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Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branchedchain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and acetohydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km r ). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ⌬ilvA ⌬panB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.Corynebacterium glutamicum is used for the production of a number of amino acids, including valine. Acetohydroxy acid synthase (AHAS) (EC 4.1.3.18), also called acetolactate synthase, is the key enzyme of the pathways for biosynthesis of branched-chain amino acids (isoleucine, valine, and leucine) ( Fig. 1) and therefore is a potential target for metabolic engineering. The enzyme catalyzes decarboxylation of pyruvate and its condensation either with another molecule of pyruvate to produce acetolactate (a precursor of valine and leucine) or with 2-ketobutyrate to produce acetohydroxybutyrate (a precursor of isoleucine). The reactions play a key role in determining the relative fluxes to different end products (2). Three AHAS isoenzymes, differing in their genetic determinations, biochemical properties, and regulation of both biosynthesis and activity, were described for Escherichia coli and Salmonella enterica serovar Typhimurium (26). The isoenzymes AHAS I, II, and III are encoded by the genes ilvBN, ilvGM, and ilvIH, respectively. Expression of these genes is subject to different multivalent repression by branched-chain amino acids. Expression of the ilvGMEDA operon is controlled by transcriptional attenuation mediated by all three amino acids, whereas the ilvBN operon is controlled by attenuation mediated only by valine and leucine. Expression of ilvIH is regulated by Lrp (leucine-responsive protein) (29). The large subunits (IlvB, IlvG, and IlvI) are responsib...

The gene cluster catRABC, involved in catechol degradation, was isolated from Rhodococcus erythropolis CCM2595. The genes catA, catB, catC, and the divergently transcribed catR code for catechol 1,2-dioxygenase, cis,cis-muconate cycloisomerase, muconolactone isomerase, and an IclR-type transcriptional regulator, respectively. Measurements of catechol 1,2-dioxygenase activity showed that the expression of catA is induced by phenol but not by catechol or cis,cis-muconate. The activity of catechol 1,2-dioxygenase was repressed by succinate, but no repression by glucose was observed. The transcription start points of catA and catR were determined by primer extension analysis, and the respective promoters (P-catA and P-catR) were thus localized. Measurements of promoter activity during batch cultivation using transcriptional fusion with the gfpuv reporter gene showed that expression of the catR-catABC operon is regulated at the level of transcription. Both P-catR and P-catA are repressed by CatR, and the induction of P-catA by phenol is maintained in the absence of the repressor (in R. erythropolis DeltacatR). Two different potential binding sites for the IclR-type regulator and a recognition site for the cyclic AMP receptor protein (CRP) were identified within the intergenic region between catR and catA.

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Corynebacterium glutamicum is an important industrial producer of various amino acids with great potential for the production of other metabolites. The complete genome sequences of two C. glutamicum strains were determined and the use of genome-based approaches (transcriptomics, proteomics, metabolomics, and fluxomics) provided large amounts of data on the metabolism of this bacterium and its regulation. Many tools for genetic manipulations in C. glutamicum have been developed and used for the analysis of gene functions as well as for the construction and improvement of production strains. These tools include various types of plasmid vectors (cloning, promoter-probe, and expression vectors), DNA transfer methods, cloning heterologous genes, introducing protein secretion systems and gene replacement and genome rearrangement methods. Here we summarize the latest developments in the field of genetic engineering in C. glutamicum, give examples of the use of these new tools, and mention the challenges which stand in the way of fully implementing these tools and this acquired knowledge for the construction of superior production strains.

Novel shuttle promoter-probe vectors replicating in Escherichia coli, Corynebacterium glutamicum, and Rhodococcus erythropolis were constructed on the basis of the C. glutamicum plasmid pCG1. The vectors carry reporter genes coding for fluorescent proteins, which allow the measurement of promoter activities in vivo. The promoter-probe vector pPRE11 contains the rsgfp reporter gene, coding for a variant of green fluorescent protein (GFP) with a red-shifted excitation maximum. To ensure efficient expression of the gfp gene in R. erythropolis from the tested promoters, the promoterless gene gfpuv, with 5' end fusion with the initial six codons of the aph gene and upstream insertion of the aph Shine-Dalgarno sequence, was used as a reporter gene in the promoter-probe vector pEPR1. Insertion of the rfp reference gene, coding for a variant of the red fluorescent protein DsRed.T4 and cloned under the strong constitutive C. glutamicum promoter P-45, into the vector pEPR1 resulted in a new-generation promoter-probe vector (pRAG5). All vectors were tested using a set of mutant P-dapA promoters displaying various transcriptional activities. The vector pRAG5 is suitable for normalized measurements of promoter activities during the growth of bacterial batch cultures because estimation of the GFP-to-red fluorescent protein fluorescence ratio in strains carrying the plasmid pRAG5 with the tested promoters upstream of gfpuv avoids the influence of plasmid copy number variations on the promoter activity assay.