Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production.

The Gram-positive Corynebacterium glutamicum is auxotrophic for biotin. Besides the biotin uptake system BioYMN and the transcriptional regulator BioQ, this bacterium possesses functional enzymes for the last three reactions of biotin synthesis starting from pimeloyl-CoA. Heterologous expression of bioF from the Gram-negative Escherichia coli enabled biotin synthesis from pimelic acid added to the medium, but expression of bioF together with bioC and bioH from E. coli did not entail biotin prototrophy. Heterologous expression of bioWAFDBI from Bacillus subtilis encoding another biotin synthesis pathway in C. glutamicum allowed for growth in biotin-depleted media. Stable growth of the recombinant was observed without biotin addition for eight transfers to biotin-depleted medium while the empty vector control stopped growth after the first transfer. Expression of bioWAFDBI from B. subtilis in C. glutamicum strains overproducing the amino acids l-lysine and l-arginine, the diamine putrescine, and the carotenoid lycopene, respectively, enabled formation of these products under biotin-depleted conditions. Thus, biotin-prototrophic growth and production by recombinant C. glutamicum were achieved.

Biotin protein ligase from Corynebacterium glutamicum: role for growth and L: -lysine production.

Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria.

AIMS: The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. METHODS AND RESULTS: Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. CONCLUSIONS: This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population.

The global gene expression response of Escherichia coli to L-phenylalanine.

We investigated the global gene expression changes of Escherichia coli due to the presence of different concentrations of phenylalanine or shikimate in the growth medium. The response to 0.5 g l(-1) phenylalanine primarily reflected a perturbed aromatic amino acid metabolism, in particular due to TyrR-mediated regulation. The addition of 5g l(-1) phenylalanine reduced the growth rate by half and elicited a great number of likely indirect effects on genes regulated in response to changed pH, nitrogen or carbon availability. Consistent with the observed gene expression changes, supplementation with shikimate, tyrosine and tryptophan relieved growth inhibition by phenylalanine. In contrast to the wild-type, a tyrR disruption strain showed increased expression of pckA and of tktB in the presence of phenylalanine, but its growth was not affected by phenylalanine at the concentrations tested. The absence of growth inhibition by phenylalanine suggested that at high phenylalanine concentrations TyrR-defective strains might perform better in phenylalanine production.

Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine.

Addition of L-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 DeltailvA DeltapanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of L-isoleucine could relieve the valine effect on VAL1 whereas L-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

DNA microarray analyses of the long-term adaptive response of Escherichia coli to acetate and propionate.

In its natural environment, Escherichia coli is exposed to short-chain fatty acids, such as acetic acid or propionic acid, which can be utilized as carbon sources but which inhibit growth at higher concentrations. DNA microarray experiments revealed expression changes during exponential growth on complex medium due to the presence of sodium acetate or sodium propionate at a neutral external pH. The adaptive responses to acetate and propionate were similar and involved genes in three categories. First, the RNA levels for chemotaxis and flagellum genes increased. Accordingly, the expression of chromosomal fliC'-'lacZ and flhDC'-'lacZ fusions and swimming motility increased after adaptation to acetate or propionate. Second, the expression of many genes that are involved in the uptake and utilization of carbon sources decreased, indicating some kind of catabolite repression by acetate and propionate. Third, the expression of some genes of the general stress response increased, but the increases were more pronounced after short-term exposure for this response than for the adaptive response. Adaptation to propionate but not to acetate involved increased expression of threonine and isoleucine biosynthetic genes. The gene expression changes after adaptation to acetate or propionate were not caused solely by uncoupling or osmotic effects but represented specific characteristics of the long-term response of E. coli to either compound.

LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli.

The function of the LysR-type regulator LrhA of Escherichia coli was defined by comparing whole-genome mRNA profiles from wild-type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent K(D) approximately 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA. The expression of flhDC (encoding FlhD(2)C(2)) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD(2)C(2) is known as the master regulator for the expression of flagellar and chemotaxis genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified. The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies. It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD(2)C(2).

Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum.

Corynebacterium glutamicum possesses both phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx) as anaplerotic enzymes for growth on carbohydrates. To analyze the significance of PCx for the amino acid production by this organism, the wild-type pyc gene, encoding PCx, was used for the construction of defined pyc-inactive and pyc-overexpressing strains and the glutamate, lysine and threonine production capabilities of these recombinant strains of C. glutamicum were tested in comparison to the respective host strains. No PCx activity was observed in the pyc-inactive mutants whereas the pyc-overexpressing strains showed eight-to elevenfold higher specific PCx activity when compared to the host strains. In a detergent-dependent glutamate production assay, the pyc-overexpressing strain showed more than sevenfold higher, the PCx-deficient strain about twofold lower glutamate production than the wild-type. Overexpression of the pyc gene and thus increasing the PCx activity in a lysine-producing strain of C. glutamicum resulted in approximately 50% higher lysine accumulation in the culture supernatant whereas inactivation of the pyc gene led to a decrease by 60%. In a threonine-producing strain of C. glutamicum, the overexpression of the pyc gene led to an only 10 to 20% increase in threonine production, however, to a more than 150% increase in the production of the threonine precursor homoserine. These results identify the anaplerotic PCx reaction as a major bottleneck for amino acid production by C. glutamicum and show that the enzyme is an important target for the molecular breeding of hyperproducing strains.

Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays.

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.

Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation.

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, approximately 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and final sigma(70)-dependent promoters.

Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose.

Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By (13)C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol. (mg of protein)(-1). min(-1), respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol. [mg of protein](-1). min(-1)) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol. [mg of protein](-1). min(-1)). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol. [mg of protein](-1). min(-1)) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.

Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria.

Escherichia coli grew in a minimal medium on propionate as the sole carbon and energy source. Initially a lag phase of 4-7 days was observed. Cells adapted to propionate still required 1-2 days before growth commenced. Incorporation of (2-13C), (3-13C) or (2H3)propionate into alanine revealed by NMR that propionate was oxidized to pyruvate without randomisation of the carbon skeleton and excluded pathways in which the methyl group was transiently converted to a methylene group. Extracts of propionate-grown cells contained a specific enzyme that catalyses the condensation of propionyl-CoA with oxaloacetate, most probably to methylcitrate. The enzyme was purified and identified as the already-known citrate synthase II. By 2-D gel electrophoresis, the formation of a second propionate-specific enzyme with sequence similarities to isocitrate lyases was detected. The genes of both enzymes were located in a putative operon with high identities (at least 76% on the protein level) with the very recently discovered prp operon from Salmonella typhimurium. The results indicate that E. coli oxidises propionate to pyruvate via the methylcitrate cycle known from yeast. The 13C patterns of aspartate and glutamate are consistent with the further oxidation of pyruvate to acetyl-CoA. Oxaloacetate is predominantly generated via the glyoxylate cycle rather than by carboxylation of phosphoenolpyruvate.

Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes.

In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.

Carbohydrate metabolism in Thermoproteus tenax: in vivo utilization of the non-phosphorylative Entner-Doudoroff pathway and characterization of its first enzyme, glucose dehydrogenase.

Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first enzyme of the ED pathway, glucose dehydrogenase, was characterized. The distribution of the 13C label in alanine synthesized by cells grown with [1-13C]glucose indicated that in vivo the EMP pathway and the modified ED pathway operate parallel, with glucose metabolization via the EMP pathway being prominent. To initiate studies on the regulatory mechanisms governing carbon flux via these pathways, the first enzyme of the ED pathway, glucose dehydrogenase, was purified to homogeneity and its phenotypic properties were characterized. The pyridine-nucleotide-dependent enzyme used both NAD+ and NADP+ as cosubstrates, showing a 100-fold higher affinity for NADP+. Besides glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the enzyme is the oxidation of glucose by NADP+. A striking feature was the influence of NADP+ and NAD+ on the quaternary structure and activity state of the enzyme. Without cosubstrate, the enzyme was highly aggregated (mol. mass > 600 kDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically active, homomeric dimers with a mol. mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarity to the respective partial sequences of alcohol dehydrogenases and sorbitol dehydrogenases, but no resemblance to the known pyridine-nucleotide-dependent archaeal and bacterial glucose dehydrogenases.

Efflux of compatible solutes in Corynebacterium glutamicum mediated by osmoregulated channel activity.

Bacteria respond to hypoosmotic stress by releasing low-molecular-mass solutes in order to maintain constant turgor pressure. We have studied the function of osmoregulated channel(s) in Corynebacterium glutamicum, which are responsible for efflux of various solutes upon sudden decrease in osmotic pressure. The channels preferentially mediated efflux of compatible solutes such as glycine betaine and proline. The release of molecules of similar size, e.g. glutamate or lysine, was restricted, ATP was completely retained even after severe osmotic shock. The cells maintained high cytoplasmic K+ and Na+ concentrations under hypoosmotic shock. Several results suggest that the solute efflux is mediated by a channel and not by a carrier, e.g. by reversal of the glycine betaine uptake systems of C. glutamicum: the release of glycine betaine and proline was extremely fast reaching an efflux rate of 6000 micromol x min(-1) x g dm(-1) or higher; the efflux was not significantly influenced by addition of external transport substrate, e.g. glycine betaine; in spite of an extremely high chemical gradient, no significant efflux under isoosmolar conditions was observed; efflux of solutes was unchanged after full uncoupling of membrane energetics by carbonylcyanide m-chlorophenylhydrazone (CCCP). These results indicate the presence of an osmoregulated channel in C. glutamicum similar to the mechanosensitive channel(s) of Escherichia coli. The activity of the channel did not depend on the growth conditions, but we observed a tight regulation on the level of activity, i.e. the mechanosensitive channel behaved as a perfect osmometer. By monitoring release of glycine betaine under slow and continuous decrease of the external osmolality, we observed continous efflux whithout a stepwise release of solutes. This resulted in a significant steady-state decrease of the membrane potential.

Accurate determination of 13C enrichments in nonprotonated carbon atoms of isotopically enriched amino acids by 1H nuclear magnetic resonance.

A method for the accurate determination of 13C enrichments in nonprotonated carbon atoms of organic compounds that makes use of unresolved 13C satellites of proton(s) bonded to the vicinal carbon atom was developed. Using glutamate as a model molecule, this 1H nuclear magnetic resonance (NMR) inverse spin-echo difference spectroscopy method was calibrated for inversion efficiency and relaxation effects which were then shown to cause only a minor loss of the measured 13C satellite amplitude (2% for glutamate C-1 and 7% for glutamate C-5). The determination of 13C enrichments in nonprotonated glutamate carbon atoms by this method was shown to be more precise than 13C NMR. As a first application, a [5-13C]glucose labeling experiment with Corynebacterium glutamicum ASK1 was performed. The labeling patterns of glutamate and arginine extracted from cellular protein were determined using the newly developed method and standard 1H NMR with and without broadband 13C decoupling. Determination of the 13C enrichment in C-5 of glutamate and arginine, respectively, by the two methods showed good agreement. From the deduced labeling pattern of 2-oxoglutarate, an in vivo carbon flux distribution within the central metabolism of C. glutamicum ASK1 was calculated. Thus, the relative flux toward oxaloacetate via the tricarboxylic acid cycle enzyme malate dehydrogenase was determined as 45%, whereas that via anaplerotic C3 carboxylation was determined as 55%.

C3-carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylase-deficient Corynebacterium glutamicum.

Phosphoenolpyruvate carboxylase (PEPCx) has recently been found to be dispensable as an anaplerotic enzyme for growth and lysine production of Corynebacterium glutamicum. To clarify the role of the glyoxylate cycle as a possible alternative anaplerotic sequence, defined PEPCx- and isocitrate-lyase (ICL)-negative double mutants of C. glutamicum wild-type and of the l-lysine-producing strain MH20-22B were constructed by disruption of the respective genes. Analysis of these mutants revealed that the growth on glucose and the lysine productivity were identical to that of the parental strains. These results show that PEPCx and the glyoxylate cycle are not essential for growth of C. glutamicum on glucose and for lysine production and prove the presence of another anaplerotic reaction in this organism. To study the anaplerotic pathways in C. glutamicum further, H13CO3--labeling experiments were performed with cells of the wild-type and a PEPCx-negative strain growing on glucose. Proton nuclear magnetic resonance analysis of threonine isolated from cell protein of both strains revealed the same labeling pattern: about 37% 13C enrichment in C-4 and 3.5% 13C enrichment in C-1. Since the carbon backbone of threonine corresponds to that of oxaloacetate, the label in C-4 of threonine positively identifies the anaplerotic pathway as a C3-carboxylation reaction that also takes place in the absence of PEPCx.