Yeast and bacteria strains, plasmids, and growth conditions
Bacterial strains and plasmids used or constructed in this study are listed in Table 1 and Table 2 respectively. Genomic DNA for PCR amplification was prepared from Pichia stipitis and Saccharomyces cerevisiae grown in YPD (10 g/l yeast extract, 20 g/l peptone, 20 g/l dextrose) broth or on YPD agar at 30°C. All bacteria were propagated at 37°C in Luria-Bertani (LB 10 g tryptone, 5 g/l yeast extract, 10 g/l sodium chloride) broth or on LB agar supplemented with appropriate antibiotics including kanamycin (25 μg/ml) and ampicillin (100 μg/ml) when needed.
Isolation of genomic DNA from Pichia stipitis and Saccharomyces cerevisiae
The yeast was grown for 20-24 h at 30°C in YPD broth. The culture was then transferred into a microcentrifuge tube and subjected to centrifugation at 13000 rpm for 5 min. The supernatant was decanted and the cell pellet was resuspended in 200 μl lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA, pH 8.0).
The suspension was immersed on ice for 5 min, heated to 95°C~100°C for 5 min and mixed with 200 μl chloroform and by vortex for 2 min.
After centrifugation at 13000 rpm for 3 min, the upper aqueous phase was transferred into a microcentrifuge tube containing 400 μl ice-cold 100% ethanol, mixed by inversion or gentle vortex and then stored at -80°C for 1 h. Finally DNA pellet was collected after precipitation by
centrifugation at 13000 rpm for 5 min and washed with 1 ml 70% ethanol.
The DNA pellet was air-dry at 37°C for 1 h, resuspended in pure H2O at 60°C, and stored at -20°C until required.
DNA manipulation
Plasmids were purified by using the High-Speed Plasmid Mini kit (Geneaid, Taiwan). All restriction endonucleases and DNA modifying enzymes were purchased from either New England Biolab (Beverly, MA) or MBI Fermentas (Hanover, MD), and were used according to the recommendation of the supplies. PCR amplifications were performed with Taq DNA polymerase (MDBio, Inc, Taiwan). PCR products or DNA fragments were purified using the Gel/PCR DNA Fragments Extraction kit (Geneaid, Taiwan). Primers used in this study were synthesized by MDBio, Inc, Taiwan.
Preparation of competent cells for electroporation
A single colony of freshly grown E. coli was inoculated in a flask containing 25 ml of LB medium and cultured overnight at 37°C with vigorous aeration (200 rpm in a rotary shaker). The overnight culture was then inoculated into 500 ml prewarmed LB medium and grown with agitation aeration (200 rpm in a rotary shaker) at 37°C. The bacterial density was measured every 20 min till the OD600 reached 0.4 and the cultural flask moved to an ice-water bath for 20 min with occasional swirl to ensure that cooling occurs evenly.
Finally, the cultures were transferred to ice-cold centrifuge bottles and
the cells were harvested by centrifugation at 13000 rpm for 20 min at 4°C.
The supernatant was decanted and the cell pellet was resuspended in 300 ml of ice-cold pure H2O and again harvested by centrifugation at 13000 rpm for 20 min at 4°C. The cell pellet was resuspended in 150 ml of ice-cold 10% glycerol and collected by centrifugation at 13000 rpm for 20 min at 4°C. The procedure was repeated once and the pellet was resuspended in 1 ml of ice-cold GYT medium (10% glycerol, 0.125%
yeast extract, and 0.25% tryptone). The cell suspension was dispensed 40 μl aliquots into sterile, ice-cold 1.5 ml microfuge tubes and stored at -80°C until required.
Software for analysis of protein structure and amino acid structural entropy
Structural predictions were performed using Swiss-Model (http://swissmodel.expasy.org//SWISS-MODEL.html) and multiple sequence alignments were performed using Vector NTI 6.0. The sequence derived structure entropy was analyzed according to the provided software at http://sdse.life.nctu.edu.tw/index.cgi.
Site-directed mutagenesis
XK mutants were produced using the QuikChange site-directed mutagenesis method (Stratagene) recommended by the manufacturer. The primers used are listed in Table 3 and the resulting plasmid was respectively termed in Table 2 and the mutant XK proteins were expressed in E. coli BL21 (DE3) or E. coli NovaBlue (DE3).
Constructions of the recombinant His6-tagged proteins
The XK expression plasmid pET30-Sc-XK was generated by PCR-amplification of the coding sequence of XK gene from S. cerevisiae using primer pair PJ036 and PJ037 (Table 3), the amplified DNA was restricted by EcoRI/SalI and then ligated into the pET-30b vector. Similar approach was used to generate other recombinant plasmids such as pET30-Ps-XK. The coding region was PCR amplified using primer pair PJ038 and PJ039 (Table 3).
Overexpression and purification of the His6-tagged proteins
Bacterial cells were incubated in 500 ml of LB medium at 37°C with shaking until OD600 reached 0.5. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was then added to a final concentration of 0.5 mM and the growth was continued for 16-20 h at 22°C.
Subsequently, the cells were harvested by centrifugation at 10000 rpm for 20 min at 4°C, resuspended in binding buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.9), the cells disrupted by sonication and then the cell debris removed by centrifugation at 13000 rpm for 10 min at 4°C.
Finally, the His6-tagged proteins were purified from the supernatant via affinity chromatography using His-Bind resin (Novagen). The washing buffer is consisted of 20 mM Tris-HCl, 500 mM NaCl, 70 mM imidazole, pH 7.9 and the elution buffer consisted of 20 mM Tris-HCl, 500 mM NaCl, 80mM imidazole, pH 7.9.
After the protein-bound resins washed several times with the wash
buffer and the protein eluted using the elution buffer, purity of the collected fractions were analyzed by SDS-PAGE. Finally, the purified His6-tagged kinase was dialyzed against 500 ml of the buffer containing 0.1 g KCl, 4 g NaCl and 1.5 g Tris-HCl, pH 7.8.
SDS-polyacrylamide gel electrophoresis
Before subjected to gel electrophoresis, the proteins in lysis buffer (0.0625 M Tris-HCl buffer pH 6.8, 4% SDS, 10% glycerol, 0.002%
bromophenol blue, 100 mM 2-Mercaptoethanol) was heated for 15 min at 95°C. Aliquots of the protein samples were applied to a 13.5% SDS polyacrylamide slab gel and electrophoresis was carried out at room temperature until the tracking dye ran off the bottom of the slab gel. The gel was then stained for 30 min with Coomassie blue (0.5 g Brilliant Blue R, 45% methanol, and 10% acetic acid) and destained briefly in destain I buffer (500 ml H2O, 400 ml methanol and 100 ml acetic acid) for 15 min and in destain II buffer (880 ml H2O, 50 ml methanol and 70 ml acetic acid) for 30 min.
Circular dichroism spectrum analysis
The CD spectra of wild-type and the mutant were recorded by using a CD spectrophotometer (AVIV 62A DS) with 1-mm path length cell, 0.5 nm wavelength step, and an averaging time of 3×10-1 s. The protein samples were adjusted to 2.5 μM before measurement. The CD spectra signals were collected from 190 nm to 260 nm at 25°C in 10 mM Tris-HCl, pH 7.4 and averaged over three scans (Lee et al., 2008).
Xylulokinase activity measurement and kinetics characterization
Activity of the purified XK was determined according to the described spectrophotometric assay (Dmytruk et al., 2008) with some modifications and the condition is summarized in Table 4. The enzymatic activity was determined by monitoring the change in absorbance at 340 nm that accompanies the oxidation of NADH to NAD+.
The XK activity assay mixture contained 5 mM MgCl2, 0.2 mM NADH, 1 mM phosphoenolpyruvate, 8.5 mM D-xylulose, 10 U lactate dehydrogenase, 1.5 U pyruvate kinase, and 2 mM ATP in 50 mM Tris-HCl, pH 7.8. The reaction at 25°C was started with addition of purified protein.
The KM and Vmax for xylulose and ATP were determined independently using standard assay conditions (Dmytruk et al., 2008). Constants for xylulose as substrate were measured at 42°C by holding constant ATP concentration and varying xylulose level with 0.0425 mM, 0.2125 mM, 0.425 mM, 2.5 mM or 4.5 mM. Similarly, ATP kinetic measurements at 42°C were made by holding constant xylulose and varying ATP concentration with 0.05 mM, 0.1 mM, 0.5 mM, 1 mM or 2 mM. KM and Vmax were calculated by fitting the data to Michaelis-Menten equation (Di Luccio et al., 2007).
Temperature effect on Xylulokinase activity
This assay was performed essentially as described above except that the reaction temperature varied at 20°C, 25°C, 37°C, 42°C, 50°C or 60°C.
Analysis of pH effect on Xylulokinase activity
This assay was performed essentially as described above except that the reaction pH varied with pH 6.5, pH 7, pH 7.5 of 50 mM Tris-HCl or pH 7.5, pH 8, pH 8.5, pH 9 of 50 mM MPOS buffer at 25°C.
Results
Genetic and sequence analysis of XK from S. cerevisiae and P. stipitis The research exhibited that the XK of P. stipitis and S. cerevisiae belong to the FGGY and FGGY family of carbohydrate kinases which included D-xylulokinase (EC 2.7.1.17), L-xylulokinase (EC 2.7.1.53), glycerol kinase (EC 2.7.1.30), glucokinase (EC 2.7.1.12), and
L-fuculokinase (EC 2.7.1.51) (Jin et al., 2002). The XK gene XYL3 from P. stipitis encodes a polypeptide of 624 amino acids and XKS1 from S.
cerevisiae coding for a polypeptide of 600 amino acids (Fig. 2).
Approximately 41.3% sequence identity was found between the two XK, which have been demonstrated to be able to metabolize xylulose (Matsushika et al., 2009b).
As shown in Fig. 3, the N-terminal LGFDLSTQQLK peptide common to sugar kinases for phosphate binding (Jin et al., 2002) appeared to be also conserved. Comparison analysis revealed Ec-XK and Ps-XK, and Ec-XK and Sc-XK shared sequence identity of 13.6% and 16.8%
respectively. The two conserved aspartate residues, Asp17 and Asp27, located at the N-terminal domain I required for Ec-XK interaction with the ATP-associated Mg2+ (Di Luccio et al., 2007) were also present in Ps-XK and Sc-XK. In order to have a negative control for the following activity analysis, a single residue change from Asp17 to Ala on Ps-XK was generated using the site-directed mutagenesis method (Appendix 1) and the mutant named Ps-XKD17A.
Search for the critical residues of S. cerevisiae XK amino acid sequence on the base of entropy difference
We explored the relationship between structural entropy profile and protein thermal stability by the approach offered a straightforward way to compute the structural entropy directly from the query sequence. It may be used as a useful tool to screen mutant candidates for thermophilic sequences in a high throughput way (Chan et al., 2004).
The XK of S. cerevisiae and P. stipitis belong to the FGGY family, of which the FGGY region was selected for site-directed mutagenesis. After the conserved residues eliminated, the rest of Sc-XK sequences were selected for the analysis of entropy change (Fig. 4). The entropy change was calculated by replacing the target amino acid with other nineteen amino acids and the minimum entropy state was selected.
As shown in Table 5, six residue substitution including S47E, S88V, G169C, S253W, S254M, and S255W with a maximal drop of the entropy were identified. To verify if each of the residue change increases the protein thermal stability as suggested by Chan et al., (Chan et al., 2004), a Ser88 to Val88 alteration of Sc-XK was generated using the site-directed mutagenesis method (Appendix 1) and the mutant named Sc-XKS88V.
Construction, expression and purification of the recombinant XK In addition to Ps-XKD17A and Sc-XKS88V mutants, a Ps-XK mutant probably resulted from random mutation during PCR cloning of Ps-XK was obtained and named Ps-XKA55T (Fig. 3).
The wild-type and mutant XK cloned into pET30b were expressed in E.
coli BL-21 (DE3) or E. coli NovaBlue (DE3) and the proteins purified by nickel affinity chromatography. Its purity was confirmed on a SDS- polyacrylamide gel and stained with Coomassie Blue. As shown in Fig. 5, expression of Sc-XK and Sc-XKS88V could barely be detected. By contrast, overexpression of Ps-XK (Fig. 6A) and Ps-XKA55T (Fig. 6B) were observed. Nevertheless, all the recombinant proteins could be purified to homogeneity (Figs. 5 and 6).
Circular dichroism analysis of the recombinant proteins
Prior to the activity measurement of the recombinant XK, the activity alteration caused by its conformational changes has to be excluded. The applications of CD spectroscopy could be categorized in various regions of biological studies such as conformational assessments of proteins and nucleic acids. Protein secondary structure can be determined by CD spectroscopy in the 'far-UV' spectral region (190–250 nm)(Ranjbar and Gill, 2009).
As shown in Fig.7, the CD spectra of Sc-XK and Sc-XKS88V are identical. Overlapping CD spectra Ps-XK, Ps-XKA55T and Ps-XKD17A showing a minimum point at 208 nm and 222 nm, a typical spectrum of high α-helix content protein (Ranjbar and Gill, 2009), were also observed (Fig. 8). These indicate no major alteration of the secondary structure of the mutant proteins.
Activity measurement of the XK
The XK activity was determined using the assay condition summarized
in Table 4. Surprisingly, no detectable activity could be observed for Sc-XK or Sc-XKS88V. On the other hand, Ps-XK and Ps-XKA55T both exhibited enzymatic activity (Fig. 9). As shown in Fig. 9, no activity for Ps-XKD17A was found further supports that the Asp residue is essential for the XK activity.
Comparative analysis of the specific activity at 25℃, pH 7.8 revealed that Ps-XKA55T carries a 3 fold higher specific activity than Ps-XK (Fig.
10). While the activity measured at different temperatures (Fig. 11) or pH (Fig. 12), Ps-XK has maximal activity at 37℃ and pH 7 while Ps-XKA55T is at 42℃ and pH 7.5. We reason that Ps-XKA55T with a single residue change of Ps-XK increased tolerance to higher temperature which is a required property for practical use in fermentation process (Dmytruk et al., 2008).
Kinetic parameters of Ps-XK and Ps-XKA55T
The kinetic constants of Ps-XK and Ps-XKA55T were determined and compared. The kinetics was determined under various concentrations of xylulose or varying the concentration of ATP (Fig.13).
After the data fitted to Lineweave-Burk equation, the kcat / KM revealed the catalytic efficiency of Ps-XKA55T for xylulose and ATP were much higher than that of the wild type at 42°C (Table 6).
As shown in Fig. 14, the structure of Ps-XKA55T using Ec-XK as template was predicted.
Discussion
To construct a S. cerevisiae strain which can efficiently grow on D-xylose has been an intense research object. Most of the research has focused on overexpression of the genes involved in the metabolism of xylose (Wahlbom et al., 2003). However, an impaired growth of the engineered strain has been a problem for mass production. Physiological studies have shown that XK is essential for growth on xylose or xylulose and is a limiting factor for the overall rate of pentose sugar utilization.
The enzyme has been studied from several prokaryotes and lower eukaryotes to higher eukaryotes.
Rodriguez-Pena et al. first suggested the XK overexpression is toxic for S. cerevisiae cells grown on D-xylulose (Rodriguez-Pena JM, 1998).
The Sc-XK as well as Ps-XK accepted D-ribulose as a substrate and hence the increased levels of the XK activity should be designed in concert with the capacity of the surrounding metabolic network (Yong-Su Jin, 2003, Richard et al., 2000). Here, we intend to obtain large amounts of a highly efficient and specific XK protein which could be added to the fermenting yeast at a certain stage without impairing the cell growth.
However, the first attempt to engineer the recombinant Sc-XK has been failed. No activity could be detected for the heterologously expressed Sc-XK and Sc-XK (S88V). The previous study demonstrates significant post-transcriptional control of protein levels for a number of different compartments and functional modules. The availability of genome-wide data of mRNA levels, translational status, and protein abundances in yeast was performed an integrated analysis of post-transcriptional
expression regulation in a whole cell. Greenbaum and coworkers discussed three potential reasons for the lack of a perfect correlation between mRNA and protein levels: (i) translational regulation, (ii) difference of in vivo protein half-lives, and (iii) the significant amount of experimental error including differences with respect to the experimental conditions. Accordingly, we speculated that post-transcriptional modification, which lacking in the E. coli strain, is essential for the activity of the recombinant proteins (Beyer et al., 2004).
The phospho group transfer of sugar kinase has been reported to be promoted by the two highly conserved aspartate residues. As predicted, the Asp17 of Ps-XK is essential for the enzymatic activity since the recombinant Ps-XKD17A had no activity detected. It has been reported that Ps-XK compared to Sc-XK is more specific for D-xylulose (Richard et al., 2000). It is likely that the recombinant Sc-XK preferred other 5C sugars as substrate.
The Ps-XK (A55T) which carries a residue change displayed higher activity than the wild-type XK. The kinetic properties analyzed demonstrated that the substitution of Ala55 increased the XK affinity toward xylulose and ATP. Interestingly, this change also increased apparently the XK activity at high temperature. The analysis of ribbon diagram XK structure as shown in Fig. 14 revealed that the change of side-chain from –CH3 (Ala) to –OH (Thr) closed to the substrate binding site may lead to an increase activity of Ps-XK.
Circular dichroism (CD) is often used to assess the degree to which solution pH, buffers, and additives such as sugars, amino acids or salts
alter the thermal stability of target proteins. In addition, protein secondary structure can be determined by CD spectrum within 'far-UV' of 190–250 nm (Ranjbar and Gill, 2009). It is hence, the study of thermal stability of the recombinant XK could be assessed by monitoring the CD spectrum changes with increasing temperatures. Alternatively, a single wavelength can be chosen which monitors specific feature of the protein structure, and the signal at that wavelength recorded continuously as the temperature increased.
The XK protein which exhibits an increased activity under the conditions when the temperature switched from 37°C to 42°C can be very useful while applied in the xylulose utilization process. This thermostable property could also be used in developing superior yeast strains possessing integrated expression systems for the commercial production of ethanol from lignocelluloses.
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