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2005-Glucose metabolism at high density growth of E-coli B and...

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2005-Glucose metabolism at high density growth of E-coli B and...

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     Glucose Metabolism at High Density Growth of E. coli B and E. coli K: Differences in Metabolic Pathways Are Responsible for Efcient Glucose Utilization in E. coli B as Determined by Microarrays and Northern Blot Analyses

     Je-Nie Phue,1 Santosh B. Noronha,2 Ritabrata Hattacharyya,2 Alan J. Wolfe,3 Joseph Shiloach1

     1 Biotechnology Unit, NIH NIDDK, Bethesda, Maryland 20892-2715; telephone: 301-496-9719; fax: 301-451-5911; e-mail: yossi@nih.gov 2 Department of Chemical Engineering IIT-Bombay, Powai, Mumbai 400076 India 3 Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 60153

     Received 1 June 2004; accepted 18 January 2005 Published online 1 April 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20478

     Abstract: In a series of previous reports it was established by implementing metabolic ux, NMR/MS, and Northern blot analysis that the glyoxylate shunt, the TCA cycle, and acetate uptake by acetyl-CoA synthetase are more active in Escherichia coli BL21 than in Escherichia coli JM109. These differences were accepted as the reason for the differences in the glucose metabolism and acetate excretion of these two strains. Examination of the bacterial metabolism by microarrays and time course Northern blot showed that in addition to the glyoxylate shunt, the TCA cycle and the acetate uptake, other metabolic pathways are active differently in the two strains. These are gluconeogenesis, sfcA shunt, ppc shunt, glycogen biosynthesis, and fatty acid degradation. It was found that in E. coli JM109, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase-acetate kinase (PtaAckA) system which uses acetyl-CoA. The inactivation of the gluconeogenesis enzyme phosphoenolpyruvate synthetase (ppsA), the activation of the anaplerotic sfcA shunt, and low and stable pyruvate dehydrogenase (aceE, aceF) cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs, and aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose concentration was high, no transcription of these genes was observed and acetate concentration increased, but at low glucose concentrations these genes were expressed and the acetate concentration decreased. It is possible that there is a major regulatory molecule that controls not only ppsA and aceBAK but also acs. The gluconeogenesis pathway (fbp, pckA, and ppsA) which leads to glycogen accumulation is constitutively

    active in E. coli BL21 regardless of glucose feeding strategy.

     Published 2005 Wiley Periodicals, Inc.{

     INTRODUCTION One of the major considerations during high-density growth of E. coli on glucose is acetate accumulation, particularly important when the growth is performed for recombinant protein biosynthesis, since acetate concentrations above 40 mM (2.4 g/L) affect growth and possibly recombinant protein production (Kleman and Strohl, 1994; Ko et al., 1995). The metabolism of glucose and acetate has been intensively investigated during the past 50 years and are well characterized (Cozzone, 1998). Briey, when E. coli is grown on glucose, acetate is produced from acetyl-CoA by phosphotransacetylase (pta) and acetate kinase (ackA) and from pyruvate by pyruvate oxidase (poxB) (Abdel-Hamid et al., 2001; El-Mansi and Holms, 1989; Kleman and Strohl, 1994). The acetate formed can be converted back to acetylCoA by acetyl-CoA synthetase (acs) and by reversing the pta-ackA pathway (Fig. 1). The acetyl-CoA is metabolized through the TCA cycle and the glyoxylate shunt pathway by isocitrate lyase (aceA) and malate synthetase (aceB) (ElMansi and Holms, 1989; Kornberg, 1966). The common explanation for acetate accumulation is the high carbon ux through glycolysis which exceeds the TCA cycle capacity, especially when glucose is in excess. Gradual addition of glucose to the culture using fed-batch techniques and the development of mutant strains with altered metabolic patterns have been implemented to limit acetate accumulations during high density growth (Contiero et al., 2000; Lee, 1996; Riesenberg and Guthke, 1999). Acetate is utilized by the glyoxylate shunt (Kornberg, 1966; Oh et al., 2002), a well-studied pathway that is regulated by the glyoxylate shunt operon (aceBAK) that

     Keywords: Escherichia coli; acetate; glucose; Northern blot; gluconeogenesis

     Correspondence to: J. Shiloach

     Published 2005 Wiley Periodicals, Inc. { This article is a US government work and, as such, is in the public domain in the United States of America.

     Figure 1. The glucose and acetate metabolism in E. coli (Brown et al., 1977; Kumari et al., 1995). FadR?ªregulator of fatty acid metabolism, FruR?ªfructose repressor, IHF?ªintegration host factor.

     contains aceK (isocitrate dehydrogenase kinase/phosphatase), aceA (isocitrate lyase), and aceB (malate synthetase) (Chung et al., 1988). Isocitrate dehydrogenase has a vital role in the regulation of the glyoxylate shunt (Thorsness and Koshland, 1987), since the protein itself is regulated by phosphorylation and dephosphorylation through isocitrate dehydrogenase kinase/phosphatase (aceK) (Borthwick et al., 1984; Cozzone, 1998; Stueland et al., 1988). The expression of the glyoxylate operon is under the negative control of two regulatory

    proteins: isocitrate lyase represor (iclR), which inhibits the expression of the glyoxylate operon, and the regulator of fatty acid metabolism (fadR), which activates the expression of the iclR gene (Gui et al., 1996; Sunnarborg et al., 1990). The glyoxylate operon expression is also under the positive control of two regulatory proteins: fructose repressor (fruR) and integration host factor (IHF), both stimulate the transcription of aceBAK (Ramseier et al., 1995; Resnik et al., 1996). Another pathway that plays an essential role when utilizing acetate is gluconeogenesis. This pathway synthesizes glucose from nonhexose precursors, by converting pyruvate to phosphoenolpyruvate through phosphoenolpyruvate synthase, and fructose 1,6 diphosphate to fructose 6 phosphate through fructose biphosphatase. Conversion of oxaloacetate directly to phophoenolpyruvate through phophoenolpyru-

     vate carboxykinase is also possible (Krebs and Bridger, 1980; Yang et al., 2003). Gluconeogenesis was characterized initially in the early 1970s (Soling et al., 1970) and recently was investigated by microarray method (Oh et al., 2002). We have previously reported that E. coli BL21, a derivative of E. coli B, excretes very little acetate even in the presence of high glucose concentration, while E. coli JM109, a K12 derivative, excretes acetate even under fed-batch conditions when the glucose concentration is kept low (Shiloach et al., 1996; van de Walle and Shiloach, 1998). The existence of an acetate control mechanism that operates in E. coli BL21 when acetate concentration reaches beyond 1 g/L was suggested. The glyoxylate shunt has been proposed as a route for acetyl-CoA consumption since higher isocitrate lyase (ICL) activity was observed in E. coli BL21 compared to E. coli JM109 in batch cultures with high levels of glucose. This observation was conrmed by measuring the carbon ux through the TCA cycle and the glyoxylate shunt in E. coli BL21 and JM109 using 13C-NMR and mass spectrometric analysis (Noronha et al., 2000). These studies showed that the glyoxylate shunt in E. coli BL21 is active at 22% of the ux through the TCA cycle, but there is no ux through the glyoxylate shunt in E. coli JM109. The metabolic patterns of these two strains became clearer when Northern blot analyses of key genes were performed (Phue and Shiloach, 2004). No apparent transcription of isocitrate lyase (aceA) and malate synthetase (aceB) was observed in E. coli JM109, while in E. coli BL21 there is constitutive transcription of aceA and aceB. In addition, the transcription level of acs is higher in E. coli BL21 and lower in the E. coli JM109, while the reverse is true for poxB transcription. More gene expression differences between the strains are likely and these can be determined by microarray analysis. Unlike Northern blots or enzyme analysis, this method gives the researcher an opportunity to look at metabolic pathways not directly linked to specic activity such as glucose utilization. Microarray analysis is an efcient and powerful tool to follow gene

    transcription at various growth and physiological conditions. In E. coli this method was used for several purposes: to analyze genes during high cell density culture (Gill et al., 2000; Yoon et al., 2003), to detect metabolic response to protein over production (Oh and Liao, 2000b), to study gene expression on growth on different carbon sources (Oh and Liao, 2000a), and to learn about the effect of physiological and genetical changes on tryptophan metabolism (Khodursky et al., 2000). In the work presented in this article microarray analysis together with Northern blot analysis is utilized for understanding the differences in glucose metabolism between E. coli K and E. coli B. MATERIALS AND METHODS Bacterial Strains The two E. coli strains studied were BL21(lDE3) (F, ompT, hsdSB (rB,mB), dcm, gal, (DE3), Cmr) and

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     JM109(DE3) (endA1, recA1, gyrA96, thi, hsdR17 (r,m), k k relA1, supE44, l, D(lac-proAB), [F0, traD36, proAB, lacIqZDM15], lDE3). Both strains were obtained from Promega Corp. (Madison, WI). Fermentation and Sample Preparation Both strains were grown at 378C in modied LB medium containing 10 g/L tryptone, 5 g/L yeast extract (15 g/L for JM109), 5 g/L NaCl, and 5 g/L K2HPO4. After sterilization, 10 mM MgSO4, 1 mL/L trace metal solution, and glucose at a different concentration for each experiment (40 g/L for batch fermentations and 2.0 g/L for the fed-batch fermentations) were added. Overnight cultures grown at 378C were used to inoculate 4.0 L of medium in a B. Braun fermentor equipped with data acquisition and a control system. The cultures were grown to high cell density, the pH was controlled at 7.0 by the addition of 50% NH4OH, and dissolved oxygen was kept above 30% of saturation at all times. Glucose feeding was conducted in response to the dissolved oxygen concentration as described previously (Shiloach et al., 1996). Samples for acetic acid analysis were collected at regular intervals, centrifuged and the supernatant was kept at 208C for further analysis. Samples for total RNA purication were collected and centrifuged at 14,000g for 10 min at 44C, the supernatant was removed and the cell pellets were quickly frozen by dry ice and stored at 808C. Analytical Methods Acetic acid in the culture supernatant was detected using a Boehringer Mannheim Kit 148621. Acetate determination is based on the formation of NADH, while acetate is converted to citrate and acetyl-CoA in the presence of acetylCoA synthetase. Glucose in the culture supernatant was determined using a YSI glucose analyzer (YSI Inc., Yellow Spring, OH). Preparation of Probe DNA and DNA Amplication Escherichia coli BL21 genomic DNA was used as template for amplication of fragments of aceA, aceE, acs, ackA, fbp, glgA, glgP, iclR, pckA, poxB, ppc, ppsA, pta, and scfA. The gene sequence was identied using BLAST search of E. coli K12 genome, and the following primers have been

    made. (a) aceA: 50 -CCAGTTCATCACCCTGGCAGGTATC-30 (Forward), 50 -GATTCTTCAGTGGAGCCGGTC-30 (Reverse). (b) aceE: 50

    -ATCGGTCATCCGTGAAGAAG-30 (Forward), 50 -CAGCGGATAGCTGAACGAAT-30 (Reverse). (c) ackA: 50 -CAGCTGACTGCTATCGGTCAC-30 (Forward), 50 -CAGGTTGTAAGGCAGGGCGTAGAG-30 (Reverse). (d) acs: 50

    -CCGCCATCTGCAAGAAAACGGC-30 (Forward), 50 -CACACCTTCGTCGGAAGTGATC-30 (Reverse).

     (e) fbp: 50 -ACCCCGTTCGGAAACTTAAT-30 (Forward), 50

    -CCTCCAGCCTGGTAACAAAC-30 (Reverse). (f) glgA: 50 -

    CGGCACTTTATCGTCAACCT-30 (Forward), 50 -GTCTTTCCGGCGTACTGAAC-30 (Reverse). (g) glgP: 50 -TTGATCAGCGAATCAACCAG-30 (Forward), 50 -ACAGCGGAAGAAGTGGAAGA-30 (Reverse). (h) iclR: 50

    -GTACGCATCTGATGCGAATGTCCG-30 (Forward), 50

    -GTACGCCAGCGTCACTTCCTTC-30 (Reverse). (i) pckA: 50

    -ACAACCCAAGCTACGACCTG-30 (Forward), 50 -GTCACCAGGCCTTTCABATG-30 (Reverse). (j) poxB: 50 -CGATGGAGATGAAAGCTGGT-30 (Forward), 50 -CTGAAACCTTTGGCCTGTTC-30 (Reverse). (k) ppc: 50

    -CCGGCTGGTTTTTCAGTTTA-30 (Forward), 50 -ATGATGCTAACCGCCAGGAG -30 (Reverse). (l) ppsA:50 -AAAGAGGCGTTTTCGTCATC-30 (Forward), 50 -GCGTAAACCAGCGCATTTAT-30 (Reverse). (m) pta: 50

    -CTGATCCCTACCGGAACCAG-30 (Forward), 50 -GCAGACCTTCAACGTAGCTC-30 (Reverse). (n) sfcA: 50 -GTCCTGGATGTTACGCAGGT-30 (Forward), 50 -GCCCTGTACTGC-30 (Reverse). DNA amplication was performed in 50 mL mixtures using 5 U of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN), 20 ng of genomic DNA/mL, 100 mM of each deoxynucleoside triphosphate, 1 mM of each primer, and Taq buffer (Roche, Indianapolis, IN) containing MgCl2. Polymerase chain reaction (PCR) was carried out for 30 cycles, each comprising 1 min of denaturing at 948C, 1 min of annealing at 648C, and 3 min of extension at 728C. The PCR products were puried by using gel extraction kit (Qiagen, Valencia, CA). The PCR products were labeled with 32 P using Ready-To-Go DNA Labeling Beads (Amersham Pharmacia Biotech, Piscataway, NJ) and were puried by Probe Quant G-50 Micro column (Amersham Pharmacia Biotech, Piscataway, NJ). Northern Blot Analysis Total RNA was isolated with MasterPure RNA Purication Kit (Epicentre Technologies, Madison, WI) according to the manufacturer??s protocol (Kit MCR 85102). The concentration of RNA was determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. The purity of RNA was determined by running a 1% agarose/formadehyde denaturing gel and measuring the absorbance ratio of at 260 nm and 280 nm (A260/A280). The isolated RNA had an A260/A280 ratio of 1.85?C1.95. The isolated RNA (5 mg/well) was separated using a 1% agarose/formadehyde denaturing gel at 75 V. The gels were blotted to Nytran Super Charge membrane (11 14 cm) (Schleicher & Schuell, Keene, NH) at room temperature (RT) in 20 SSC. The membranes

    were xed by UV-induced crosslinking. The hybridization with 32 P-labeled DNA probes were performed with Quickhyb solution (Stratagene, La Jolla, CA) as recommended by the manufacturer??s protocol. Northern blots were repeated to verify reproducibility of results, quantization was carried out by phosphor image scanning.

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     Microarray Experiments Total RNA was isolated with MasterPure RNA Purication Kit MCR 85102 (Epicentre Technologies, Madison, WI). Puried RNA was isolated with RNAeasy Kit 75144 (Qiagen Inc., Valencia, CA.). RNA concentration was determined by measuring the absorbance at 260 nm (A260). RNA purity was determined by running a 1% agarose/formadehyde denaturing gel and measuring the absorbance ratio at 260 and 280 nm, the isolated RNA had an A260/A280 ratio of 1.85?C 1.95. The isolated total RNA was used to synthesize uorescently (Cy-5 or Cy-3) labeled cDNAs that was hybridized to a microarray. Glass DNA microarrays (University of Wisconsin, Gene Expression Center), consisting of full-length PCR products from all E. coli ORFs according to Blattner et al. (1997) were used. Twenty micrograms total RNA were labeled for Cy-3 or Cy-5 using indirect labeling methods. Briey, total RNA was mixed with 4 mg of random primer (hexamer) in total volume of 25 mL, denatured at 708C for 10 min, primed while cooling to RT. To this primed total RNA, 2.5 mL of 20 dNTP mix [10 mM each of dATP, dGTP, dCTP, 4 mM of dTTP, and 6 mM aminoallyl-dUTP (Sigma, St. Louis, MO)], 10 mL of 5 rst strand buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 5 mL of 0.1M DDT, 1 mL of SUPERase. InTM (10,000 unit; Ambion, Austin, TX), and 3 mL of Superscript II reverse transcriptase (200 mg/mL; Invitrogen, Carlsbad, CA) were added. After 60 min incubation at 428C, the RNA was degraded by adding 2 mL of RNase H (2 mg/mL) and incubating for 20 min at 378C. The cDNAs were precipitated by ethanol and resuspended in 10 mL of 0.1M NaHCO3. For chemical coupling of aminoally groups in cDNAs with Cy3 or Cy5 dyes, monoreactive Cy3 or Cy5 Dye (Amersham Pharmacia Biotech, Piscataway, CA) was dissolved in 0.1M NaHCO3 and mixed with cDNAs. The mixture was incubated for 1 h at RT. Labeled cDNAs were precipitated with ethanol and puried by Nucleospin Extraction Kit (Clontech, Palo Alto, CA). The labeled cDNA probes were combined and concentrated to 11 mL by a Microcon-YM30 (Millipore, Bedford, MA). The hybridization mix consisted of salmon sperm DNA (0.56 mg/mL, BRL), yeast tRNA (0.22 mg/mL, Sigma), 5 SSC, 0.1% SDS, and 25% formamide in nal volume of 28 mL. Before hybridization, the cDNA microarray slides were incubated with prehybridization buffer (5 SSC, 0.1% SDS, and 1% BSA) for 1 h at 428C and washed in water and isopropanol. Hybridization was carried out at 458C overnight. Before scanning,

    slides were washed in 2 SSC containing 0.1% SDS for 2 min, then 1 SSC, 0.2 SSC, and 0.05 SSC, sequentially for 1 min. Hybridized arrays were scanned at 10 mm resolution on a GenePix 4000A scanner (Axon Instrument, Union City, CA) at variable PMT voltage to obtain maximal signal intensities with less than 1% probe saturation. Resulting images were analyzed via GenePix Pro v3.0 (Axon Instrument) as described in the manufacturer??s manual. Each sample was examined at least twice by switching the uorescent dye. The variance in the measured

     duplicated uorescence ratio approached the minimum when the uorescence signal was greater than approximately 0.4% of the measurable total signal dynamic range above background in both hybridization channels. The brightness of each spot was normalized by the ratio of total intensities of Cy3 and Cy5 signals on each slide. The subset of spots was selected by this criterion to identify well-measured spots. Average values of two experiments on each sample were obtained and values that were missing in two experiments were excluded from further analysis. The condence between forward labeled samples and reverse-labeled samples was calculated by Pearson equation. The values were ranged 0.91?C0.95. RESULTS High Glucose Escherichia coli Fermentation Bacterial biomass and acetate concentrations during batch growth of E. coli BL21 and JM109 at high glucose are shown in Figure 2. The growth parameters conrmed previously published observations (Shiloach et al., 1996): E. coli BL21 grew faster and did not accumulate acetate, the nal concentration was 0.1 g/L, but at one point during the growth it peaked at 3.7 g/L. In contrast, at the same growth conditions E. coli JM109 accumulated acetate throughout the growth process up to a concentration of 11 g/L. The specic growth rate of E. coli BL21 was 0.55 compared with 0.37 for E. coli JM109, the glucose consumption rate of E. coli JM109 was higher: 1.18 g/h compared with 0.66 g/h for BL21. The glucose concentration in the BL21 culture after 6 h growth when the OD was 63.6, was similar to that of the E. coli JM109 culture after 8 h growth when the OD was 35. Glucose concentration at this time was 0.096 g/L for E. coli BL21 and 0.087 g/L for E. coli JM109. Microarray Analysis Microarray analysis results of samples taken at late log phase when the culture entered the stationary phase (indicated with an arrow in Fig. 2), from both strains expressed as log 2 ratios

     Figure 2. Growth and acetate production during the high glucose batch fermentation of E. coli BL21 and JM109.

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     Figure 3. Gene expression in E. coli BL21 and JM109 during high glucose batch fermentation by microarray analysis. (A) Glyoxylate shunt pathway; (B) acetate production and uptake; (C) glycogen, gluconeogenesis, and anaplerotic pathway; (D) glycolysis pathway; (E)

TCA cycle; and (F) fatty acid pathway.

     are shown in Figure 3. A positive ratio indicates that BL21 expressed the gene to a higher level than did JM109; a negative ratio indicates the opposite. The data are clustered in the following metabolic pathway groups: the glyoxylate shunt and its regulators (panel A), acetate production and utilization (panel B), glycogen biosynthesis and degradation, gluconeogenesis, and anaplerosis (panel C), glycolysis

     (panel D), the TCA cycle (panel E), and fatty acid biosynthesis and degradation (panel F). The following picture emerged when these gene groups were analyzed: Examining the glyoxylate shunt pathway genes (Fig. 3A) aceA, aceB, and aceK, and the positive regulators fruR, himA, and him D are upregulated in E. coli BL21 compared with E. coli JM109 while the iclR is

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     downregulated. Concerning the acetate production and uptake gene group (Fig. 3B), ackA, pta, aceE, aceF, andacs are upregulated while poxB is downregulated in E. coli BL21, compared with E. coli JM109. In the gluconeogenesis and anaplerotic metabolism group (Fig. 3C), fructose bisphosphatase (fbp), pyruvate carboxykinase (pckA), phosphoenolpyruvate synthetase (ppsA), and the anaplerotic enzyme phosphoenolpyruvate carboxylase (ppc) are upregulated in E. coli BL21, while NAD-linked malate dehydrogenase (scfA) is upregulated in E. coli JM109. As for the glycogen biosynthetic pathway, glgA, glgB, and glgC were upregulated in E. coli BL21 except for glgS which was downregulated. The glycolysis pathway genes were upregulated in E. coli JM109 (Fig. 3D), and the TCA cycle and the fatty acid pathways were upregulated in E. coli BL21 (Fig. 3E and F). The assumption is that there is a correlation between the corresponding protein and the mRNA level and the difference in the gene transcription is translated to enzyme level.

     Time Course Northern Blot Analysis To understand the dynamics of extracellular acetate production based on the information obtained from the microarray evaluations, a time course Northern blot analysis of individual genes from the two strains grown at high glucose fermentation was conducted.

     Time Course Transcription of the Acetate Producing and Consuming Genes Time course transcription of acetate producing and consuming genes is shown in Figure 4. Figure 4A shows the acetate producing genes ackA and pta (acetate kinase and phosphotransacetylase), Figure 4B shows poxB (pyruvate oxidase) and aceE (pyruvate dehydrogenase), and Figure 4C shows the acetate consuming gene acs (acetyl-CoA synthetase). The transcription of the acetate-producing genes ackA (acetate kinase) and pta (phosphotransacetylase) decreased

     Figure 4. Transcription patterns in E. coli BL21 and JM109 during high glucose batch fermentation by Northern blot analysis. (A) ackA and pta, (B) aceE and poxB, (C) acs.

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     in both strains (Fig. 4A), and the transcription of the acetateconsuming gene acs (acetyl-CoA synthetase) increased after 5 h of growth in BL21 and remained at zero in JM109 (Fig. 4C). It is interesting that the transcription of the aceE gene (pyruvate dehydrogenase) that converts pyruvate to acetyl-CoA decreased in E. coli BL21 and slightly increased in E. coli JM109, there was hardly any transcription of the poxB gene (pyruvate oxidase) in E. coli BL21 and there was increased transcription in E. coli JM109 (Fig. 4B). Time Course Transcription of the Glyoxylate Shunt Associated Genes Time course transcription of the glyoxylate shunt associated genes, aceA (isocitrate lyase) and iclR (repressor of the glyoxylate operon), is shown in Figure 5. An expected difference was observed in the transcription of aceA (isocitrate lyase), its transcription increased in E. coli BL21 and was zero in E. coli JM109. Transcription of iclR (repressor of the glyoxylate operon) was slightly higher in E. coli JM109 at the early growth phase; it then went down, while the transcription of the repressor in E. coli BL21 was more or less stable throughout the growth. The Transcription of the Glucoengenesis and the Anaplerotic Pathways Genes Figure 6 summarizes the transcription of the gluconeogenesis and the anaplerotic pathways genes. Figure 6A shows the gluconeogenesis genes pckA (phosphoenolpyruvate carboxykinase), ppsA (phosphoenolpyruvate synthetase), and fbp (fructose bisphosphatase). Figure 6B shows the glycogen syththase (glgA); Figure 6C shows the transcription pattern of

     anaplerotic genes, ppc (phosphoenolpyruvate carboxylase), and sfcA (NAD-linked malate dehydrogenase). The difference in the transcription of the three gluconengenesis genes, pckA (phosphoenolpyruvate carboxykinase), ppsA (phosphoenolpyruvate synthetase), and fbp (fructose bisphosphatase) between the two strains was dramatic. All three genes were increasing in E. coli BL21 but were decreasing or not detectable, except for pckA which increased in E. coli JM109 at the last sample taken after 8 h of growth. Glycogen synthetase (glgA) transcription was increasing in both strains but it was two times higher in E. coli BL21 (Fig. 6B). The transcription of the anaplerotic gene ppc decreased in both strains but its level in E. coli BL21 was 5 times higher at the early log phase. sfcA transcription was more or less stable before it started decreasing towards the end of the growth, but the level in E. coli BL21 was as high as the ppc gene. The results suggest that increased gluconeogenesis and glycogen synthesis may help

    acetyl-CoA synthetase and the glycoxylate shunt to reduce the acetate concentration. Low Glucose Fermentation Bacterial biomass and acetate concentrations during fed batch growth of E. coli BL21 and JM109 at low glucose is shown in Figure 7. The growth parameters were conrmed in previously published observations (Shiloach et al., 1996), E. coli BL21 grew faster and did not accumulate acetate more than 1.6 g/L acetate throughout the growth process. While at the same growth conditions, E. coli JM109 accumulated acetate to a maximum concentration of 3.4 g/L which decreased to less than 1 g/L. Microarray Analysis Analysis of microarray results of samples taken at late log phase (indicated with an arrow in Fig. 7) expressed as log 2 ratio, from both strains is shown in Figure 8. As was done in the case of high glucose the data were clustered in the following metabolic pathway groups: TCA cycle, glycolysis, glyoxylate shunt, acetate production and uptake, glycogen biosynthesis, gluconeogenesis and fatty acid metabolism. The results are almost the opposite from those obtained at the high glucose fermentation (Fig. 2). The examination of the glyoxylate shunt pathway genes (Fig. 8A) indicated that the positive regulators are upregulated in E. coli BL21 compared with E. coli JM109 but unexpectedly, the negative regulators fadR and iclR and the glyoxylate shunt enzymes are upregulated in E. coli JM109 compared with E. coli BL21. Concerning the acetate production and uptake genes, ack, pta, aceE, ace, and acs are upregulated in E. coli JM109, while poxB is upregulated in E. coli BL21 (Fig. 8B). In the gluconeogenesis and anaplerotic metabolism groups, except for ppc, all the genes are upregulated in E. coli JM109, as for the glycogen biosynthetic pathway only the glgS upregulated in E. coli JM109, the rest of the genes (glgA, glgB, and

     Figure 5. Ttranscription patterns of aceA and iclR in E. coli BL21 and JM109 during high glucose batch fermentation by Northern blot analysis.

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     Figure 6. Transcription patterns in E. coli BL21 and JM109 during high glucose batch fermentation by Northern blot analysis. (A) pckA, ppsA, and fbp; (B) glgA; and (C) ppc and sfcA.

     glgC) were downregulated in E. coli JM109 (Fig. 8C). As was indicated in the high glucose section, assuming correlation between the corresponding mRNA and the protein it is expected that higher transcription is translated to higher enzymatic activity. Time Course Northern Blot Analysis To understand the microarray results, at a fed-batch growth where E. coli BL21 maintained acetate accumulation to a minimum while E. coli JM109 behaves more like E. coli BL21 under batch conditions, a time course Northern blot analysis was performed.

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