The strains used were E. coli BW25113 (F⁻ λ⁻ rph⁻¹ ΔaraBAD_AH33 lacI^q ΔlacZ_WJ16 rrnB_T14 ΔrhaBAD_LD78 hsdR514) and its single gene knockout mutant lacking cra (JW0078), which was obtained from Keio collection [20]. All the strains were first precultured in the Luria-Bertani (LB) medium. The second preculture and the main culture were carried out using M9 synthetic medium containing the following components (per liter): 6.81 g Na₂HPO₄, 2.99 g KH₂PO₄, 0.58 g NaCl and 5.94 g (NH₄)₂SO₄. The carbon source was a mixture of glucose, fructose, and xylose each contained 5 g/l, or some of the combinations among them for the batch and continuous culture. The following components were filter sterilized and then added (per liter) with 1 ml of 1 M MgSO₄·7H₂O, 1 ml of 0.1 mM CaCl₂·2H₂O, 1 ml of 1 mg/L thiamine HCl and 10 ml of trace element solution containing (per liter): 0.55 g CaCl₂·2H₂O, 1.67 g FeCl₃·6H₂O, 0.1 g MnCl₂·4H₂O, 0.17 g ZnCl₂·2H₂O, 0.043 g CuCl₂·2H₂O, 0.06 g CoCl₂·2H₂O, and 0.06 g Na₂M_OO₄·2H₂O.

The first pre-cultivation was performed by transferring 0.1 ml glycerol stock to a 10 ml L tube. After 8 h cultivation, the second pre-cultivation was performed by transferring 1 ml of culture broth to a 100 ml T tube. Batch and continuous cultures were conducted in a 2-l fermentor (Venus 25, Biott Co.) with a working volume of 1 l. The pH was controlled at 7.0 ± 0.1 using 2 N HCl or 2 N NaOH, and the temperature was set at 37˚C. The air flow rate was 1 vvm (air volume/working volume/ min), and the agitation speed was 400 rpm to maintain the dissolved oxygen concentration to be at 35% - 40% (v/v) of air saturation for the case of aerobic cultivation [21]. In the case of anaerobic condition, aeration was stopped and the agitation speed was reduced to 100 rpm. The dilution rate in the continuous culture was 0.1 h⁻¹. The triplicate samples were collected at the steady state which was confirmed by the constant cell density. It generally took 3 - 4 residence times to achieve the steady state.

Bacterial growth was monitored by measuring the optical density of the culture broth at 600 nm (OD₆₀₀) using a spectrophotometer (Ubet-30, Jasco, Tokyo, Japan). After fermentation, E. coli cells from 200 ml of culture were harvested by centrifugation and dried. After confirming that the weight of the dried material was constant, the dry cell weight per liter of culture was calculated from the measured weight. It was then converted to dry cell weight (DCW) based on the OD₆₀₀-DCW relationship. Glucose, fructose, xylose, lactate, acetate, formate, and ethanol concentrations in the medium were measured using commercially available kits (Wako Co., Osaka, Japan for glucose, fructose and xylose; Roche, Molecular Biochemical, Mannheim, Germany for others).

Total RNA was isolated from E. coli cells by Qiagen RNeasy Mini Kit (QIAGEN K.K., Tokyo, Japan) according to the manufacture’s recommendation. The quantity and purity of RNA were determined by the optical density measurements at 260 and 280 nm and by 1% formaldehyde agarose gel electrophoresis. The primer sequences for respective genes used in this study were reported elsewhere [22]. Criteria for the design of the gene-specific primer pairs were followed according to Sambrook and Russel [23]. The primers used in this study were synthesized at Hokkaido System Science Co. (Sapporo, Japan). In all cases, the primer-supplied company confirmed the purity and absolute specificity of the primers.

RT-PCR reactions were carried out in a Takara PCR Thermal Cycler (Takara TP240, Japan) using Qiagen one-step RT-PCR Kit (QIAGEN K.K., Japan). The reaction mixture was incubated for 30 min at 50˚C for reverse transcription (cDNA synthesis) followed by 15 min incubation at 95˚C for initial PCR activation. Then, the process was subjected to 30 cycles of amplification which consisted of a denaturing step (94˚C for 1 min), an annealing step (approximately 5˚C below melting temperature of primers for 1 min) and an extension step (72˚C for 1 min), and finally the reaction mixture was incubated for 10 min at 72˚C for final extension. To check for nucleic acid contamination, one negative control was run in every round of RT-PCR. This control lacks the template RNA in order to detect possible contamination of the reaction components. 5 ml of amplified products were run on a 1% agarose gel. Gels were stained with 1 μg/ml of ethidium bromide, photographed using a Digital Image Stocker (DS-30, FAS III, Toyobo, Osaka, Japan) under UV light and analyzed using GelPro Analyzer 3.1 (Toyobo) software. Although the PCR products obtained for all the genes showed the predicted sizes on agarose gel, the identity of amplified fragments of some genes was demonstrated by DNA sequencing. In order to determine the optimal amount of input RNA, the two-fold diluted template RNA was amplified in RTPCR assays under identical reaction conditions to construct a standard curve for each gene product. When the optimal amount of input RNA was determined for each gene product, RT-PCR was carried out under identical reaction conditions to detect differential transcript levels of genes. The gene dnaA, which encodes DnaA, a replication initiation factor in E. coli and is not subjected to variable expression, i.e. abundant expression at relatively constant rate in most cells, was used as an internal control for the RT-PCR determinations. To calculate the standard deviation, RT-PCR was independently performed three times for each gene under identical reaction condition. To ensure that the observed changes were statistically significant, the measurements were made in triplicate, and the Student’s t-test was applied.

Figure 1(a) shows the batch cultivation result of wild type E. coli under aerobic condition for the case of using a mixture of glucose and fructose as a carbon source, while Figure 1(b) shows the case of the wild type using a mixture of glucose and xylose as a carbon source. As can be seen from the figure, glucose was first consumed, while fructose or xylose was consumed after glucose was depleted. This is due to the catabolite repression caused by the low level of cAMP or cAMP-Crp when glucose was present.

Figure 1(c) shows the aerobic batch cultivation result of cra mutant for the case of using a mixture of glucose and fructose as a carbon source, where it indicates an interesting phenomenon that both glucose and fructose were consumed simultaneously with fructose to be consumed a little faster as compared to glucose. Figure 1(c) shows, however, that acetate was more produced and less consumed after glucose was depleted as compared to the wild type (Figure 1(a)). This may be due to the repressions of aceA and icdA gene expressions caused by cra gene knockout, while pfkA and pykF gene expressions were activated by cra gene knockout [19].

Figure 2 shows the batch aerobic cultivation result of the wild type (Figure 2(a)) and cra mutant (Figure 2(b)) for the case of using a mixture of glucose, fructose, and xylose as a carbon source. Figure 2(a) shows that fructose and xylose were both consumed after glucose was depleted due to catabolite repression in the wild type. Figure 2(b) indicates that fructose and glucose were consumed simultaneously with fructose to be consumed faster than glucose as also observed in Figure 1(c), whereas xylose was consumed after glucose was depleted, where the latter phenomenon may be considered to be due to the catabolite repression by the lower level of cAMP-Crp in cra mutant.

Figure 3 shows the batch anaerobic cultivation results of the wild type (Figure 3(a)), and cra mutant (Figure 3(b)) for the case of using a mixture of glucose, fructose, and x-ylose as a carbon source. Figure 3(a) shows that after glucose was depleted, fructose and xylose were consumed, and that xylose was little consumed, where the latter phenomenon is different from the case of aerobic condition (Figure 2(a)). The fermentation products were formed in the order of formate, acetate, ethanol, as well as a little lactate, where the biomass formation was mainly made during the period when glucose and fructose were used as carbon sources. In the case of cra mutant as shown in Figure 3(b), fructose and glucose were first consumed where fructose was consumed faster as compared to glucose, which was also observed under aerobic condition (Figure 2(b)), and xylose was little consumed as also observed above for the wild type. The metabolite formation patterns were nearly the same as the case of the wild type (Figure 3(a)).

In order to investigate these phenomena in particular under anaerobic condition in more detail, the anaerobic continuous cultures of the wild type and cra mutant, were performed at the dilution rate of 0.1 h⁻¹ using a mixture of glucose, fructose, and xylose as a carbon source (Table 1). Glucose concentration was below detectable level, and the fructose concentration was less than xylose concentration for the wild type, consistent with the batch cultivation result (Figure 3(a)). On the other hand, fructose concentration was below detectable level, while glucose concentration was lower as compared to xylose concentration for cra mutant, consistent with the batch cultivation result (Figure 3(b)).

Figure 4 shows the comparison of the specific total carbon source consumption rates together with specific metabolite production rates obtained based on Table 1. It may be of practical interest that total carbon source consumption rate was higher for cra mutant as compared to the wild type. This contributes to the increase in the total metabolite production rate (Figure 4), which is attractive from the efficient metabolites production point of view.

Figure 5 shows the comparison of gene transcript levels for the case of continuous cultures as shown in Table 1. Figure 5 indicates that fruBKA as well as manX and mtlA genes were up-regulated in cra mutant as compared to the wild type (P < 0.01 for all these genes). In accordance with the global regulator-metabolic pathway genes relationship (Appendix A), the transcript levels of glycolysis genes such as pfkA, pykF, PP pathway genes such as edd and eda, together with anaplerotic pathway gene ppc, were all up-regulated (P < 0.01, P < 0.05, P < 0.01, P < 0.01, P < 0.01, and P < 0.01, respectively), while gluconeogenic pathway genes such as pckA, ppsA, fbp, and glyxoyalte pathway gene aceA, as well as the TCA cycle gene icdA were all down-regulated (P < 0.01 for all genes) for the cra mutant as compared to the wild type. The down-regulation of ptsG transcript level (P < 0.01) may be due to down-regulation of crp (P < 0.01) (Appendix A). The down regulation of aceA transcript level (P < 0.01) is also consistent with up-regulation of iclR (P < 0.01). The down-regulation of crp gene transcript level may be due to higher glucose concentration for cra mutant as compared to the wild type (Table 1). The xylose assimilating pathway genes such as xylR, xylFGH, xylAB, and xylE were all down regulated (P < 0.01 for all genes). Moreover, in accordance with higher production rate of acetate (Table 1), pta and ackA genes were up-regulated (P < 0.01 for both genes), and in accordance with higher ethanol production rate, adhE gene transcript level was up-regulated (P < 0.01) for cra mutant as compared to the wild type. The pflA gene transcript level was increased (P < 0.01), which corresponds to the higher production rate of formate (Table 1). Note that rpoS transcript level was decreased (P < 0.01) for cra mutant as compared to the wild type, which might be due to higher glucose concentration, and this also corresponds to the down regulations of acnA and fumC, but not acnB gene transcript level [21] (Appendix A).