Glucose Catabolite Repression Research Articles

Improvement ofYarrowia lipolyticalipase production by fed‐batch fermentation

Two different types of fed-batch fermentation were investigated to improve production yields of the Lip2 extracellular lipase in Y. lipolytica mutant-strain LgX64.81 grown in a 20l bioreactor. Compare to batch cultures, culture feeding with the complete medium led to a 2-fold increased lipase production (2016 +/- 76 U ml(-1)) whereas addition of a combination of glucose and olive oil led to a 3-fold increase. The high level of lipase production obtained on glucose media with Y. lipolytica LgX64.81 could be related to its phenotype, i.e., a lower sensibility to glucose catabolite repression due to a modification in the level of HXK1 expression.

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37 degrees C. At 42 degrees C, its plasmid content was ninefold higher than JM101 at 37 degrees C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp(uv), under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.

α-Chitinase activity among lactic acid bacteria

Chitin is a polysaccharide widely distributed in nature. Among 115 strains from 29 species of lactic acid bacteria only strains belonging to Carnobacterium divergens and Carnobacterium maltaromaticum hydrolyzed α-chitin. This activity was not affected by temperature (10 °C versus 30 °C) and in most cases not subject to glucose catabolite repression.

Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R

Corynebacterium glutamicum R adhA gene encodes a homodimeric, NAD-dependent, 345 amino acid residue alcohol dehydrogenase with two zinc ions per subunit. Chromosomal inactivation of the adhA gene rendered the strain incapable of growth on either ethanol or n-propanol as the sole carbon source. RNA hybridization analysis revealed that adhA transcription was not only induced by these two substrates, but it was also subject to glucose catabolite repression. Accordingly, both induction of AdhA activity and ethanol utilization were detected only after depletion of glucose. Deletion of either or both of potential cyclic adenosine monophosphate (cAMP) receptor binding site and an inverted repeat of sequence 5'-GCAATTGATG-N (8)-CACAATTGC-3' in the promoter region of adhA strongly suggested that IR, which does not share significant similarity with other regulatory DNA elements of C. glutamicum, represents a transcriptional repressor binding site. Purified recombinant AdhA displayed the highest substrate specificities towards ethanol and n-propanol and their corresponding aldehydes.

Impairing and Monitoring Glucose Catabolite Repression in l‐Carnitine Biosynthesis

Signal transduction pathways are usually avoided when optimizing a biotransformation process because they require complex mathematical formulations. The aim of this work was to use a Systems Biology approach to optimize and monitor the biotransformation of L-carnitine using signal transduction pathways. To this end, a dynamic model was constructed, integrating the metabolic pathways of L-carnitine biosynthesis as well as the expression of this metabolism by means of its regulation by transcription factors such as cAMP-CRP and CaiF. The model was validated using different C-sources as well as different reactor feeding approaches. A linear relationship between the external cellular cAMP and the L-carnitine production levels was predicted before being experimentally confirmed in several scenarios. Moreover, results of the model simulations and subsequent experimental findings demonstrated that the addition of exogenous cAMP was able to restore the L-carnitine production when glucose was used as C-source. Additionally, a way to monitor the L-carnitine biosynthesis by using the level of cAMP as a marker of the biotransformation state was in silico and experimentally demonstrated.

The Production of Bacillus cereus Enterotoxins Is Influenced by Carbohydrate and Growth Rate

Enterotoxin production is a key factor in Bacillus cereus food poisoning. Herein, the effect of the growth rate (mu) on B. cereus toxin production when grown on sucrose was studied and the Hemolytic BL enterotoxin (HBL) and nonhemolytic enterotoxin (Nhe) production by B. cereus was compared according to carbohydrate at mu = 0.2 h(-1). The anaerobic growth was carried out on continuous cultures in synthetic medium supplemented with glucose, fructose, sucrose, or an equimolar mixture of glucose and fructose. Concerning the HBL and Nhe enterotoxin production: (1) the highest enterotoxin production has occurred at mu = 0.2 h(-1) when growing on sucrose; (2) HBL production was repressed when glucose was consumed and the presence of fructose (alone or in mixture) cancelled glucose catabolite repression; (3) the consumption of sucrose increased Nhe production, which was not affected by the catabolite repression. Furthermore, analysis of the fermentative metabolism showed that whatever the mu or the carbon source, B. cereus used the mixed acid fermentation to ferment the different carbohydrates. The enterotoxin productions by this strain at mu = 0.2 h(-1) are highly influenced by the carbohydrates that do not involve any fermentative metabolism changes.

The Saccharomyces cerevisiae acetyl-coenzyme A synthetase encoded by the ACS1 gene, but not the ACS2-encoded enzyme, is subject to glucose catabolite inactivation.

In Saccharomyces cerevisiae, the structural genes ACS1 and ACS2 each encode an isoenzyme of acetyl-CoA synthetase (ACS; EC 6.2.1.1). Involvement of glucose catabolite repression in regulation of the two isoenzymes was investigated by following ACS activity after glucose pulses (100 mM) to ethanol-limited chemostat cultures. In wild-type S. cerevisiae and in an isogenic strain in which ACS2 had been disrupted, ACS activity decreased after a glucose pulse. No such inactivation was observed in a strain in which ACS1 was disrupted. Western blots demonstrated that the ACS1 product, but not the ACS2 product, was degraded after a glucose pulse. Inactivation kinetics of the ACS1 product resembled those of isocitrate lyase.

Production and characterization of a type B feruloyl esterase from Fusarium proliferatum NRRL 26517

A novel feruloyl esterase (or ferulic acid esterases, FAEs) was discovered from a Fusarium proliferatum strain (NRRL 26517) known to be capable of utilizing corn fiber xylan. Nitrogen sources and carbon sources in growth medium strongly influence the enzyme production. The feruloyl esterase production requires an inorganic nitrogen salt. Common complex organic nitrogen sources, such as yeast extract and peptone, severely suppress the production of FAE. The nature of the anion of inorganic salts is also an important factor in determining the level of production. The biosynthesis of feruloyl esterase may be subject to glucose catabolite repression. Growth using xylose as the sole carbon source does not support the enzyme production. The induction of the enzyme production apparently requires the presence of an esterified ferulic acid, and free ferulic acid does not stimulate the production of the enzyme. Under optimal conditions, the strain produced the feruroyl esterase with a titer of 334.6 units/l. The enzyme was purified to homogeneity by ion-exchange chromatography. The molecular weight of the enzyme was determined to be 31 kDa. It exhibits a pH optimum at 6.5–7.5. Unlike other feruloyl esterases, it is stable over a broad pH range (5–9). Its unusual insensitivity to pH makes it a potentially useful enzyme in applications that require acidic or alkaline conditions. The optimal temperature is 50 °C and it is stable at a temperature up to 50 °C. The crude enzyme preparation from the strain supplemented with a commercial xylanase can release about 32% of the alkaline-extractable ferulic acid from corn bran.

Involvement of Hexokinase Hxk1 in Glucose Catabolite Repression of LIP2 Encoding Extracellular Lipase in the Yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. However, very little is known about the mechanisms controlling its expression, especially on glucose media. In this work, the involvement of hexokinase Hxk1 in the glucose catabolite repression of LIP2 was investigated in a lipase overproducing mutant less sensitive to glucose repression. This mutant has a reduced capacity to phosphorylate hexose compared with the wild-type strain, but no differences could be observed between the HXK1 sequences in the two isolates. This suggested that the reduced phosphorylating activity of the mutant strain probably resulted from a modification in the level of HXK1 expression. However, overexpression of the HXK1 gene in this mutant led to a decrease of both LIP2 induction and extracellular lipase activity, suggesting that the hexokinase is involved in the glucose catabolite repression of LIP2 in Y lipolytica.

A Hexose Transporter Homologue Controls Glucose Repression in the Methylotrophic Yeast Hansenula polymorpha

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser(85)) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.

The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate.

A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km 102 and 10 s(-1) mM(-1), respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.

Cloning and transcriptional analysis of the Thermoanaerobacter ethanolicus strain 39E maltose ABC transport system.

Thermoanaerobacter ethanolicus strain 39E is a Gram-positive thermophile that converts sugars resulting from plant carbohydrate polymer degradation into ethanol. A putative maltose ATP-binding cassette (ABC) transport operon was isolated with genes encoding for the integral membrane components (malF and malG); the ATP-binding protein (malK); and a partial gene for the maltose-binding protein (malE). This operon is unlike most other maltose transport operons, which do not contain a contiguous malK gene. Sequence analysis showed that the individual genes in the putative operon possessed a considerable range of similarities to their respective homologs in other eubacteria and archaea. MalK had 52% amino-acid identity and over 70% similarity with its homolog from the archaeon Thermococcus litoralis, while the membrane components and binding protein exhibited much less similarity with a range of other thermophilic eubacteria. Transcript was not detected in maltose-, glucose-, or xylose-grown cells using Northern blotting, but RT-PCR showed that malFGK were expressed in cells grown on maltose or xylose. Based on these results, the strain 39E maltose operon may be subject to glucose catabolite repression.

The ptsH gene from Bacillus thuringiensis israelensis. Characterization of a new phosphorylation site on the protein HPr.

The ptsH gene from Bacillus thuringiensis israelensis (Bti), coding for the phosphocarrier protein HPr of the phosphotransferase system has been cloned and overexpressed in Escherichia coli. Comparison of its primary sequence with other HPr sequences revealed that the conserved His15 and Ser46 residues were shifted by one amino acid and located at positions 14 and 45, respectively. The biological activity of the protein was not affected by this change. When expressed in a Bacillus subtilis ptsH deletion strain, Bti HPr was able to complement the functions of HPr in sugar uptake and glucose catabolite repression of the gnt and iol operons. A modified form of HPr was detected in Bti cells, and also when Bti ptsH was expressed in E. coli or B. subtilis. This modification was identified as phosphorylation, because alkaline phosphatase treatment converted the modified form to unmodified HPr. The phosphoryl bond in the new form of in vivo phosphorylated HPr was resistant to alkali treatment but sensitive to acid treatment, suggesting phosphorylation at a histidine residue. Replacement of His14 with alanine in Bti HPr prevented formation of the new form of phosphorylated HPr. The phosphorylated HPr was stable at 60 degrees C, in contrast with HPr phosphorylated at the N delta 1 position of His14 with phosphoenolpyruvate and enzyme I. (31)P-NMR spectroscopy was used to show that the new form of P-HPr carried the phosphoryl group bound to the N epsilon 2 position of His14 of Bti HPr. Phosphorylation of HPr at the novel site did not occur when Bti HPr was expressed in an enzyme I-deficient B. subtilis strain. In addition, P-(N epsilon 2)His-HPr did not transfer its phosphoryl group to the purified glucose-specific enzyme IIA domain of B. subtilis.

The Saccharomyces cerevisiae ICL2 gene encodes a mitochondrial 2-methylisocitrate lyase involved in propionyl-coenzyme A metabolism.

The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologous ICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced, ICL2 cannot complement the growth defect of icl1 null mutants. It has therefore been suggested that ICL2 encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whether ICL2 encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-type S. cerevisiae and of isogenic icl1, icl2, and icl1 icl2 null mutants. Isocitrate lyase activity was absent in icl1 and icl1 icl2 null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and single icl mutants but not in the icl1 icl2 mutant. This demonstrated that ICL2 encodes a specific 2-methylisocitrate lyase and that the ICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that the ICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that of ICL1, transcription of ICL2 is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction of ICL2 mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.

Role of flhDC in the Expression of the Nuclease Gene nucA, Cell Division and Flagellar Synthesis in Serratia marcescens

We investigated in Serratia marcescens the functions of the flhDC operon, which controls motility and cell division in enteric bacteria. Included in our evaluations were investigation of cell division, flagellar synthesis and regulation of the expression of nuclease (encoded by the nucA_Sm gene, one of the virulence factors). Interruption of the chromosomal flhDC operon in S. marcescens CH-1 resulted in aberrant cell division and loss of nuclease and flagella. Expression of nucA_Sm and other mutated phenotypes was restored in the flhDC mutant by the induction of overexpression of flhDC in a multicopy plasmid. Multicopied flhDC also induced the formation of differentiated cells (polyploid aseptate cells with oversynthesis of peritrichous flagella) in broth culture using minimal growth medium. Expression of the flhDC operon showed positive autoregulation, and was growth phase dependent (upregulated in early log phase). In addition, flhDC expression was inhibited when the temperature increased from 30 to 37°C, and when osmolarity was increased, but was not influenced by glucose catabolite repression. These results show that FlhD/FlhC is a multifunctional transcriptional activator involved in the process of cell differentiation, swarming and virulence factor expression.

Role of flhDC in the expression of the nuclease gene nucA, cell division and flagellar synthesis in Serratia marcescens.

We investigated in Serratia marcescens the functions of the flhDC operon, which controls motility and cell division in enteric bacteria. Included in our evaluations were investigation of cell division, flagellar synthesis and regulation of the expression of nuclease (encoded by the nucA(Sm) gene, one of the virulence factors). Interruption of the chromosomal flhDC operon in S. marcescens CH-1 resulted in aberrant cell division and loss of nuclease and flagella. Expression of nucA(Sm) and other mutated phenotypes was restored in the flhDC mutant by the induction of overexpression of flhDC in a multicopy plasmid. Multicopied flhDC also induced the formation of differentiated cells (polyploid aseptate cells with oversynthesis of peritrichous flagella) in broth culture using minimal growth medium. Expression of the flhDC operon showed positive autoregulation, and was growth phase dependent (upregulated in early log phase). In addition, flhDC expression was inhibited when the temperature increased from 30 to 37 degrees C, and when osmolarity was increased, but was not influenced by glucose catabolite repression. These results show that FlhD/FlhC is a multifunctional transcriptional activator involved in the process of cell differentiation, swarming and virulence factor expression.

Manipulation of carbon assimilation with respect to expression of the pac gene for improving production of penicillin acylase in Escherichia coli

A strategy of genetically manipulating carbon assimilation with respect to expression of the pac gene was employed for overproduction of recombinant penicillin acylase (PAC). Two expression plasmids of pCLL2902 and pCLL3201, which contain the pac coding region but differ in the pac regulatory region, were constructed for the production experiments. Expression of the pac gene was subjected to phenyl acetic acid (PAA-) induction and glucose catabolite repression for pCLL3201, whereas it was subjected to neither of the two transcriptional regulations for pCLL2902. The specific PAC activity for strains harboring pCLL2902 was significantly higher than that for strains harboring pCLL3201 due to an improved transcription efficiency. In addition, no inclusion bodies were observed upon production of PAC using the current expression systems. The results suggest that using the native pac promoter instead of a strong promoter such as tac for regulation is a feasible approach for production of PAC. The impact of the current expression systems is also significant from a process viewpoint since, using strains harboring pCLL2902, not only could glucose replace PAA as a carbon source of Escherichia coli cultures for production of PAC but also the volumetric PAC activity was highly improved.

Catabolite repression of the xylanase gene (xynA) expression in Bacillus stearothermophilus no. 236 and B. subtilis.

Catabolite repression of the Bacillus stearothermophilus No. 236 xynA gene, encoding an extracellular xylanase, was investigated in this work. Expression of the xynA gene in the B. stearothermophilus strain was found to be subject to glucose catabolite repression, and the level of repression was about 50-fold when the relative amounts of xynA transcript synthesized on different carbon sources were analyzed. The experiments with the B. subtilis MW15 strains carrying plasmids containing the xynA::aprA fusion gene showed that the cloned xynA gene did not require any specific carbon source for its induction. Nevertheless, the expression of the cloned gene was repressed by the presence of glucose. From the nucleotide sequence of the cloned xynA gene, we found two potential catabolite responsive elements (cre) within its reading frame region (cre-1: nucleotides +160 to +173 and cre-2: +173 to +186). Furthermore, by using various deletion derivatives of the xynA::aprA fusion plasmid (pMGW23), we suggested that only the cre-2 element might play a role in the glucose catabolite repression. Repression level of the xynA gene expression in the recombinant B. subtilis strain was estimated to be about 3-fold by analysis of the amounts of xynA transcript.

The Saccharomyces cerevisiae acetyl-coenzyme A synthetase encoded by the ACS1 gene, but not the ACS2-encoded enzyme, is subject to glucose catabolite inactivation

In Saccharomyces cerevisiae, the structural genes ACS1 and ACS2 each encode an isoenzyme of acetyl-CoA synthetase (ACS; EC 6.2.1.1). Involvement of glucose catabolite repression in regulation of the two isoenzymes was investigated by following ACS activity after glucose pulses (100 mM) to ethanol-limited chemostat cultures. In wild-type S. cerevisiae and in an isogenic strain in which ACS2 had been disrupted, ACS activity decreased after a glucose pulse. No such inactivation was observed in a strain in which ACS1 was disrupted. Western blots demonstrated that the ACS1 product, but not the ACS2 product, was degraded after a glucose pulse. Inactivation kinetics of the ACS1 product resembled those of isocitrate lyase.

Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism.

The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized. The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent Km of 6 microM for gluconate. Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. Thus, it was concluded that gntT is monocistronic and is regulated by two promoters. Both of the promoters have - 10 and -35 sequence elements typical of sigma70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence. A gntT::lacZ operon fusion was constructed and used to examine gntT expression. The results indicated that gntT is maximally induced by 500 microM gluconate, modestly induced by very low levels of gluconate (4 microM), and partially catabolite repressed by glucose. The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein. Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.