1 Roche Vitamins AG, Department VFB, Building 203/112a, CH-4070 Basel, Switzerland, 3 Novozymes A/S, Smørmosevej 25, DK-2880 Bagsværd, Denmark and 4 Waldshuterstrasse 15, CH-4310 Rheinfelden, Switzerland
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Abstract |
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Keywords: consensus sequence/phytase/protein engineering/thermostabilization
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Introduction |
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Besides the rather successful directed evolution approaches, stabilizing amino acid substitutions have been predicted successfully using various rational concepts and/or by comparison with the sequence or 3D-structure of a homologous protein from a thermophilic organism. The success rate in most of these cases has been rather low. One of the most prominent exceptions has been reported by van den Burg et al. (van den Burg et al., 1998) who applied different rational and semi-rational approaches to the stabilization of a thermolysin-like protease. They came up with a mutant showing a 21°C increase in thermostability which allowed the protease to be active at 100°C. Furthermore, a computational method is now available that combines (i) potential functions that model a protein sequence's compatibility with a structure, and (ii) fast optimization tools that can search the huge number of sequence possibilities (Dahiyat, 1999
). This method was used, for example, to predict seven stabilizing point mutations, combination of which enhanced the unfolding temperature of the ß1 domain of streptococcal protein G from 81°C to >100°C (Malakauskas and Mayo, 1998
).
Recently, we presented a new semi-rational `consensus' approach for increasing the thermostability of proteins (Lehmann et al., 2000). The approach is based on the comparison of amino acid sequences of homologous proteins and the subsequent calculation of a consensus amino acid sequence using one of the available standard programs. Applying this approach to the family of fungal phytases, which at that time consisted of sequences from 13 different ascomycetes (Table I
), resulted in a consensus phytase that exhibited a 1522°C increase in its unfolding temperature compared to each of its parent phytases. The catalytic properties of the novel consensus phytase-1 resembled most those of Emericella nidulans and Aspergillus fumigatus phytase. A possible explanationbased on statistical thermodynamicsas to why more frequently occurring amino acids at a given position of an amino acid sequence alignment have a larger stabilizing effect than less frequently occurring amino acids, has been provided by Steipe et al. (Steipe et al., 1994
). This approach of using sequence comparisons to predict individual thermostabilizing `consensus' amino acid substitutions, and the subsequent combination of experimentally proven thermostabilizing single mutations in a multiple mutant have been employed successfully for improving the thermostability of immunoglobulin domains (Ohage et al., 1997
, 1999
; Ohage and Steipe, 1999
; Wirtz and Steipe, 1999
), an SH3 domain of a tyrosine kinase (Maxwell and Davidson, 1998
), the tumor suppressor p53 DNA binding domain (Nikolova et al., 1998
), GroEL minichaperones (Wang et al., 1999
, 2000
), as well as a WW domain (Jiang et al., 2001
).
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Materials and methods |
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Phytic acid (dodecasodium salt) was purchased from Sigma (St Louis, MO) and p-nitrophenyl phosphate from Merck (Darmstadt, Germany). Unless specified otherwise, A.fumigatus and consensus phytases were purified from the supernatants of transformed Hansenula polymorpha or Saccharomyces cerevisiae strains. Despite extensive investigations (Wyss et al., 1999a,b
; and unpublished data), there is no indication that the temperature optima, TmS and specific activities of the phytases under investigation are affected by the host strain used for phytase production.
Design of the consensus phytases
The amino acid sequence alignments used for the design of the different consensus phytases (Table I) were calculated with the program PILEUP from the GCG Sequence Analysis Software Package, Release 9.0 (Devereux et al., 1984
) by using standard parameters (gap creation penalty 12, gap extension penalty 4). The location of the gaps was refined using a text editor. The consensus sequences were calculated with the program PRETTY which is also part of the GCG package. Plurality was set at 2.0, threshold at 3, and the scoring matrix used was prettypep.cmp. The origin and the vote weight of the individual phytase amino acid sequences used in the respective calculations are shown in Table I
. The impact of very similar amino acid sequences that originate from different strains of one fungal species was restricted to 1 by assigning appropriate vote weights to them. The design of the consensus sequence was restricted to the mature protein. For secretion of the protein, the first 26 amino acid residues of Aspergillus terreus cbs116.46 phytase were used as signal peptide and fused to the N-termini of consensus phytases-1 and -10.
Gene construction
Calculation of the DNA sequences, construction of the synthetic genes, cloning of the consensus phytase genes into the expression vector pFP, transformation of the methylotrophic yeast H.polymorpha, and purification of the consensus phytases expressed in H.polymorpha were performed as described previously (Lehmann et al., 2000).
For expression in S.cerevisiae, the consensus phytase genes were ligated into the EcoRI site of the plasmid RO11, which contains a shortened version of the constitutive GAPFL (glyceraldehyde-3-phosphate dehydrogenase) promoter and the terminator of the pho5 gene from S.cerevisiae, as described by Janes et al. (Janes et al., 1990). The correct orientation of the genes was checked by PCR. Transformation of S.cerevisiae strains (e.g. INVSc1; Invitrogen, Carlsbad, CA) was done according to Hinnen et al. (Hinnen et al., 1978
). Single colonies harboring the phytase gene under the control of the GAPFL promoter were picked and cultivated in 5 ml of dextrose medium lacking uracil [SD-(ura); Sherman et al., 1986
] at 30°C under vigorous shaking (250 r.p.m. LAB-Shaker, Kühner AG, Basel, Switzerland) for 1 day. The pre-culture was then added to 500 ml of YPD medium (Sherman et al., 1986
) and grown under the same conditions. After 4 days of incubation, the culture broth was centrifuged (8000 g, 15 min, 5°C) to remove the cells, and the supernatant was concentrated by way of Ultrafiltration in Amicon 8400 cells (PM30 membranes; Grace AG, Wallisellen, Switzerland) and Ultrafree-15 centrifugal filter devices (Biomax-30K; Millipore, Bedford, MA). The concentrate (10 ml) was desalted on a 40 ml Sephadex G-25 Superfine column (Amersham Pharmacia Biotech, Freiburg, Germany), with 10 mM sodium acetate, pH 5.0, serving as elution buffer. The desalted sample was brought to 2 M (NH4)2SO4 and directly loaded onto a 1 ml butyl-Sepharose 4 Fast-Flow hydrophobic interaction chromatography column (Amersham Pharmacia Biotech) which was eluted with a linear gradient from 2 to 0 M (NH4)2SO4 in 10 mM sodium acetate, pH 5.0. Phytase was eluted in the flow-through, concentrated and loaded on a 120 ml Sephacryl S-300 gel permeation chromatography column (Amersham Pharmacia Biotech). The consensus phytases thus purified eluted as homogeneous symmetrical peaks and were shown by SDSPAGE to be ~95% pure.
Site-directed mutagenesis
Mutations were introduced using the Quick ExchangeTM Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA) with appropriate primers. Plasmids harboring the desired mutation(s) were identified by DNA sequence analysis.
Activity measurements
Standard phytase activity assays were performed as described previously (Wyss et al., 1999b) with 5 mM phytic acid as substrate at pH 5.0 and 37°C and by measuring the release of inorganic phosphate. One unit of activity is defined as the amount of enzyme that releases 1 µmol of phosphate per minute. pHactivity profiles were recorded by measuring phytase activity at 37°C in appropriate buffer solutions at pH values between 2.5 and 9.0 (Wyss et al., 1999b
). Substrate specificity was addressed by measuring the specific activities of a given phytase with a range of phosphate compounds. For that purpose, phytic acid in the standard phytase assay was replaced with 5 mM concentrations of the phosphate compounds listed in the legend to Figure 3
. For determination of the temperature optima, enzyme and substrate solution (100 µl each) were pre-incubated separately for 5 min at a series of temperatures between 37 and 85°C and then mixed. After 15 min of incubation, the reaction was stopped and the amount of liberated inorganic phosphate determined according to the standard activity assay.
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For determination of the unfolding temperature (Tm), the proteins purified from H.polymorpha culture supernatants were extensively dialyzed against 10 mM sodium acetate, pH 5.0, and concentrated to 5060 mg/ml. DSC experiments were performed on a Mettler DSC 821e heat flux calorimeter equipped with the STARe control and evaluation software (Version 4.00). Tightly sealed 40 µl aluminum crucibles were used for the protein samples, while the reference cell was empty. The instrument was calibrated using indium as standard. The sample was cooled down from room temperature to 5°C, the starting temperature of the experiment. After equilibration for 5 min at this temperature, a constant heating rate of 10°C/min was applied up to 9095°C.
Other methods
Protein concentrations were determined using the enzyme extinction coefficients at 280 nm calculated according to Pace et al. (Pace et al., 1995). One absorption unit (1 OD) at 280 nm corresponds to 1.10 mg/ml of consensus phytase-1 and 1.04 mg/ml of consensus phytase-10.
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Results |
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Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases from three Aspergillus niger isolates, two isolates of A.terreus, five isolates of A.fumigatus, and one isolate each of the ascomycete fungi E.nidulans, Talaromyces thermophilus, and Myceliophthora thermophila (Table I). A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 1526°C more thermostable than all parent phytases used in its design (Lehmann et al., 2000
). In the present study, the consensus approach was refined by adding more fungal phytase sequences to the alignment.
In the case of consensus phytase-10, two more sequences were added: (i) the phytase sequence from the ascomycete Thermomyces lanuginosus (Berka et al., 1998); (ii) the conbasidio phytase sequence, which itself is the consensus sequence of five phytase sequences from the basidiomycete fungi Peniophora lycii, Trametes pubescens, Agrocybe pediades, and cf. Ceriporia (Lassen et al., 2001
). The subsequent calculation of the new consensus phytase-10* sequence by PRETTY left 21 positions undefined (shown in small letters in Figure 1
). Nine of these positions (46, 78, 265, 277, 297, 356, 366, 393, 423) were filled by the respective amino acid obtained from a calculation in which the conbasidio phytase sequence was omitted and the threshold was lowered from 3 to 2. At positions 18, 85, 130, 163, 166, 167 and 248, for which also in the latter calculation no consensus amino acid was defined, the corresponding amino acid residue of consensus phytase-1 was chosen. For the remaining five open positions (165, 181, 197, 223, 268), an amino acid occurring at those alignment positions in one of the wild-type phytases, but not the amino acid of consensus phytase-1, was selected arbitrarily.
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The differences above also might have been caused by the increased threshold used for the PRETTY session. However, changing the threshold from 2 to 3 and leaving all other conditions unchanged resulted in a consensus phytase-10threshold3 sequence that differed in eight positions (H6S, L191T, V204A, S250-, Q269A, -297V, -366I, K421T; a dash means that no amino acid was calculated by PRETTY) from the consensus phytase-10* sequence (the actual sequence calculated by PRETTY that still contains open positions; see above); however, none of those positions matched one of the actual amino acid changes discussed above.
In the calculation of consensus phytase-11, all five basidiomycete phytase sequences rather than the conbasidio phytase sequence were included in the alignment, however with a vote weight of 0.2 each to limit the impact of this sequence group in total to a vote weight of 1. Consensus phytase-11 contains 19 differences in comparison to consensus phytase-10*. Most of the changes affect positions for which no amino acid was defined in the calculation of either consensus phytase-11 (G13-, E367-) or consensus phytase-10* (-46K, -77E, -130N, -167H, -181S, -197D, -248R, -265D, -356K, -366I, -393A, -423L). At five positions, the procedure resulted in an amino acid replacement (Q111N, E199T, V204A, H264A, E440A).
While no thermostability data are available for consensus phytase-11 which only is a theoretical construct, consensus phytase-10 displayed a 7.4 and 9.0°C increase in unfolding temperature and temperature optimum, respectively, in comparison to consensus phytase-1 (Figure 2 and Table II
).
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The impact on thermostability of most of the 38 residues that differed between consensus phytase-1 on one hand and consensus phytases-10 and/or -11 on the other hand was tested by site-directed mutagenesis in consensus phytase-1 (Table III). In addition, a number of substitutions were made that were based on a comparison with the basidiomycete phytases (B in Table III
). Some of the introduced amino acid residues were non-consensus residues, but were present in at least one of the parent phytases (* in Table III
). Changes in thermostability were measured by recording temperatureactivity profiles with the supernatants of the respective S.cerevisiae transformants.
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Even though only 28 of the 32 amino acid differences between consensus phytase-1 and consensus phytase-10 were tested individually by site-directed mutagenesis, the fact that consensus phytase-10 is 7.4°C more thermostable than consensus phytase-1 somewhat contrasts with the finding of an equal number (i.e. 10 each) of stabilizing and destabilizing amino acid substitutions. This may indicate that the absolute effects of the stabilizing amino acid substitutions are larger than those of the destabilizing ones, or that at least some consensus amino acids provided synergistic rather than additive thermostabilization effects.
It might be objected that the higher thermostability of consensus phytase-10 as compared to consensus phytase-1 may be due to inclusion of a slightly more stable phytase (from T.lanuginosus) in the alignment rather than to the alignment of more amino acid sequences which should make determination of the consensus amino acids more reliable. However, out of the 10 amino acid substitutions depending on T.lanuginosus phytase, only three were stabilizing (30% success rate), while two were neutral and five were destabilizing. Therefore, the T.lanuginosus phytase sequence is not likely to skew the analysis.
Additive effect of stabilizing mutations in consensus phytase-1
Eight (E35A, D174N, E244D, R268I, R306H, S341T, A356K, G381A) and 11 (E35A, D46K, D174N, T191L, E199T, E244D, R268I, R306H, S341T, A356K, G381A) of the stabilizing mutations listed in Table III were jointly introduced into consensus phytase-1, yielding variants termed consensus phytase-1-thermo[8] and consensus phytase-1-thermo[11], respectively. In addition, in some constructs, the Q27T and K68A mutations were introduced, which have virtually no or only a minor effect on the thermostability of the protein, but increase the specific activity and broaden the pH optimum (data not shown). The 6.7 and 10.5°C increases in Tm for consensus phytase-1-thermo[8] Q27T and -thermo[11] Q27T relative to consensus phytase-1 (Table II
) demonstrate that the thermostabilizing effects of the individual mutations are additive.
Thermostabilization of consensus phytase-10 through back-mutation of destabilizing residues
Not all amino acid exchanges introduced into consensus phytase-10 relative to consensus phytase-1 had a stabilizing effect as evidenced by site-directed mutagenesis in consensus phytase-1 (Table III). Therefore, the most destabilizing of these residues were reverted to the original (consensus phytase-1) residues. In consensus phytase-10-thermo[3], residues K71, V135 and A373 were replaced by A, I and S, respectively. These alterations yielded a 3.6°C increase in Tm, from 85.4 to 89.0°C (Figure 2
and Table II
). Reversion of another destabilizing residue (G414A) and introduction of the stabilizing mutation E199T (suggested by consensus phytase-11) led to consensus phytase-10-thermo[5] which displayed a further increased Tm of 90.4°C (Figure 2
and Table II
). A215P had a destabilizing effect in consensus phytase-1. However, the reverse mutation in consensus phytase-10 did not provide increased thermostability (data not shown). Again, the Q27T and K68A mutations positively affected the catalytic properties of the enzyme (higher specific activity between pH 2.5 and 7) while having, if at all, only small effects on the thermostability of the protein (data not shown).
Thermostabilizing effects of a subset of the consensus residues when introduced into A.fumigatus phytase
To evaluate the stabilizing effect of a subset of clear consensus residues in a different background, six positions in the phytase amino acid sequence alignment were selected that are dominated each by a clear consensus residue, but at which A.fumigatus phytase contains a rare non-consensus residue. Simultaneous replacement of these six non-consensus residues with the respective consensus residues (F28Y, V73I, F87Y, A220L, S242P, N271D) in the A.fumigatus ATCC 13073 phytase Q27T variant yielded A.fumigatus phytase 13073thermo (Lehmann et al., 2000). The latter displayed a 5°C increase in temperature optimum and a 4.5°C increase in Tm relative to A.fumigatus ATCC 13073 wild-type phytase (Table II
). Introduction of four more amino acid exchanges (E35A, R306H, S341T, G381A) that were shown to be stabilizing as single mutations in consensus phytase-1 (Table III
) yielded A.fumigatus phytase 13073hythermo. This mutant also contained the S126N mutation which previously was shown to reduce protease susceptibility of A.fumigatus phytase (Wyss et al., 1999a
). Both A. fumigatus phytase 13073hythermo and its K68A variant displayed a further 3°C increase in temperature optimum as compared to A.fumigatus phytase 13073thermo (Table II
).
Catalytic properties of consensus phytases
The catalytic properties of consensus phytase-1 resemble those of E.nidulans and A.fumigatus phytase (Lehmann et al., 2000). Mutations Q27T/Q27L and K68A, which were previously shown to have pronounced effects on the catalytic properties of A.fumigatus phytase (Tomschy et al., 2000
, 2002
), also increased the specific activity and broadened as well as shifted the pH optimum into the more acidic range in the various consensus phytases (data not shown). In addition, the Q27T and Q27T, K68A variants of consensus phytase-10-thermo[3] had an even higher specific activity over the entire pH range than the corresponding, less thermostable consensus phytase-1 variants (Figure 3
). This is despite the fact that besides residue 71, none of the replaced amino acids is located in or close to the active site.
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Discussion |
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When most of the 32 differing residues were tested as single mutations in consensus phytase-1, 10 replacements had a positive effect on the protein's stability, 10 had a negative effect, and eight replacements were neutral. Four residues were not tested. All amino acid exchanges affected the thermostability by <3°C in our test system. These results indicate that the observed 7.4°C increase in unfolding temperature of consensus phytase-10 relative to consensus phytase-1 is the combined effect of multiple stabilizing amino acid replacements that are distributed over the entire protein. It is, thus, not the result of a single dominant mutation that was introduced just by chance. This interpretation in all likelihood also applies to the 1526°C increase in unfolding temperature of consensus phytase-1 over the proteins used for its design.
We have shownin accordance with findings from Steipe et al. (Steipe et al., 1994)that the consensus approach does not always select the most stabilizing amino acid for a given position of an amino acid sequence alignment. As shown by site-directed mutagenesis (Table III
), 10 amino acid substitutions newly introduced in consensus phytase-10 as compared to consensus phytase-1 had a destabilizing effect relative to the original amino acid residue. Combining only the amino acid exchanges in consensus phytase-1 that showed a stabilizing effect resulted in a protein, consensus phytase-1-thermo[11] (that also harbors a substitution derived from consensus phytase-11), that had a 3°C higher unfolding temperature than consensus phytase-10. Taking this approach further, we back-mutated four of the identified destabilizing residues of consensus phytase-10 and introduced one stabilizing residue derived from consensus phytase-11 (see above). The resulting protein, consensus phytase-10-thermo[5], had an unfolding temperature of 90.4°C which is another 5°C higher than that of consensus phytase-10.
It needs to be emphasized that the amino acid replacements summarized in Table III affect positions of the alignment for which the determination of a consensus residue was ambiguous for both consensus phytase-1 and consensus phytase-10, either because of high variability or because two or more amino acids are found at those positions with more or less the same frequency. However, the fact that consensus phytase-10 is more thermostable than consensus phytase-1 demonstrates that ambiguous positions can be optimized, in terms of their contribution to phytase thermostability, by including more sequences in the alignment. Alternatively, if additional sequences are not available, site-directed mutagenesis can be used to address the impact of ambiguous consensus residues on the stability of the protein, as shown in Table III
.
To address whether both clear and ambiguous consensus amino acids have a more general effect on the thermostability of fungal phytases, six clear consensus residues were introduced into A.fumigatus phytase, either alone or in combination with four ambiguous amino acids identified in the present study to have a stabilizing effect on the consensus phytases. The resulting variants A.fumigatus phytase 13073thermo (Lehmann et al., 2000; note that by mistake, it was not mentioned in the previous publication that A.fumigatus phytase 13073thermo also contains the Q27T mutation) and A.fumigatus phytase 13073hythermo (this study) displayed increases in temperature optima relative to A.fumigatus wild-type phytase of 5 and 8°C, respectively. Thus, typical consensus residuesand even ambiguous consensus residues identified to be stabilizingimprove the intrinsic stability not only in the consensus phytase environment, but also in A.fumigatus phytase and most probably in other homologous phytases. This is a further indication that the consensus approach relies on more general rules that are valid beyond the family of fungal phytases.
In some respect, thermostabilization of proteins by DNA family shuffling (Ness et al., 1999) and by our consensus approach resemble each other. Both methods are able to combine amino acids, the stability contribution of which is higher than average, in a new variant that exhibits increased intrinsic stability. In the case of gene shuffling, homologous genes are digested to small pieces and newly assembled by PCR. Variants that display increased intrinsic stability are then identified by screening or by applying a selective pressure. It is interesting to note that assembly statistics are likely to bias gene shuffling experiments towards consensus sequences since in any given generation the probability of extending a fragment by a consensus residue is larger than choosing a non-consensus residue. The size of the fragments to be reassembled limits the generation of possible new combinations in gene shuffling experiments. In the case of the consensus approach, no restrictions exist per se for the recombination of amino acid residues. Every single residue is `optimized' independently. Thus, the consensus approach describes a clearly defined way to determine, ideally, one final sequence for a more thermostable variant that basically consists of the most frequent amino acid at every position of a sequence alignment of homologous proteins.
The consensus approach for stabilizing proteins has the following advantages: (i) a given residue is replaced only by an amino acid that has already proven its evolutionary fitness at the corresponding position of at least one of the other homologous wild-type proteins, thereby reducing the risk of deleterious mutations; (ii) no 3D-structure is required for the method to be applicable to a given protein family; (iii) no analytical assay suitable for high-throughput screening is required.
In conclusion, through a combination of our consensus approach with limited site-directed mutagenesis work, we were able to increase the unfolding temperature of phytase from 48 to 69°C, as observed for the parental wild-type fungal phytases, to up to 90.4°C for consensus phytase-10-thermo[5]. This 2142°C increase in intrinsic thermostability is remarkable, even when compared to some of the most impressive examples of protein stabilization reported in literature: using directed evolution, subtilisin E, p-nitrobenzyl esterase from Bacillus subtilis, and a thermostable kanamycin-resistance gene product from Thermus thermophilus were all stabilized by 1720°C (Hoseki et al., 1999; Spiller et al., 1999
; Zhao and Arnold, 1999
). Using a more rational approach, van den Burg et al. (van den Burg et al., 1998
) enhanced the temperature optimum of an already thermostable bacterial protease by a further 21°C. In addition to other lines of evidence, the higher thermostability of consensus phytase-10, calculated from an amended sequence alignment, relative to consensus phytase-1 strengthens the idea that our consensus approach is generally applicable, even and well beyond phytases.
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Notes |
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Acknowledgments |
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Received August 17, 2001; revised February 1, 2002; accepted February 19, 2002.