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Table 3. Selected ANEH variants as catalysts in the hydrolytic kinetic resolution of rac-1 favoring (S)-2, obtained previouslyin the case of substrate rac-3 bysaturation mutagenesis at site B byusing NDT codon degeneracy. Variant Mutations E DK-04 Leu215Ser/Ala217Ile/Arg219Arg 16 DK-27 Leu215Ser/Ala217Val/Arg219Leu 35 DK-35 Leu215His/Ala217Tyr/Arg219Val 25 fourth cycle of iterative CASTing at four different sites (B, C, D, and F) along the upward pathwaywhen using NNK codon ACHTUNGTRENUNGdegeneracy. [21] Conclusions We have addressed once more the numbers problem in directed evolution byconsidering statistical aspects of librarygeneration and performing appropriate laboratoryexperiments based on saturation mutagenesis. The model studyinvolves the epoxide hydrolase from Aspergillus niger (ANEH) as the enzyme with enhancement of the reaction rate (substrate acceptance) and enantioselectivity in the hydrolytic kinetic resolution of a chiral substrate serving as the catalytic parameters. Whereas previous studies have demonstrated the merits of targeted randomization, [12,13,16, 19] culminating in iterative saturation mutagenesis (ISM), [22,23] this contribution provides a tool that is useful in the quest to generate even “smarter” enzyme libraries. Its basis is the optimal choice of the codon degeneracywhen tailoring saturation mutagenesis experiments. In the model study, we have compared systematically two different codon degeneracies that relate to two differentlysized aa alphabets, specificallythe conventional NNK (32 codons) encoding 20 aa versus NDT (12 codons) encoding only12 aa’s. This led to the discoverythat vastlydifferent libraryqualities result. Upon comparing identicallysized NNK and NDT libraries each limited to 5000 transformants, the frequencyof hits in the NDT case turned out to be much higher. The catalytic profiles of the best hits in terms of activityand enantioselectivityalso proved to be different, those resulting from NDT codon usage again being superior. These experimental results are in line with a statistical analysis based on algorithms proposed earlier. [24,25] The basic prediction regarding the main trends is not expected to change byconsidering possible aa bias. Nevertheless, this aspect is the subject of an ongoing project in our laboratories. We conclude that it is possible to decrease the size of focused libraries generated bytargeted mutagenesis, while increasing their qualityin terms of the frequencyof beneficial variants and the degree of catalyst improvement. This is possible for two reasons. The correct choice of codon degeneracy ensures the proper minimal set of aa’s in terms of structural and electronic characteristics and allows for nearlycomplete coverage of a given size-restricted library. Both factors working together maximize the probabilityof hits and reduce the number of “junk” transformants. These features have been ACHTUNGTRENUNGuncovered experimentallyfor the first time in a model case, but we expect the trends to be general. NDT is not the onlycodon degeneracyfor constructing reduced aa alphabets, others can likewise be considered depending upon the nature of the protein propertyto be engineered (catalytic profile of enzymes or binding property of proteins). Excluding those aa’s that are similar to those that are needed minimallyis one important recommendation. The other general guideline for achieving more efficient and therefore faster directed evolution is derived from our latest data and from the statistical analysis, namely that the number of codons should be equal to the number of aa’s being used as building blocks when performing saturation mutagenesis. This approach is not limited to CASTing, other forms of targeted mutagenesis as in the B-Fit method [22,23] are also relevant. Moreover, reduced aa alphabets as described herein can also be used when combining site randomization with DNA shuffling [16] or synthetic shuffling. [36] Finally, we point out that the screening/selection problem when applying targeted mutagenesis becomes even more acute when randomizing sites composed of five or more aa positions. Even if assaysystems capable of rapid evaluation of 10 8 –10 9 transformants for activity/enantioselectivity were to be developed in a general way, [37] this would not suffice to cover the relevant protein sequence space when applying saturation mutagenesis based on the conventional NNK approach (Table 1). We suggest that in this kind of broadlytargeted mutagenesis, which might be beneficial in some systems (for example, randomization at aa positions of an enzyme domain), the strategydescribed herein could provide the experimenter with an effective tool for handling the numbers problem in ACHTUNGTRENUNGdirected evolution. [38] Experimental Section M. Reetz et al. Vector construction: The PCR-amplified ANEH genes were cloned into vector pET22b(+) (Novagen, Madison, USA) between the EcoRI and MscI restriction sites, and E. coli BL21GoldACHTUNGTRENUNG(DE3) (Novagen, Madison, USA) was transformed with the resulting plasmids. Oligonucleotides: The NDT and NNK libraries at site B (215, 217, 219) were prepared byusing the complementaryprimers (Invitrogen) NDT-B-for (5’-CGGTTCATTTGAACNDTTGCNDTATGNDTGCTCC- ACHTUNGTRENUNGCCCTGAG-3’) and NDT-B-rev (5’-CTCAGGGGGAGCAHNCATAHNGACHTUNGTRENUNGC- ACHTUNGTRENUNGAAHNGTTCAAATGAACCG-3’) as well as NNK-B-for (5’-CGGTTCATT- ACHTUNGTRENUNGTGACHTUNGTRENUNGAACNNKTGCNNKATGNNKGCTCCCCCTGAG-3’) and NNK-B-rev (5’- CTCAGGGGGAGCMNNCATMNNGCAMNNGTTCAAATGAACCG-3’). Primers for NDT libraryat site A (193, 195, 196) were: NDT-A-for (5’- GGGAGGTGATNDTGGTNDTNDTGTTGGACGACTGTTGG-3’) and NDT- A-rev (5’-CCAACAGTCGTCCAACAHNAHNACCAHNATCACCTCCC-3’). For NDT libraryat site F (244, 245, 247), NDT-F-for (5’-CGAATGGA- GAAGNDTNDTACCGATGGCNDTGCTTATGCCATG-3’) and NDT-F-rev (5’-CATGGCATAAGCAHNGCCATCGGTAHNAHNCTTCTCCATTCG-3’) were used. Library construction: Saturation mutagenesis libraries were generated bythe QuikChange TM mutagenesis method [12] with the above degenerate primers. Briefly, each PCR contained (25 mL final volume): 10”KOD buffer (Novagen; 2.5 mL), MgCl 2 (1 mL, 25 mm), dNTP (5.0 mL, 2 mm each), appropriate degenerate primers (5.0 mL, 1802 www.chembiochem.org 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2008, 9, 1797 – 1804
The Numbers Problem in Directed Evolution 2.5 mm), template plasmid (0.5 mL, 10 ngmL 1 ) and 0.5 units of KOD Hot start polymerase. PCRs were carried out on a Biometra Thermocycler (Whatman Biometra, Gçttingen, Germany). Thermal cycling consisted of an initial denaturation of 3 min at 948C, followed by25 cycles of 60 s at 52 8C, 60 s at 72 8C, 480 s at 72 8C, and a final elongation of 14 min at 72 8C. Residual template in each PCR reaction was removed bydouble digestion with 1 unit of DpnI (New England Biolabs) in 1” manufacturer’s buffer for 2 h at 378C, followed byfurther addition of 1.0 unit of DpnI for 1 h. The PCR product was used to transform competent BL21GoldACHTUNGTRENUNG(DE3) (Novagen) cells, the cells were plated on LB CB/TET plates. Gene sequencing: Mutant genes were sequenced byusing the standard T7 and T7-Ter primer (Medigenomix, Martinsried, Germany). Expression for screening: Colonies were picked with a colony picker QPIX (Genetix, New Milton, UK), and precultures were grown overnight at 38C and 800 rpm in deep-well plates with lactose-free 505 medium (800 mL per well; formula per liter: 20 mL of 50 ”505 solution (250 g L 1 glycerol, 25 gL 1 glucose, and distilled water), 50 mL of 20”NPS solution (66 gL 1 (NH 4) 2SO 4, 136 g L 1 KH 2PO 4 and 142 g Na 2HPO 4), 2 mL of 1 mm MgSO 4, and filled to a volume of 1 L with ZY medium (10 gL 1 tryptone, 5 gL 1 yeast extract)). The preculture (50 mL) was used to inoculate an expression culture with 5052 medium (800 mL per well) containing lactose as inducer (formula per liter: 20 mL of 50 ”5052 solution (100 gL 1 a-lactose, 250 gL 1 glycerol, 25 gL 1 glucose, and distilled water), 50 mL of 20”NPS solution, 2 mL of 1 mm MgSO 4, and filled to a volume 1 L with ZY medium)). In both media, carbenicillin was used as anti- ACHTUNGTRENUNGbiotic (100 mgL 1 ). After being grown at 378C and 800 rpm for 6 h, cultures were centrifuged at 4000 rpm for 5 min. Cells were washed bycentrifuging for 3 min at 4000 rpm and 4 8C with PBS buffer (800 mL; Na 2HPO 4 1.1 gL 1 , NaH 2PO 4 0.3 gL 1 , NaCl 9.0 gL 1 ). Supernatants were discarded, and cells were resuspended in PBS buffer (800 mL). Screening: Stock solutions of rac-1-phenyl-2,3-epoxy butane (rac- 3; 72 mmol substrate in acetonitrile) were stored at 208C. The ACHTUNGTRENUNGreactions were performed as follows: washed expression culture (20 mL) was added to PBS buffer (150 mL, 57 mmol, pH 7.2), and the substrate was dissolved in acetonitrile (10 mL, 72 mmol). The reaction suspension was incubated for 10 h at 800 rpm and 378C. The hydrolytic reaction was monitored by using a cell-based adrenaline assayfor high-throughput screening. [33] The change of absorption was obtained with a Spectramax UV/Vis spectrophotometer (Molecular Devices Corp.). Initial activitywas assessed with a threshold value of 10 %, incorporating procedural variabilities and the expected conversion. Active clones were collected and reproduced in triplicate based on the aforementioned preculture. Mutant genes were sequenced byusing the standard T7 and T7-Ter primers (Medigenomix). A similar procedure was used for 20% and 40% conversions. Variant expression and purification: For all variants, expression was performed in a ZY 5052 expression culture (100 mL) supplemented with carbenicillin (100 mg L 1 ). The culture was incubated at 308C and 250 rpm overnight. The cells were chilled on ice for 20 min, harvested (5 min, 10 000 rpm, 4 8C), washed with PBS buffer (57 mmol, pH 7.2), and centrifuged again (3 min, 10 000 rpm, 4 8C). The cell pellet was typically resuspended in PBS buffer (8 mL, 57 mmol, pH 7.2) containing DNase I (0.5 mg mL 1 ; Applichem, Darmstadt, Germany) and lysozyme (1.0 mg mL 1 ?; GERBU Biotechnik, Gaiberg, Germany). The suspension was chilled on ice for 1 h before sonication (Bandelin, 2 ”30 s, 40% pulse, on ice). After cen- trifugation (14 000 rpm, 45 min, 4 8C) a clear supernatant was collected. The variants were purified byion-exchange chromatography(HiTrap Q HP, 5 mL, GE Healthcare) with a step-gradient and a 5 mL min 1 flow rate (starting buffer A: 57 mm phosphate buffer pH 7.4; elution buffer B: 57 mm phosphate buffer pH 7.4 with 1m NaCl). HPLC analysis: The chiral analyses of the hydrolytic kinetic resolution reaction of rac-3 were performed byusing Chiracel AD-RH chiral column (2.5 m, 4.6 mm i.d., Daicel Chemical Industries, Tokyo, Japan). Conditions: methanol/H 2O, 0.5 mLmin 1 , UV 210 nm. Acknowledgements This work was supported by the Fonds der Chemischen Industrie (FCI) and the DFG (SSP 1170 “Directed Evolution to Optimize and Understand Molecular Biocatalysis”; Project RE 359/13-1). D.K. thanks the FCI for a KekulØ Stipend. We thank A. Vogel, J. D. Carballeira, F. Schulz, J. 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