Miroslav Pátek

Biodegradation potential of the genus Rhodococcus.

A large number of aromatic compounds and organic nitriles, the two groups of compounds covered in this review, are intermediates, products, by-products or waste products of the chemical and pharmaceutical industries, agriculture and the processing of fossil fuels. The majority of these synthetic substances (xenobiotics) are toxic and their release and accumulation in the environment pose a serious threat to living organisms. Bioremediation using various bacterial strains of the genus Rhodococcus has proved to be a promising option for the clean-up of polluted sites. The large genomes of rhodococci, their redundant and versatile catabolic pathways, their ability to uptake and metabolize hydrophobic compounds, to form biofilms, to persist in adverse conditions and the availability of recently developed tools for genetic engineering in rhodococci make them suitable industrial microorganisms for biotransformations and the biodegradation of many organic compounds. The peripheral and central catabolic pathways in rhodococci are characterized for each type of aromatics (hydrocarbons, phenols, halogenated, nitroaromatic, and heterocyclic compounds) in this review. Pathways involved in the hydrolysis of nitrile pollutants (aliphatic nitriles, benzonitrile analogues) and the corresponding enzymes (nitrilase, nitrile hydratase) are described in detail. Examples of regulatory mechanisms for the expression of the catabolic genes are given. The strains that efficiently degrade the compounds in question are highlighted and examples of their use in biodegradation processes are presented.

Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif.

Relatively limited information about promoter structures in Corynebacterium glutamicum has been available until now. With the aim of isolating and characterizing such transcription initiation signals, random Sau3A fragments of C. glutamicum chromosomal DNA and of the corynebacterial phage phi GA1 were cloned into the promoter probe vector pEKplCm and selected for promoter activity by chloramphenicol resistance of transformed C. glutamicum cells. The nucleotide sequence of ten chromosomal and three phage fragments was determined and the transcriptional start (TS) sites were localized by primer extension analyses. Additionally, the promoters of five previously isolated C. glutamicum genes were cloned and mapped. All of the isolated promoters were also functional in the heterologous host Escherichia coli. A comparative analysis of the newly characterized promoter sequences together with published promoters from C. glutamicum revealed conserved sequences centred about 35 bp (ttGcca) and 10 bp (TA.aaT) upstream of the TS site. The position of these motifs and the motifs themselves are comparable to the -35 and -10 promoter consensus sequences of other Gram-positive and Gram-negative bacteria, indicating that they represent transcription initiation signals in C. glutamicum. However, the C. glutamicum consensus hexamer of the -35 region is much less conserved than in E. coli, Bacillus, Lactobacillus and Streptococcus.

Promoters of Corynebacterium glutamicum.

Regulation of gene expression in Corynebacterium glutamicum represents an important issue since this Gram-positive bacterium is a notable industrial amino acid producer. Transcription initiation, beginning by binding of RNA polymerase to the promoter DNA sequence, is one of the main points at which bacterial gene expression is regulated. More than 50 transcriptional promoters have so far been experimentally localized in C. glutamicum. Most of them are assumed to be promoters of vegetative genes recognized by the main sigma factor. Although transcription initiation rate defined by many of these promoters may be affected by transcription factors, which activate or repress their function, the promoter regions share common sequence features, which may be generalized in a consensus sequence. In the consensus C. glutamicum promoter, the prominent feature is a conserved extended -10 region tgngnTA(c/t)aaTgg, while the -35 region is much less conserved. Some commonly utilized heterologous promoters were shown to drive strong gene expression in C. glutamicum. Conversely, some C. glutamicum promoters were found to function in Escherichia coli and in other bacteria. These observations suggest that C. glutamicum promoters functionally conform with the common bacterial promoter scheme, although they differ in some sequence structures.

Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum

LysE of Corynebacterium glutamicum belongs to a large new superfamily of translocators whose members are probably all involved in the export of small solutes. Here, the transcript initiation site of lysE, and its divergently transcribed regulator gene, lysG, are identified. Single-copy transcriptional fusions of lysE with lacZ, and titration experiments, show that LysG is the positive regulator of lysE expression enabling its up to 20-fold induction. This induction requires the presence of a coinducer, which is either intracellular L-lysine, or L-arginine. A competition experiment showed that LysE exports these two basic amino acids at comparable rates of about 0.75 nmol min(-1) (mg dry wt)(-1). Although L-histidine and L-citrulline also act as coinducers of lysE expression, these two amino acids are not exported by LysE. As is evident from the analysis of a lysEG deletion mutant, the physiological role of the lysEG system is to prevent bacteriostasis due to elevated L-lysine or L-arginine concentrations that arise during growth in the presence of peptides or in mutants possessing a deregulated biosynthesis pathway. C. glutamicum has additional export activities other than those of LysE for exporting L-histidine, L-citrulline and L-ornithine.

Export of l-Isoleucine from Corynebacterium glutamicum: a Two-Gene-Encoded Member of a New Translocator Family

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of L-leucine and L-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in alpha-proteobacteria but not in other prokaryotes analyzed.

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

ABSTRACT Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain 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 aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Kmr). 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 ΔilvA ΔpanB 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.

Sigma factors and promoters in Corynebacterium glutamicum.

The Corynebacterium glutamicum genome codes for 7 sigma subunits (factors) of RNA polymerase (RNAP): primary sigma factor SigA (σ(A)), primary-like SigB and 5 other alternative sigma factors (SigC, SigD, SigE, SigH and SigM). Each sigma factor is responsible for recognizing promoters of genes belonging to a regulon (sigmulon) involved in specific functions of the cell. Most promoters of C. glutamicum housekeeping genes are recognized by RNAP+σ(A), whereas σ(B) is involved in transcription of a large group of genes active during the transition phase between the exponential and stationary growth phases when various stress factors threaten to damage the cell. The σ(H) regulon consists of the genes involved in heat shock response including those coding for regulators and other sigma factors. It seems therefore that σ(H) occupies a central position in the cross-regulated network of sigma factors and controls their concerted response to various stress conditions in C. glutamicum. The σ(M) factor was found to regulate genes responding to oxidative stress. The main role of σ(E) is to activate genes involved in response to a cell surface stress. Promoters of individual classes recognized by different sigma factors are compiled and the respective consensus sequences of their key recognition motifs (-35 and -10 regions) are derived. In a number of genes, two or more promoters controlled by the same or different sigma factors were discovered. These multiple, overlapping or dual promoters contribute to a complex gene transcription control mechanisms that integrate internal and external signals and tune gene expression in cells as required by environmental and physiological conditions.

Integrative and autonomously replicating vectors for analysis of promoters in Corynebacterium glutamicum

Autonomously replicating shuttle (Escherichia coli – Corynebacterium glutamicum) promoter-probe vector pET2 and integrative promoter-probe vector pRIM2 for C. glutamicum were constructed. Transcriptional fusions of promoter-carrying fragments to the promoterless chloramphenicol acetyltransferase gene (cat) carried by the vectors can be used to determine position, strength and regulation of the respective promoters in multicopy system (pET2) and in single-copy system (pRIM2) and to perform deletion and mutation studies of the promoters. Utility of the vectors was shown on three C. glutamicum promoters.

Rational Design of a Corynebacterium glutamicum Pantothenate Production Strain and Its Characterization by Metabolic Flux Analysis and Genome-Wide Transcriptional Profiling

ABSTRACT A “second-generation” production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, dl-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity.

Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation.

The previously constructed strain Corynebacterium glutamicumilvNM13 with acetohydroxy acid synthase, resistant to inhibition by all three branched-chain amino acids (L-valine, L-isoleucine and L-leucine), was used as a basis to develop a new type of valine producer by genetic engineering. The main strategy was to modulate expression of the genes involved in the biosynthesis of branched-chain amino acids. The activity of the promoters P-ilvD (dihydroxyacid dehydratase) and P-ilvE (transaminase) was up-modulated and the activity of the promoters P-ilvA (threonine deaminase) and P-leuA (isopropylmalate synthase) was down-modulated by site-directed mutagenesis. A constructed weak promoter of ilvA (or leuA), which was introduced into the C. glutamicum chromosome via a gene-replacement technique reduced the biosynthetic rate of isoleucine (or leucine), which lowered the mutant growth rate and increased valine production. Overexpression of ilvD and ilvE driven by the strong mutant promoters P-ilvDM7 and P-ilvEM6 resulted in an even higher level of valine production. Thus, the strain C. glutamicum ilvNM13 DeltapanB P-ilvAM1CG P-ilvDM7 P-ilvEM6, having all mutations constructed within the chromosome, produced 136 mM valine in a 48-h cultivation.