid are produced with this microorganism annually and used as a flavoring agent (Leuchtenberger, 1996). During the past 40 years, various mutants of C. glutamicum have been isolated with the capacity to produce significant amounts of different l-amino acids. Today, l-lysine is produced with mutants deregulated in the biosynthetic pathway on a scale of 4.5×105 tons per year. This amino acid is mainly used as a feed additive.
The common practice of developing amino acid-overproducing strains by mutagenesis and selection is a very well established technique (Rowlands, 1984). Mutagenic procedures were optimized in terms of the mutagen used and the dose applied. Selection procedures were designed to allow maximum expression and detection of the desirable mutant types. So far the improvement of amino acid-producing C. glutamicum strains has mainly been carried out by an iterative procedure of mutagenesis and selection. However, the precise genetic and physiological changes resulting in an increased overproduction of amino acids in various C. glutamicum strains remained unknown. Future success in attempts to further increase the productivity and yield of already highly productive strains will depend on the availability of detailed information on the metabolic pathways, their regulations, and their mutations. In recent years, genetic engineering has become a fascinating alternative to mutagenesis and random screening procedures (Sahm et al., 1995). Overexpression or deletion of genes in microorganisms via recombinant DNA techniques is the most powerful method for the construction of strains with the desired genotype. Furthermore, this approach avoids the complication of uncharacterized mutations that are often obtained with classical mutagenesis.
Since the mid-1980s, several genes from the biosynthetic pathways leading to the aspartate-derived amino acids l-lysine, l-threonine, and l-isoleucine, as well as to the vitamin D-pantothenate in C. glutamicum have been cloned and analyzed (Sahm et al., 2000). These genes were mainly identified by heterologous complementation of Escherichia coli mutants, and occasionally, in the homologous system by conferring an amino acid-analog resistance. These studies already led to a general understanding of metabolic pathways, but a complete picture of the complex interactions could not be achieved due to the lack of detailed genetic information. Genomic sequencing followed by automatic and manual annotation turned out to represent the ideal method to obtain the missing genetic information for the development of industrial C. glutamicum strains. For this reason, we decided in 1998 to sequence the genome of C. glutamicum (Hodgson, 1998), sometimes also referred as Brevibacterium divaricatum, B. flavum, B. lactofermentum, or C. melassecola (Liebl et al., 1991, Kämpfer and Kroppenstedt, 1996). The sequencing strategy was to use large-insert libraries, e.g. cosmid- and BAC-clones for establishing the complete genome sequence (Tauch et al., 2002a). We now report on the completed genomic sequence of the type strain C. glutamicum ATCC 13032. The genome data provide a rich source for metabolic reconstruction of the pathways leading to industrially important products derived from the amino acid l-aspartate.
During our sequencing work, we learned that due to its outstanding biotechnological relevance, the genome of C. glutamicum was sequenced independently by different groups. The Japanese company Kyowa Hakko Kogyo Co., Ltd. established a sequence independently from our project and put it into the public databases (GenBank NC_003450). Its market competitor, Ajinomoto Co. sequenced a close relative, C. efficiens, an organism isolated by researchers of this company (Fudou et al., 2002). The sequence of this strain was released recently in the GenBank database (NC_004369).
Section snippets
Assembly and annotation of the C. glutamicum ATCC 13032 genome sequence
The complete genome sequence of C. glutamicum ATCC 13032 was determined from 116 overlapping genomic clones. Of these, 95 were isolated from an ordered SuperCos I cosmid library (Bathe et al., 1996), and 21 were selected from a set of 2304 bacterial artificial chromosomes (BACs) upon mapping to cosmid contig ends by colony hybridization and terminal BAC sequencing (Tauch et al., 2002a). The cosmid library alone covered only 86.6% of the C. glutamicum genome and the ordered BAC library was
The structure of the C. glutamicum ATCC 13032 genome
General features of the C. glutamicum genome sequence are shown in Table 1 and Fig. 1. The C. glutamicum genome is represented by a circular chromosome of 3 282 708 bp, which is smaller than the genome of the taxonomically related bacterium M. tuberculosis (4.2 Mb), but larger than that of its close relative C. diphtheriae (2.5 Mb). The G+C content of the genome is 53.8%, which is close to that of E. coli and rather unusual for the taxonomic class of the Actinobacteria referred to as ‘high G+C
Annotation of coding regions
Gene finding tools in conjunction with homology searches in databases and an additional expert annotation with the genome annotation tool GenDB (Meyer et al., 2003) revealed 3002 potential protein-coding genes in the C. glutamicum genome sequence (Table 1). To 2489 of these, at least putative functions or localizations could be assigned by similarity analyses. Of the remaining predicted genes, 250 are similar to hypothetical proteins in other organisms (conserved hypothetical proteins) and only
Metabolic reconstruction of the biosyntheses of aspartate-derived amino acids and vitamins from glucose
A number of metabolites of biotechnological importance are derived from the amino acid l-aspartate. These are l-lysine, l-threonine, l-methionine and l-isoleucine. Two others compounds, the amino acid l-valine and the vitamin D-pantothenate, are strongly interconnected to the synthesis of aspartate-derived amino acids and were, therefore, included into this study. For the reconstruction of the formation of all these compounds from glucose, several functional complexes have to be considered.
Conclusions
The establishment of a completely annotated C. glutamicum genome sequence is a big leap forward to the understanding of the biology of this organism and will boost metabolic engineering to overproduce compounds of biotechnological relevance. It helped to identify missing genes to close the respective biosynthetic pathways directly or by providing a limited number of candidate genes to be tested. The complete genome sequence is the basis for extensive expression analyses by proteome and
Acknowledgements
The C. diphtheriae sequence data were produced by the C. diphtheriae Sequencing Group at the Sanger Institute and obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/cdip/. The work was supported by grant 031U213D of the Bundesministerium für Bildung und Forschung.
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