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directed evolution of aspartate transaminasefor example. As reclon-ing of a library is laborious and prone to loss of diversity, and the effect evolving highly efficient enzymes on selection pressure only qualitatively predictable, inducible systems that provide a tightly controlled and graded transcriptional response toan external inducer represent potentially attractive alternatives.
Regulable promoters that use arabinoseor tetracyclias indu- cer compounds have been well characterized. Both allow homo- Caroline Heintz, Peter Kast & Donald Hilvert geneous gene expression over a broad dynamic range. To constructselection plasmids that combine inducer dose-dependent gene expres- .com/naturebiotec Combining tunable transcription with an enzyme-degradation
sion with the convenience of high-copy plasmids, we opted for the tag affords an effective means to reduce intracellular enzyme tetracycline-inducible Ptet systemwhich does not require specifically concentrations from high to very low levels. Such fine-tuned engineered host strains. We replaced the weak, constitutive bla .nature control allows selection pressure to be systematically
promoter on our selection plasmid pKECMB(Fig. 1a) with a increased in directed-evolution experiments. This facilitates modified Ptet promoter cassette (Fig. 1b), which includes a down- identification of mutants with wild-type activity, as shown stream T7 promoter, to simplify protein overproduction of selected here for an engineered chorismate mutase. Numerous selection variants. As expected, auxotrophic KA12/pKIMP-UAUC cells harbor- http://www formats and cell-based screening methodologies may benefit
ing the gene for the very weakly active hEcCM under the control of from the large dynamic range afforded by this easily this promoter system (on plasmid pKT) did not grow under selective oup implemented strategy.
conditions in the absence of inducer, but they regained prototrophy athigh tetracycline concentrations (Table 2). In contrast, cells containing Genetic selection can greatly facilitate the search for rare catalysts in the more active tEcCM and EcCM variants grew even in the absence of lishing Gr very large protein libraries. Auxotrophic strains, which grow only if
inducer. Thus, background transcription affords sufficient amounts of provided with a protein that functionally replaces a missing cellular these more active catalysts to fully satisfy the metabolic needs of the enzyme, are frequently used for this purpose. However, growth cell. Clearly, in this case, tight transcriptional control alone is not represents an indirect (and imperfect) readout of catalyst activity.
sufficient to reduce protein concentrations to a level low enough to Even mediocre catalysts may provide sufficient activity for cells to allow discrimination between a moderately active catalyst (tEcCM) Nature Pub grow at wild-type levels, making it difficult to distinguish the most
active variants from their less effective counterparts. As a result, it can To reduce protein concentration further, we fused an 11-amino-acid 200 be difficult or impossible to optimize relatively inefficient enzymes SsrA degradation signalto the C terminus of the catalyst (Fig. 1c).
through multiple rounds of mutagenesis and selection.
The SsrA tag targets the catalyst for rapid degradation by the We faced this problem when we tried to improve an engineered intracellular ClpXP protease. The efficacy of this strategy was estab- chorismate mutase. The dimeric helical bundle chorismate mutase lished using green fluorescent protein as a reporter (Supplementary from Escherichia coli (EcCM) was successfully converted into a func- Fig. 1 and Supplementary Methods online). The presence of the tional hexamer (hEcCM) by inserting a five-amino-acid hinge loop into degradation tag in plasmid pKTS also increased the dynamic range of the middle of the long H1 helix spanning the parent dimerbut the the chorismate mutase selection system, as demonstrated by topological change was accompanied by a 2- to 3-order-of-magnitudedecrease in activity (Table 1). Activity was partially recovered whenhEcCM was subjected to two rounds of random mutagenesis and Table 1 Catalytic parameters of the evolved hinge-loop variants selection in a chorismate mutase–deficient E. coli strain (KA12/pKIMP-UAFig. 1a)Nevertheless, the best variant, tEcCM, which contained three mutations and possessed a trimeric quaternary structure, still had a 14-fold lower kcat value than the parent EcCM dimer (Table 1), and further improvements were not possible because tEcCM already conferred wild-type levels of growth to its host In theory, selection pressure in a complementation assay can be aThe sequences of the variants are provided in Supplementary Figure 4 online. bRef. 4, increased by decreasing the intracellular catalyst concentration.
pH 6.5. The catalytic parameters of EcCM are similar at neutral and acidic pH. cThis work.
This might be accomplished by switching to a weakly active promoter, Protein production and characterization is described in Supplementary Methods.
Kinetic measurements were performed at 20 1C in PBS (10 mM Na low gene dose or inefficient ribosomal binding sites for catalyst KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) supplemented with 0.1 mg/ml bovine production. Such strategies have been profitably exploited for the Laboratory of Organic Chemistry, ETH Zurich, Ho¨nggerberg HCI F 339, CH-8093 Zurich, Switzerland. Correspondence should be addressed to D.H.
Received 21 March; accepted 24 August; published online 16 September 2007; Figure 1 Selection plasmid design. (a) The catalyst gene (cm) onplasmid pKECMBis constitutively expressed under control of the bla promoter. E. coli strain KA12 is deficient in chorismate mutase activity and also requires plasmid pKIMP-UAUC, which encodes two prephenate- processing enzymes, for growth on minimal medium(b) Selection plasmid pKT provides graded and homogeneous transcriptional control of catalyst production from promoter PtetA. The tetR gene and its promoter region are located upstream of cm, so that the TetR repressor simultaneouslyregulates transcription of catalyst and TetR repressor geneThe tetracycline-resistance determinant of the Tn10 transposon (tetA) isintegrated in the KA12 chromosome. (c) Selection plasmid pKTS permits graded transcriptional control and limited enzyme half-life. The ssrA sequence is incorporated as a downstream genetic fusion to the catalyst gene. The resulting enzyme carries the degradation tag at its C terminus, and is directed to the ClpXP protease, where it is degraded. Plasmid construction details are provided in Supplementary Methods online.
(400 ng/ml tetracycline), an H66R mutation occurred frequently .com/naturebiotec
and was further enriched at a complementation frequency of 2 ± 1% (200 ng/ml tetracycline). An S42L mutation was also enriched under the latter conditions, whereas the R44C mutation, which was prevalent before selection, occurred less frequently, presumably because itprovides no catalytic benefit. As selection pressure further increased(r100 ng/ml tetracycline), the complementation frequency dropped http://www
to o0.1% and only false positives were observed. The latter had lost complete suppression of cell growth in the absence of inducer for all the degradation tag mainly through frameshift mutations, and were oup tested chorismate mutase variants (Table 2). Restoration of growth
therefore presumably produced at elevated concentrations. The fact upon addition of specific tetracycline concentrations on solid media that the most stringent conditions only yielded false positives illus- roughly correlated with specific activity, distinguishing the most trates the importance of fine-tuning the selection pressure in these weakly active variant, hEcCM, from the more active tEcCM and experiments to maximize the yield of highly active variants.
lishing Gr EcCM variants. The correlation between growth rate, tetracycline
The genes of six clones selected at a complementation frequency of concentration and specific activity was even more apparent in liquid 2 ± 1% were retransformed, and the transformants grew faster than six culture (Fig. 2a). At the highest tetracycline concentrations tested, out of seven variants selected at a complementation frequency of 14 ± wild-type growth rates were achieved with the tEcCM and EcCM 5% (Supplementary Fig. 3 online). For six fast-growing variants, the variants, but not with the weakly active hEcCM. Moreover, at degradation tag was replaced with a (His)6 tag for protein purification, Nature Pub
7
intermediate tetracycline concentrations, cells harboring tEcCM
and the enzymatic activity of the four variants that could be produced grew more slowly than those with wild-type EcCM. The ability to in soluble form was determined in vitro. They showed uniformly high 200
control selection stringency simply by adjusting tetracycline concen-
Vmax values comparable to EcCM (Supplementary Table 1 online).
tration raises the possibility of evolving topologically novel catalysts The highest catalytic efficiency was exhibited by variant EcCM-200/4, which contained four mutations relative to hEcCM (A9V, S42L, H66R, To test the utility of this system in a directed evolution experiment, T87I; see also Supplementary Fig. 4 online). It eluted from a gel we inserted library fragments that encode the first 93 residues of filtration column as a trimer and catalyzed the rearrangement of hEcCM, diversified by error-prone PCR and DNA shuffliinto the chorismate to prephenate with a kcat of 12 s–1 and a kcat/Km of 45,000 pKTS acceptor vector in place of a stuffer fragment, in-frame with the M–1s–1 (Table 1). The turnover number, which represents a 75-fold last seven residues of hEcCM fused to the SsrA tag. After transforma- improvement over hEcCM and a tenfold improvement relative to the tion of the KA12/pKIMP-UAUC selection strain (1.5 Â 107 transfor- best previously characterized variant tEcCM, is similar to that of mants), library clones were picked randomly and sequenced to check wild-type EcCM (Table 1). This result is notable as high kcat values are library quality. With the exception of the R44C substitution, whichoccurred in 40% of the sequences because of an apparent DNA Table 2 Benchmark complementation assays with chorismate shuffling artifact, mutations were evenly distributed over the entire hEcCM gene. Aliquots of the library were plated onto selective M9c plates containing varying amounts of tetracycline to identify activecatalysts based on their ability to complement the chorismate mutase deficiency. The number of complementing clones decreased withdecreasing tetracycline concentration (Fig. 2b), consistent with the hypothesis that reducing intracellular enzyme concentration increases Sequence analysis of 96 clones revealed a mutational bias in active variants that correlates roughly with selection stringency. The +, cell growth; 0, no cell growth. Streak-outs of KA12/pKIMP-UAUC cells containing the emergence and disappearance of specific mutations upon increasing indicated selection plasmids were evaluated after 2 d of incubation at 30 1C on M9cmedium plates[tc] is the tetracycline concentration in M9c medium. pKT places the selection pressure is illustrated in Figure 2c (see also Supplementary gene under control of the tetracycline-inducible Ptet system, pKTS additionally encodes Fig. 2 online). At a complementation frequency of 14 ± 5% a degradation tag fused to the catalyst.
Figure 2 Tetracycline-dependent growth in selective M9c medium and influence of tetracycline concentration on the selection process. (a) KA12/pKIMP- hnology UAUC cells were transformed with the pKTS selection plasmid encoding wild-type EcCM, tEcCM or hEcCM. Growth curves were determined for each
transformant. Error bars indicate the s.d. of the curve fit in each growth experiment. (b) Complementation frequency among gene library members onselective M9c plates as a function of tetracycline concentration. (c) Mutation bias as a function of complementation frequency, determined using 24sequences originating from three independent selection experiments for each selection regime. The occurrence of false positives lacking the degradation tagand the relatively frequent mutations H66R, S42L and R44C is plotted for different selection regimes. Clones grown on nonselective rich medium plates(100% complementation) were examined to assess library size, quality and sequence diversity before selection. See Supplementary Figure 2 online foralignments of all 96 sequences used for this analysis and the Supplementary Methods for detailed experimental protocols for the liquid growth tests, library .com/naturebiotec construction and selection experiments.
important for industrial biocatalysis, where high conversion of sub- Reprints and permissions information is available online at In conclusion, pairing a tunable promoter with a degradation tag http://www can provide very low but adjustable catalyst concentrations within
cells. By providing systematic control over selection stringency, this oup strategy facilitates the evolution of substantially more active enzymes
1. Taylor, S.V., Kast, P. & Hilvert, D. Angew. Chem. Int. Ed. Engl. 40, 3310–3335 than is possible with systems reliant on weak constitutive gene 2. MacBeath, G., Kast, P. & Hilvert, D. Protein Sci. 7, 325–335 (1998).
expression. Extension of this approach for regulating stringency 3. Kast, P., Asif-Ullah, M., Jiang, N. & Hilvert, D. Proc. Natl. Acad. Sci. USA 93, should benefit any cell-based selectior screeninsystem in 4. Vamvaca, K., Butz, M., Walter, K.U., Taylor, S.V. & Hilvert, D. Protein Sci. 14, lishing Gr which the setting of a threshold for minimum activity allows the best
variants to be distinguished from less interesting ones.
5. Yano, T., Oue, S. & Kagamiyama, H. Proc. Natl. Acad. Sci. USA 95, 5511–5515 6. Khlebnikov, A., Risa, O., Skaug, T., Carrier, T.A. & Keasling, J.D. J. Bacteriol. 182, Note: Supplementary information is available on the website.
7. Hansen, L.H., Ferrari, B., Sorensen, A.H., Veal, D. & Sorensen, S.J. Appl. Environ.
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7 This work was generously supported by the Schweizerischer Nationalfonds and
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11. Turner, N.J. in Enzyme Assays (ed. J.L. Reymond) 139–161, (Wiley-VCH, Weinheim, M.N., P.K. and D.H. designed research; M.N., M.B. and C.H. performed the 12. Jestin, J.L. & Kaminski, P.A. J. Biotechnol. 113, 85–103 (2004).
experiments; M.N., M.B., P.K. and D.H. analyzed data; M.N., P.K. and D.H.
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