Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli
- First Online:
- Cite this article as:
- Sarkar, D., Siddiquee, K.A.Z., Araúzo-Bravo, M.J. et al. Arch Microbiol (2008) 190: 559. doi:10.1007/s00203-008-0406-2
- 417 Downloads
To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved.
KeywordsEscherichia coli cra mutant cra.edd mutant cra.edd.iclR mutant DNA microarray
The central metabolic pathways of Escherichia coli are controlled by a number of global regulators depending on the carbon sources available and the culture condition. Among them, the catabolite repressor/activator protein (Cra) initially characterized as the fructose repressor, FruR plays an important role in the control of carbon flow in E. coli (Moat et al. 2002; Saier and Ramseier 1996; Saier et al. 1997). The genes such as ptsHI, pfkA, pykF, acnB, edd–eda, fruBKA, mtlADR, gapB are reported to be negatively controlled, while ppsA, fbp, pckA, acnA, icd, aceA, aceB, and cydA,B are positively controlled by the Cra protein (Saier and Ramseier 1996; Cunningham et al. 1997; Moat et al. 2002; Perrenoud and Sauer 2005). It has been known that the mutant defective in cra gene is unable to grow on the gluconeogenic substrates such as pryruvate, acetate, and lactate. This phenomenon appears to be due to the deficiency in the gluconeogenic enzymes such as PEP synthase, PEP carboxykinase, some TCA cycle enzymes, the two glyoxylate shunt enzymes, and certain electron transport carrier (Saier et al. 1997). The gluconeogenic pathway will be deactivated by cra gene knockout as such, and the carbon flow toward catabolism or the glucose consumption rate may increase, since the glycolysis pathway genes such as ptsHI, pfkA, and pykF are derepressed. However, the regulation mechanism is complex and the story is not so simple. The details of such complex regulation network have not yet been fully investigated for the cra mutant (Saier et al. 1997). Some studies on the cra mutant have been performed based on the molecular level approaches using the lacZ-transcriptional fusion, flux analysis, etc. (Ryu et al. 1995; Mikulskis et al. 1997; Prost et al. 1999; Ramseier et al. 1996; Cortay et al. 1994; Perrenoud and Sauer 2005; Phue et al. 2005).
In the present research, we investigated the effect of cra gene knockout on the metabolism in E. coli based on gene expressions obtained by DNA microarray together with some of the enzyme activities. Moreover, based on the metabolic analysis of cra gene knockout mutant, we constructed cra.edd double genes knockout mutant and cra.edd.iclR triple genes knockout mutant, and analyzed the phenotypes of those mutants as compared with the wild type in relation to the activation of the glycolysis, thus improving the glucose consumption rate.
Materials and methods
Strains and cultivation conditions
Bacterial strains and plasmids
Strain or plasmid
cra mutant (for competent cell)
cra.edd double mutant (cassette free)
cra.edd.iclR triple mutant
Ampicillin(amp)r and chloramphenicol(cm)r
kanamycin(kan)r and ampr
ampr and kanr
ampr, helper plasmid
ampr and cmr
The M9 minimal medium was used for all the experiments as described before (Siddiquee et al. 2004a, b). The cultivation was made at 37°C in a 20-l reactor (M-100, Tokyo, Rikakiki Co., Tokyo, Japan) with the working volume of 1 l equipped with pH, dissolved oxygen (DO) and temperature sensors. The air flow was maintained at 1 l/min, and the DO concentration was kept around 30–40% of air saturation. The pH of the culture was maintained at 7.0 by automatic addition of 2.0 M HCl or 2.0 M NaOH with a pH controller. Continuous cultivation was conducted with the working volume of 500 ml in a 1-l reactor at the dilution rate of 0.2 and 0.6 h−1, where the feed glucose concentrations were either 4 or 10 g/l. The working volume was kept constant by removal of effluent by use of precalibrated peristaltic pumps.
Cell concentration was measured by the optical density (OD) of the culture with a spectrophotometer (Ubet-30, Jasco Co., Tokyo, Japan), and then converted to dry cell weight (DCW) per liter based on the relationship between OD and DCW. Glucose concentration was measured using enzymatic kit (Wako Co., Osaka, Japan). The concentrations of the metabolites such as acetic acid were measured using enzymatic kits (Boehringer Co., Mannhiem, Germany). Oxygen and carbon dioxide concentrations in the bioreactor off gas were measured by the off-gas analyzer (LX-750, Iijima Electronics Co., Japan).
The preparation of crude cell extract and the analyses of enzyme activities involved in the main metabolic pathways such as phosphoglucosetransferase system (PTS), phosphofructo kinase (Pfk), pyruvate kinase (Pyk), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), acetate kinase (Ack), citrate synthase (CS), aconitase (Acn), isocitrate dehydrogenase (ICDH), isocitrate lyase (Icl), malate synthase (MS), malic enzyme (Mez), phosphoenol pyruvate carboxylase (Ppc), phosphoenol pyruvate carboxykinase (Pck), and ED pathway enzymes were made as described previously, where specific ED pathway activity was determined from the combined Edd and Eda reactions (Peng and Shimizu 2003). Each measurement was performed in triplicate from three samples of the same culture.
RNA isolation and semi-quantitative RT-PCR
Total RNA was isolated from E. coli cells by Qiagen RNeasy Mini Kit (QIAGEN K.K., Japan) according to their protocol. The quantity and purity of RNA were determined by measuring the optical density at 260 and 280 nm and by 1% formaldehyde agarose-gel electrophoresis. Criteria for the design of the gene-specific primer pairs were followed according to Sambrook and Russel (2001). The primers used in the present study were synthesized at Hokkaido System Science Co. (Sapporo, Hokkaido, Japan), where the primers used for the gene expressions by RT-PCR are described elsewhere (Kabir and Shimizu 2003).
RT-PCR reactions were carried out in a Takara PCR Thermal Cycler (Takara TP240, Japan) using a Qiagen one-step RT-PCR kit (Qiagen K.K., Japan) as described previously (Kabir and Shimizu 2003). We determined the optimal amount of input RNA using twofold dilution of RNA for RT-PCR assays under identical reaction condition to construct a standard curve for each gene product. After the optimal amount of input RNA was determined for each gene product, RT-PCR was carried out under identical reaction condition to detect differential transcript levels of genes. The gene dnaA, which is expressed at relatively constant rate (Kabir and Shimizu 2003), was used as internal control. To calculate the standard deviation, RT-PCR was independently performed three times for each gene under identical condition.
The isolation of RNA for the DNA microarray was similar to RT-PCR as mentioned above. Instead of mini kit, the Qiagen Maxi kit and 10 ml of bacterial cultures were used. The DNA microarray experiment needs high purity of RNA. Therefore, after isolation, the RNA was treated with the DNaseI from Qiagen using a protocol of Gross laboratory of University of California, San Francisco (http://www.microarrays.org/pdfs/Total_RNA_from_Ecoli.pd), that was modified in our laboratory (Rahman et al. 2006). The original RNA sample was diluted 100-fold with 10 mM TAE buffer (pH 7.5), and the sample with the absorbance at 260 and 280 nm was taken to determine the concentration of RNA and protein in the sample.
Takara IntelliGene E. coli chips Version 2 and Takara labeling kit were used for this study, and the Cy3-dUTP and Cy5-dUTP dyes were supplied from Amersham Bioscience Co. The labeled cDNA probes were prepared according to Takara protocol, where 300 pmol/μl of random hexamer, 10 μg of total RNA, 5× reaction buffer, 10× dNTP mixture for Cy3/Cy5, 1 μl of Cy3/Cy5-dUTP, RNase inhibitor (40 U/μl), and M-MLV reverse transcriptase (2,000 U/μl) were added. Before starting the cyber-labeling experiment, 1 μl (1 ng/μl) of human TFR RNA was added to the reaction mixture as a negative control. Then the labeled cDNA probes were purified by the columns supplied from Takara Co., and after that further purified by phenol and ethanol precipitation. After drying the precipitates, these were dissolved in a 25 μl of hybridization buffer containing 6× SSC, 0.2% SDS, 5× Denhardt’s solution, 0.1 mg/ml denatured salmon sperm DNA. The intensity of the Cy3 and Cy5 labeling cDNA were checked by scanning the gel at 532 and 635 nm in fluorescent image analyzer (FLA-8000, Fuji Photo Film Co.). The preparation for the set-up of the chamber and hybridization was performed as described by Takara protocol. After hybridization and washing, the slides were dried by low-speed centrifugation at 500×g for 2 min. The fluorescent signal of each spot was read with a DNA microarray scanner (Fluorescent image analyzer, FLA-8000, Fuji photo film Co.) at 532 and 635 nm. After scanning of the image data, it was analyzed by microarray software Array vision version 6.0 (Amersham Bioscience Co.).
For genes whose expression ratios are reproducible and the values are >2.0 or <0.5 (>1 or <−1 in log2 representation).
In good consistency (p < 0.05) under Student’s t test.
Fermentation characteristics of cra gene knockout mutant
Growth parameters for E. coli BW25113 and its cra mutant cultivated at the dilution rate of 0.2 h−1 where feed glucose concentration was 4 g/l
Biomass yield (g/g)
0.44 ± 0.01
0.30 ± 0.02
Glucose uptake rate [mmol/(g h)]
2.54 ± 0.11
3.61 ± 0.03
Acetate production rate [mmol/(g h)]
0.02 ± 0.01
0.84 ± 0.02
O2 uptake rate [mmol/(g h)]
10.24 ± 0.52
8.88 ± 0.63
CO2 evolution rate [mmol/(g h)]
8.51 ± 0.35
8.57 ± 0.8
Gene expressions by DNA microarray
Gene expressions of cra mutant as compared with the wild type strain
Logarithmic ratio, Log2 (cra/parent strain)
(a) Carbon and energy related genes
Isocitrate lyase (EC 18.104.22.168)
Isocitrate dehydrogenase kinase/phosphatase (EC 22.214.171.124) (EC 3.1.3.-)
Alcohol dehydrogenase (EC 126.96.36.199)
Cytochrome d ubiquinol oxidase subunit II (EC 1.10.3.-)
Cytochrome d ubiquinol oxidase subunit I (EC 1.10.3.-)
Fructose-1,6-bisphosphatase (EC 188.8.131.52)
PTS system, fructose-specific IIA/FPR component (EIIA-Fru)
1-Phosphofructokinase (EC 184.108.40.206) (fructose 1-phosphate kinase)
l-Fuculose phosphate aldolase (EC 220.127.116.11)
Fucose isomerase FucI (EC 5.-.-.-)
l-Fuculokinase (EC 18.104.22.168) (l-fuculose kinase)
Lactaldehyde reductase (EC 22.214.171.124)
Fucose operon FucU protein
Glyoxylate carboligase (EC 126.96.36.199) (tartronate-semialdehyde synthase)
Citrate synthase (EC 188.8.131.52)
l-Glycerol 3-phosphate dehydrogenase
Malate dehydrogenase (EC 184.108.40.206)
Phosphoenolpyruvate synthase (EC 220.127.116.11)
Pyruvate kinase (EC 18.104.22.168)
Transketolase 1 (EC 22.214.171.124) (tk 1)
Xylose isomerase (EC 126.96.36.199) (version 1)
Glucose-6-phosphate 1-dehydrogenase (EC 188.8.131.52)
(b) Metabolic transport related genes
Glutamate/aspartate transport ATP-binding protein gltL
Maltose/maltodextrin transport ATP-binding protein MalK
Phosphotransferase system enzyme II (EC 184.108.40.206), mannose-specific, factor III
Galactoside transport system permease protein
Glycine betaine/l-proline transport ATP-binding protein ProV
PTS system, phosphocarrier protein HPr (histidine-containing protein)
d-Xylose-proton symporter (d-xylose transporter)
(c) Fatty acids, purine and pyrimidine metabolism related genes
3-Hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC 220.127.116.11)
3-Oxoacyl-[acyl-carrier-protein] synthase I (EC 18.104.22.168)
3-Oxoacyl-[acyl-carrier-protein] synthase (EC 22.214.171.124) III
3-Deoxy-manno-octulosonate cytidylyltransferase (EC 126.96.36.199)
Endonuclease III (EC 188.8.131.52) (DNA-(apurinic or apyrimidinic site) lyase)
Phosphoribosylaminoimidazole carboxylase catalytic subunit (EC 184.108.40.206) (AIR carboxylase) (AIRC)
Amidophosphoribosyltransferase (EC 220.127.116.11)
(d) Amino acid metabolism genes
Chorismate synthase (EC 18.104.22.168).
Phospho-2-dehydro-3-deoxyheptonate aldolase, Phe-sensitive (EC 22.214.171.124) (phospho-2-keto-3-deoxyheptonate aldolase)
Arginine-binding periplasmic protein 1 precursor
Shikimate kinase (EC 126.96.36.199) II
Aromatic amino acid transport protein aroP (general aromatic amino acid permease)
Cysteine synthase A (EC 188.8.131.52)
Histidine biosynthesis bifunctional protein hisIE [includes: phosphoribosyl-AMP cyclohydrolase (EC 184.108.40.206) (PRA-CH); phosphoribosyl-ATP pyrophosphatase (EC 220.127.116.11) (PRA-PH)]
Acetolactate synthase (EC 18.104.22.168) II large chain
4-Hydroxy-2-oxovalerate aldolase (EC 4.1.3.-)
X-his dipeptidase (EC 22.214.171.124) precursor
Trimethylamine-n oxide reductase precursor (EC 126.96.36.199)
Anthranilate synthase component I (EC 188.8.131.52)
Tryptophan biosynthesis protein trpCF [includes: indole-3-glycerol phosphate synthase (EC 184.108.40.206) (IGPS); N-(5′-phospho-ribosyl)anthranilate isomerase (EC 220.127.116.11) (PRAI)]
Probable glutaminase ybaS (EC 18.104.22.168)
(e) Global and metabolic regulatory genes
Acetate operon repressor (repressor protein IclR)
DNA-binding protein fis (factor-for-inversion stimulation protein) (HIN recombinational enhancer binding protein)
l-Fucose operon activator
Transcriptional activator protein lysR
Purine nucleotide synthesis repressor
RNA polymerase sigma-stationary phase
Table 3(a) shows that the expression of the glycolytic pathway gene pykF was up-regulated (while pykA expression was slightly down-regulated (0.88 which corresponds to −0.184 in log2 representation), data not shown in Table 3), and those of the gluconeogenic pathway genes such as ppsA and fbp were down-regulated as expected. The gene expression related to TCA cycle such as acnA was down-regulated, and the glyoxylate pathway related genes such as aceA and aceK were down-regulated in the mutant as expected. The pentose phosphate (PP) pathway related genes such as zwf were up-regulated in the mutant, which will be discussed later in the present paper. The adhE gene was also up-regulated in the mutant as compared with the parent strain, consistent with the result of Mikulskis et al. (1997). The respiratory pathway related genes such as cydA and cydB genes were down regulated, which is known to be positively controlled by Cra (Ramseier et al. 1996). Several other genes were also differentially regulated as explained in Appendix A.
Verification of DNA microarray result by RT-PCR
Some of the DNA micro-array results are consistent with the previously published data, while several others seem to be under control of Cra as well. This was investigated by detecting the Cra-binding sites (Appendix B).
Efect of cra.edd and cra.edd.iclR genes knockout on the metabolism
In order to reduce the acetate production, we further knocked out iclR gene so that the glyoxylate pathway is activated. Figure 4b shows the batch cultivation result for the cra.edd.iclR triple genes knockout mutant, where the figure indicates that acetate production was reduced and the cell concentration became the highest among the four strains used in the present research.
In Escherichia coli, Crp and Cra are known to control transcriptional responses to carbon availability (Saier and Ramseier 1996). While cAMP–Crp complex primarily controls the initiation of carbon source utilization, Cra influences the direction of carbon flux (Ramseier et al. 1995). It is quite important to understand the regulation mechanism for the carbon flow in terms of the global regulatory genes, and it is useful to utilize such information for the improvement of fermentation characteristics. Since Cra represses the glycolysis pathway genes such as ptsHI, pfkA, pykF, and activates the gluconeogenic pathway genes such as fbp, ppsA, pckA, etc., cra gene knockout may activate glycolysis, and the glucose consumption rate may be increased as shown in Table 2.
As stated above in “Results”, although the glucose consumption rate could be increased by cra gene knockout mutant (Table 2), the acetate was more produced and less consumed as compared with the wild-type strain (Fig. 1), resulting in the decrease in the cell yield (Table 2). This may be due to the activation of ED pathway (Fig. 3) and the repression of the TCA cycle [Table 3(a), Fig. 3]. We then blocked the ED pathway by constructing cra.edd double genes knockout mutant. Noting that aceA,B and icd genes were repressed in cra and cra.edd mutants, we constructed cra.edd.iclR mutant to reduce acetate formation. Usually, iclR is activated by fadR and induces aceBAK operon upon growth on either acetate or fatty acids.
Let us consider in more detail one by one about the TCA cycle regulation. The first enzyme of the TCA cycle, citrate synthase (CS) catalyzes the condensation of OAA and AcCoA to produce citrate plus coenzyme A. This enzyme is encoded by gltA in E. coli, which is under control of ArcA, while its regulation is independent of fnr, crp, and cra gene products (Park et al. 1994). On the other hand, aconitase genes (acnA and acnB) are transcriptionally regulated by a variety of global regulatory genes, where acnB expression is activated by Crp and repressed by ArcA, Fis, and Cra, while acnA expression is initiated by σ38, activated by Crp, Fur, SoxR/S, Cra and repressed by ArcA and Fnr (Cunningham et al. 1997). Thus, roughly speaking, acnB predominantly expresses during exponential growth phase, while acnA expresses at the stationary phase. In the case of cra gene knockout mutant, acnB is activated, while acnA is repressed as shown in Table 3(a), which indicates that the aconitase activity may be enhanced during the cell growth phase, or in the continuous culture at the dilution rate of 0.6 h−1(due to the activation of acnB gene), while its activity may be repressed at the stationary phase or in the continuous culture at the dilution rate of 0.2 h−1(due to the repression of arcB gene). The latter phenomenon can be observed in Fig. 3, while the former phenomenon cannot be seen in Fig. 5, which might be due to other global regulatory proteins such as Crp, etc.
The ace operon of E. coli contains three structural genes such as aceB, aceA, and aceK, which are transcribed in this order. The aceB and aceA encode two such enzymes of the glyoxylate pathway as malate sysnthase (MS) and isocitrate liase (Icl), respectively, while aceK encodes the bifunctional enzyme ICDH kinase/phosphatase, which regulate the activity of ICDH by reversible phosphorylation. The activity of this enzyme determines the fluxes of the junction point between the TCA cycle and the glyoxylate pathway. The ace operon is negatively regulated by iclR, which is located downstream from the aceK gene. Moreover, since Cra positively regulate ace operon, the glyoxylate pathway is repressed in cra gene knockout mutant. Figure 5 indicates that ICDH activity decreased for cra and cra.edd mutants as compared with the wild type, which is due to cra gene knockout. Moreover, the ICDH activity further decreased in cra.edd.iclR genes knockout mutant, which may be caused by the phosphorylation of ICDH due to iclR gene knockout. As for Icl activity, it decreased for cra and cra.edd mutants as compared with the wild type, which is due to cra gene knockout, while the Icl activity increased for cra.edd.iclR mutant (Fig. 5), which is due to the release from the inhibition by iclR. The fadR gene knockout (Farmer and Liao 1997; Peng and Shimizu 2006) also shows the reduction of acetate formation, giving the cell yield improvement by reducing the CO2 production via glyoxylate pathway utilization. However, the current cra.edd.iclR mutant shows also the activation of the glycolysis pathway genes such as ptsH, pfkA, and pykF (data not shown), thus increasing the glucose consumption rate, which is different from fadR (or iclR) gene knockout mutant. Although glucose consumption rate can be increased by the reduction of ATP production (Koebman et al. 2002), the present phenomenon is different from such mechanism.
Escherichia coli possesses two terminal oxidases Cyo and Cyd; whereas Cyo functions under fully oxidizing condition, Cyd functions under microaerophilic condition. The cydA,B operon is known to be controlled by ArcA and Fnr in response to oxygen limitation, and also controlled by Cra by carbon source availability (Ramseier et al. 1996). As shown in Table 3(a), cydA,B gene expressions were down-regulated for cra gene knockout mutant. In the case of continuous culture, enough oxygen is supplied and Cyo plays the role, and the effect of down-regulation of cyd is the least, while its effect may become significant at the stationary phase of batch culture. Although it has been reported that cyd operon is regulated by the interdependency of Cra, Fnr, and ArcA (Ramseier et al. 1996), the down-regulation of this operon was due to Cra in the present case since fnr and arcA changed little for the continuous culture at the present aerobic condition.
Table 3(a) shows the up-regulation of adhE gene. Cra is reported to repress adhE gene in the aerobic condition (Mikulskis et al. 1997). Although this did not result in the ethanol production, this may become important for the microaerobic or anaerobic condition, since this enzyme is allostelically affected by NADH/NAD+, which is not high in the current aerobic condition.
Cra-controlled operons used to construct the Cra recognition weight matrix
Putative Cra-binding site
Ramseier et al. (1993)
Mikulskis et al. (1997)
Ramseier et al. (1995)
Ramseier et al. (1995)
Ramseier et al. (1993)
Ramseier et al. (1993)
Ramseier et al. (1993)
Ramseier et al. (1995)
Tyson et al. (1997)
Ramseier et al. (1995)
Nègre et al. (1996)
Ramseier et al. (1993)
Ramseier et al. (1993)
Ramseier et al. (1995)
In conclusion, the present research shows the importance of the metabolic regulation analysis for the construction of multiple genes knockout mutants. Namely, we paid attention to the role of global regulatory gene cra for changing the direction of the carbon flow by knockout of this gene. As expected, the important glycolysis enzymes such as PTS, Pfk and Pyk were activated, and thus glucose consumption rate was increased. Since Cra activated ED pathway, and repressed TCA cycle and the glyoxylate pathway, we constructed cra.edd.iclR genes knockout mutant to reduce the acetate production by activating the glyoxylate pathway, thus improving the cellular performance. If we compare the activities of PTS, Pfk, and Pyk between Fig. 3 (dilution rate at 0.2 h−1) and Fig. 5 (dilution rate at 0.6 h−1), the extent of the increased activities were less for the latter. This may be due to the fact that cra gene is more expressed at the stationary phase (or at low dilution rate in the continuous culture) as compared with the exponential growth phase (or at high dilution rate in the continuous culture) (Rahman and Shimizu 2008).
Marcos J. Araúzo-Bravo would like to acknowledge Japanese Society for Promotion of Science (JSPS) for supporting him for this research. Dayanidhi Sarkar would like to acknowledge Japanese Government Scholarship, Monbukagakusho (Ministry of Education, Culture, Sports, Science and Technology, Japan) for supporting his research.