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Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês

10.1074/jbc.m401447200

1083-351X

Melissa L. Geddie, Ichiro Matsumura,

Monoclonal and Polyclonal Antibodies Research

Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli β-glucuronidase (GUS) variants with increased β-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331–339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-β-d-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a ∼200-fold improvement over the wild type in reactivity (kcat/Km) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique. Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli β-glucuronidase (GUS) variants with increased β-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331–339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-β-d-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a ∼200-fold improvement over the wild type in reactivity (kcat/Km) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique. Adaptive evolution is arguably the most fundamental biological process, although it remains poorly understood. A better understanding of molecular adaptation would facilitate the engineering of enzymes with novel specificities. Theoreticians have proposed that a relatively small number of catalytically inefficient, broad specificity proto-enzymes diversified through gene duplication and adaptive evolution into the multitude of efficient and specialized catalysts present in the contemporary biosphere (2Kacser H. Beeby R. J Mol. Evol. 1984; 20: 38-51Crossref PubMed Scopus (113) Google Scholar, 3Lazcano A. Diaz-Villagomez E. Mills T. Oro J. Adv. Space Res. 1995; 15: 345-356Crossref PubMed Scopus (19) Google Scholar, 4Jensen R.A. Annu. Rev. Microbiol. 1976; 30: 409-425Crossref PubMed Scopus (817) Google Scholar, 5Ycas M. J. Theor. Biol. 1974; 44: 145-160Crossref PubMed Scopus (160) Google Scholar, 6Waley S.G. Comp. Biochem. Physiol. 1969; 30: 1-11Crossref PubMed Google Scholar). This supposition cannot be proven nor refuted through experimentation, but recapitulation of the diversification process in the laboratory would support its feasibility and reveal possible underlying structural mechanisms. The glycoside hydrolases exemplify enzyme diversification. Natural selection has matched the great structural diversity of carbohydrates with a multitude of enzymes that selectively catalyze their cleavage. These enzymes have been classified into 91 families based on amino acid sequence similarity (7Bourne Y. Henrissat B. Curr. Opin. Struct. Biol. 2001; 11: 593-600Crossref PubMed Scopus (359) Google Scholar). The GH-A clan (sometimes called the 4/7 superfamily) comprises >3000 GenBank™ sequences from Families 1, 2, 5, 10, 17, 26, 35, 39, 42, 53, 59, 72, 79, and 86 (8Henrissat B. Davies G. Curr. Opin. Struct. Biol. 1997; 7: 637-644Crossref PubMed Scopus (1400) Google Scholar). All GH-A enzymes retain the same (β/α)8-barrel fold and catalytic mechanism (9Henrissat B. Callebaut I. Fabrega S. Lehn P. Mornon J.P. Davies G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7090-7094Crossref PubMed Scopus (512) Google Scholar) and are thought to have diverged from a common ancestor (10Henrissat B. Callebaut I. Fabrega S. Lehn P. Mornon J.P. Davies G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5674PubMed Google Scholar). Consider the specific example of two well characterized glycoside hydrolases: human β-glucuronidase (GUS, 1The abbreviations used are: GUS, β-glucuronidase; XYL, β-xylosidase; PSQ, β-glucuronidase mutants containing Pro557/Ser566/Gln568; PAF, β-glucuronidase mutants containing Pro557/Ala566/Phe568; pNP-gluc, p-nitrophenyl-β-d-glucuronide; pNP-xyl, p-nitrophenyl-β-d-xylopyranoside; StEP, staggered extension process; LB, Luria broth.1The abbreviations used are: GUS, β-glucuronidase; XYL, β-xylosidase; PSQ, β-glucuronidase mutants containing Pro557/Ser566/Gln568; PAF, β-glucuronidase mutants containing Pro557/Ala566/Phe568; pNP-gluc, p-nitrophenyl-β-d-glucuronide; pNP-xyl, p-nitrophenyl-β-d-xylopyranoside; StEP, staggered extension process; LB, Luria broth. Family 2) and Thermoanaerobacterium saccharolyticum β-xylosidase (XYL, Family 39). These distant homologs catalyze the hydrolysis of similar substrates that differ only in their C5 substituents (carboxylate for β-glucuronides, hydrogen for β-xylosides), but overlap very little in specificity. Their amino acid sequences are too divergent to align, but six conserved active site residues are superimposable (Fig. 1) (11Yang J.K. Yoon H.J. Ahn H.J. Il Lee B. Pedelacq J.D. Liong E.C. Berendzen J. Laivenieks M. Vieille C. Zeikus G.J. Vocadlo D.J. Withers S.G. Suh S.W. J. Mol. Biol. 2004; 335: 155-165Crossref PubMed Scopus (63) Google Scholar, 12Jain S. Drendel W.B. Chen Z.W. Mathews F.S. Sly W.S. Grubb J.H. Nat. Struct. Biol. 1996; 3: 375-381Crossref PubMed Scopus (201) Google Scholar). Our goal is to understand how enzymes like GUS and XYL evolved to become so specific. X-ray crystallography is essential to understanding the structural basis of enzyme specificity, but the complexity of protein structures necessitates the development of complementary functional approaches. We randomly mutate genes or parts of genes, and express the resulting libraries in populations of microorganisms. High throughput screening enables the functional evaluation of thousands of sequence variants in parallel. Iterative cycles of mutagenesis, point mutant recombination, and high throughput screening lead to the accumulation of beneficial mutations in selected clones (13Rowe L.A. Geddie M.L. Alexander O.B. Matsumura I. J. Mol. Biol. 2003; 332: 851-860Crossref PubMed Scopus (57) Google Scholar). Directed evolution has been used to alter properties of enzymes including substrate specificity (reviewed in Refs. 14Farinas E.T. Bulter T. Arnold F.H. Curr. Opin. Biotechnol. 2001; 12: 545-551Crossref PubMed Scopus (231) Google Scholar, 15Zhao H. Chockalingam K. Chen Z. Curr. Opin. Biotechnol. 2002; 13: 104-110Crossref PubMed Scopus (151) Google Scholar, 16Schmidt-Dannert C. Biochemistry. 2001; 40: 13125-13136Crossref PubMed Scopus (67) Google Scholar, 17Harris J.L. Craik C.S. Curr. Opin. Chem. Biol. 1998; 2: 127-132Crossref PubMed Scopus (47) Google Scholar, 18Powell K.A. Ramer S.W. Del Cardayre S.B. Stemmer W.P. Tobin M.B. Longchamp P.F. Huisman G.W. Angew Chem. Int. Ed. Engl. 2001; 40: 3948-3959Crossref PubMed Scopus (188) Google Scholar). The Escherichia coli GUS is a good model system for the study of adaptive evolution. As noted above, the human GUS protein has been crystallized; the amino acid sequences of the E. coli and human GUS homologs are 50% identical, with highly conserved active sites (12Jain S. Drendel W.B. Chen Z.W. Mathews F.S. Sly W.S. Grubb J.H. Nat. Struct. Biol. 1996; 3: 375-381Crossref PubMed Scopus (201) Google Scholar). The E. coli β-glucuronidase gene (gusA, formerly uidA) is more amenable to directed evolution, because it can be overexpressed at high levels in E. coli, enabling the detection of weak catalytic activities in reactions with β-xylosides and other non-native substrates. We have previously directed the evolution of GUS variants with increased β-galactosidase activity. We expressed a library of randomly mutated gusA alleles in a lacZ-E. coli strain. Clones exhibiting increased β-galactosidase activity on LB agar plates containing 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside (X-gal) were isolated. After two additional rounds of DNA shuffling (point mutation recombination, Ref. 19Stemmer W.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10747-10751Crossref PubMed Scopus (1077) Google Scholar) and screening, clones exhibiting 500-fold increases in β-galactosidase activity were isolated. All clones were sequenced, and the fittest allele contained 13 mutations. We dissected the effects of these mutations through site-directed mutagenesis and found that four amino acids changes in an active site loop (T509A, S557P, N566S, K568Q) accounted for the changes in substrate specificity (1Matsumura I. Ellington A.D. J. Mol. Biol. 2001; 305: 331-339Crossref PubMed Scopus (156) Google Scholar). Here we extend a recently developed mutagenesis strategy (20Sio C.F. Riemens A.M. van der Laan J.M. Verhaert R.M. Quax W.J. Eur. J. Biochem. 2002; 269: 4495-4504Crossref PubMed Scopus (44) Google Scholar, 21Lingen B. Grotzinger J. Kolter D. Kula M.R. Pohl M. Protein Eng. 2002; 15: 585-593Crossref PubMed Scopus (61) Google Scholar, 22Miyazaki K. Arnold F.H. J. Mol. Evol. 1999; 49: 716-720Crossref PubMed Scopus (176) Google Scholar) to direct the evolution of GUS variants with increased XYL activity. We randomized specificity-determining residues that were identified in our previous study (Ser557, Asn566, Lys568) by saturation mutagenesis (also called combinatorial cassette mutagenesis, Refs. 22Miyazaki K. Arnold F.H. J. Mol. Evol. 1999; 49: 716-720Crossref PubMed Scopus (176) Google Scholar, 23Coco W.M. Encell L.P. Levinson W.E. Crist M.J. Loomis A.K. Licato L.L. Arensdorf J.J. Sica N. Pienkos P.T. Monticello D.J. 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The selected clones were further evolved through random mutagenesis and point mutation recombination and high throughput screening (Table I).Table ISummary of evolution experiments The library was constructed in a different way for each round of evolution. The average selection coefficient (improvement in fitness over the ancestor) for each population was determined in quantitative whole cell xylosidase assays. We assayed 16 replicates of each clone, determined the average of those clones that exhibited improvement over its immediate ancestor, and divided by the xylosidase activity of the wild type.Round of evolutionDiversity generationAverage selection coefficient1Saturation mutagenesis30-fold2Random mutagenesis80-fold3DNA shuffling (catalytic domain)90-fold4StEP/shuffling100-fold Open table in a new tab Materials—Most of the materials, including the constitutive His6-gusA expression vector, were previously described (13Rowe L.A. Geddie M.L. Alexander O.B. Matsumura I. J. Mol. Biol. 2003; 332: 851-860Crossref PubMed Scopus (57) Google Scholar). The oligonucleotides (Fig. 2) were synthesized by IDT (Coralville, IA); the pNP-β-d-xylopyranoside (pNP-xyl) was from Sigma-Aldrich. The GeneAmp XL polymerase kit, including the rTth and Vent polymerases, was from Applied Biosystems (Foster City, CA). Saturation Mutagenesis—The codons encoding gusA amino acids 557, 566, and 568 were randomized by whole plasmid PCR (28Eisinger D.P. Trumpower B.L. BioTechniques. 1997; 22: 250-252, 254Crossref PubMed Scopus (7) Google Scholar) using a mixture of Tth and Vent polymerase (29Cheng S. Fockler C. Barnes W.M. Higuchi R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5695-5699Crossref PubMed Scopus (571) Google Scholar) and back-to-back 5′-phosphorylated degenerate primers: 5′-GCGCAATATGCCTTGKNNGGTCGAAAATCGG-3′ (gusA S557X) and 5′-GTTGGCGGTNNKAAGNNKGGGATCTTCACTCGC-3′ (gusA N566X,K568X), where K is T or G. The long PCR reactions contained 50 ng of the ancestral gusA expression vector, 500 nm primers, 200 nm of each dNTP, 1× ABI XL buffer II, and 1.5 mm magnesium acetate. The reactions were overlaid with light mineral oil and heated to 80 °C in a thermal cycler for a hot start. 0.5 μl of the Tth/Vent mixture was added, and the temperatures were immediately raised to 94 °C for 1 min, followed by 30 cycles of 94 °C × 15 s, 68 °C × 5 min (1 min per kb). The reaction was further incubated at 72 °C for 10 min and stored at 4 °C. The mineral oil was removed, and the PCR reaction was incubated with 0.5% SDS and 50 μg/ml proteinase K at 65 °C for 15 min to eliminate the thermostable polymerases. The PCR product was purified using the Promega Wizard PCR prep kit (Madison, WI) as directed by the manufacturer, with the final elution in 50 μl of water. The DNA was digested with restriction enzyme DpnI (to eliminate methylated ancestral template), and the desired PCR product was gel-purified using a QiaQuick spin column as directed by the manufacturer (Qiagen, Valencia, CA). The concentration of eluted DNA was estimated via agarose gel electrophoresis. For intramolecular self-ligation of blunt-ended DNA, we recommend low concentrations of DNA and high concentrations of T4 DNA ligase. 4 fmol of purified PCR product were reacted with 2.5 units of T4 DNA polymerase and 5 Weiss units of T4 DNA ligase in 20-μl reactions containing 200 nm each dNTP, 100 μm ATP, 50 mm Tris, pH 7.6, 10 mm MgCl2, 5 mm dithiothreitol, and 25 μm bovine serum albumin, for 1 h at 23 °C. The T4 DNA ligase was heat-killed at 65 °C for 10 min, and DNA was purified by butyl alcohol precipitation (30Thomas M.R. BioTechniques. 1994; 16: 988-990PubMed Google Scholar). Freshly prepared E. coli InvαF′ cells were transformed by electroporation as described by Dower et al. (31Dower W.J. Miller J.F. Ragsdale C.W. Nucleic Acids Res. 1988; 16: 6127-6145Crossref PubMed Scopus (2157) Google Scholar). The transformed bacteria were titered, distributed into 384-well plates, and evaluated in our high throughput microplate assay (32Geddie M.L. Rowe L.A. Alexander O.B. I. M. Methods Enzymol. 2004; 388: 134-145Crossref PubMed Scopus (11) Google Scholar) using pNP-xyl as the substrate. Clones associated with the highest absorbance at 405 nm after 16 h at 37 °C were streaked onto LB-ampicillin agar plates and characterized in a whole cell activity assay. Whole Cell Activity Assays (Secondary Screens)—Microcultures associated with high xylosidase activity in primary screens were streaked onto LB-ampicillin plates. Individual colonies were picked and grown to saturation in a 96-well microplate. 20 μl of the saturated culture was placed in 200 μl of 0.4 mm pNP-β-d-xylopyranoside in a 96-well plate, and the absorbance at 405 nm was monitored for 5 h. Each clone was evaluated in three independent trials, and the average values (and associated S.E.) were calculated. The activity of each clone was normalized to that of the ancestral strain. Whole Plasmid PCR—The 14 plasmids selected in the first round of evolution were purified, pooled, and PCR-amplified as described above using 5′-GGGAAGCTTCTCATTGTTTGCCTCCCTGCTGCG-3′ (3′-gusA3) and 5′-GGGAAGCTTGCGGCCGCACTCGAGCAC-3′ (3′-pE-Tout3). The amplification product was purified, digested with restriction enzymes DpnI and Hind III (to digest the sites underlined in the primer sequence), gel-purified, and self-ligated. The ligation reaction was similar to that described above, except that we used a lower concentration of T4 DNA ligase (1 unit instead of 5), no T4 DNA polymerase (nor dNTPs and bovine serum albumin), and a 16 °C incubation (instead of 23 °C). E. coli strain InvαF′ was transformed with the plasmid library, and the transformants were screened as described above. DNA Shuffling—The gusA alleles isolated in the second round of evolution were pooled and amplified in a standard PCR using 500 nm primers 5′-GGACTTTGCAAGTGGTGAATCCGCAC-3′ (gusA 720) and 3′-gusA3, 50 ng of template DNA, 60 mm Tris-HCl, pH 8.5, 15 mm (NH4)SO4, 2.0 mm MgCl2, 0.2 mm each dNTP, and 5 units of Taq polymerase. The mixture was overlaid with mineral oil and cycled 25 times between 94 °C for 30 s and 72 °C for 2 min. The resulting PCR product was purified according to the Wizard PCR procedure and randomly recombined by DNA shuffling (19Stemmer W.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10747-10751Crossref PubMed Scopus (1077) Google Scholar). We reacted the PCR product with 0.1 units of DNase I in 50 mm Tris-HCl, pH 7.6, 10 mm MgCl2, for 2 min at 23 °C. The DNA was immediately extracted in phenol/chloroform (50:50) and ethanol precipitated. The fragments were reassembled in a 45 cycle PCR-like reaction with no additional primers. The full-length recombinant products were amplified in the standard PCR reaction as described above and subcloned using overlap PCR as follows (33Temesgen B. Eschrich K. BioTechniques. 1996; 21: 828-830, 832Crossref PubMed Scopus (5) Google Scholar). The ancestral expression vector and part of the gusA gene were amplified in a long PCR (29Cheng S. Fockler C. Barnes W.M. Higuchi R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5695-5699Crossref PubMed Scopus (571) Google Scholar) with 5′-CGGTTTGTGGTTAATCAGGAACTGTTCG-3′ (gusA 900out) and 3′-pETout3. Finally, the shuffled gusA mutants and the vector were combined in a long overlap PCR using primers 3′-gusA3 and 3′-pETout3. The resulting full-length PCR product was purified, restriction-digested, and self-ligated. E. coli strain InvαF′ was transformed with the plasmid library, and the transformants were screened for clones exhibiting increased XYL activity. Staggered Extension Process (StEP)—Plasmids derived from clones isolated in the third round of evolution were purified and pooled. Three overlapping regions of the pooled gusA alleles were amplified separately in standard PCR reactions, except that 100 cycles with short (15 s) extension times were executed. This produces incomplete fragments that can recombine by switching templates (34Zhao H. Giver L. Shao Z. Affholter J.A. Arnold F.H. Nat. Biotechnol. 1998; 16: 258-261Crossref PubMed Scopus (585) Google Scholar). The 5′-end of the gusA gene pool was amplified with 5′-TAATCACCATTCCCGGCGGGATAGTC-3′ (gusA 500out) and 5′-TCTAGATCTGGCACGACAGGTTTCCCGACTG-3′ (rev137), the central region was amplified with 5′-GCCATTTGAAGCCGATGTCACGCCG-3′ (gusA 360) and 5′-CCTGTAAGTGCGCTTGCTGAGTTTCC-3′ (gusA 1150out), and the 3′-end was amplified with 5′-GGGAAGCTTATGTTTGCCTCCCTGCTGCGGTTTTTCA-3′ (3′-gusA2) and 5′-CTGCTGCTGTCGGCTTTAACCTCTCT-3′ (gusA 1080). The three amplification products combined and amplified in an overlap PCR using primers rev137 and 3′-gusA2. The vector was amplified with 5′-AGCTGTTTCCTGTGTGAAATTGTTATCC-3′ (rev0out) and 3′-pETout3. The vector and insert were then assembled in a long overlap PCR using primers 3′-pETout3 and 3′-gusA2. The resulting library was purified, restriction-digested, and self-ligated. E. coli strain InvαF′ were transformed with the resulting library, and the transformants were screened for clones with improved XYL activity. DNA Sequencing—The evolved gusA alleles were sequenced using the Applied Biosystems Big Dye protocol at the Center for Fundamental and Applied Evolution (Emory University). The 3′-end of every evolved gusA allele was sequenced using the primer 5′-GCTCAGCGGTGGCAGCAGCCAACTC-3′ (GC 3′pET). Particularly interesting alleles (namely 1.15, 2.10, 4.7, and 4.8) were sequenced in their entirety, using the following additional primers: 5′-ATGCTTCCGGCTCGTATGTTGTGTGG-3′ (rev 80), gusA 360, 5′-GGACTTTGCAAGTGGTGAATCCGCAC-3′ (gusA 720), and gusA 1080. Protein Purification and Characterization—Each GUS protein was fused to an N-terminal His6 tag, and the enzymes 1.15, 2.10, 4.7, and 4.8 were purified to homogeneity (as judged by SDS-PAGE analysis) using nickel chelate affinity chromatography. The total protein concentration was quantified using the Bradford protein assay (Bio-Rad). 5 nm to 1 μm of each purified protein were separately reacted with 1 ml of pNP-β-d-xylopyranoside (concentrations ranging from 10 μm to 1 mm) in 50 mm Tris-HCl buffer (pH 7.6), and the formation of the pNP product was followed in a spectrophotometer. The steady-state kinetic parameters were determined by fitting the initial velocity values to the Michaelis-Menten equation (35Matsumura I. Wallingford J.B. Surana N.K. Vize P.D. Ellington A.D. Nat. Biotechnol. 1999; 17: 696-701Crossref PubMed Scopus (62) Google Scholar). Each of the values (kcat,Km, kcat/Km) reported in Table III (and the associated S.E.) is an average of three independent trials.Table IIIKinetic parameters of the evolved enzymesSubstrate enzymepNP-β-d-xylopyranosidepNP-β-d-glucuronidekcatKmkcat/KmkcatKmkcat/Kms-1μ ms-1m-1s-1μ ms-1m-1WTaWT, wild type GUS0.0010 ± 0.0001360 ± 602.9 ± 0.368 ± 6200 ± 10340,000 ± 20,0001.150.054 ± 0.003660 ± 5081 ± 30.039 ± 0.001210 ± 10190 ± 102.100.063 ± 0.005110 ± 10590 ± 300.042 ± 0.002180 ± 20240 ± 204.7NDbNot determinedND240 ± 100.012 ± 0.001220 ± 1054 ± 24.8NDND190 ± 100.021 ± 0.004280 ± 9080 ± 10a WT, wild typeb Not determined Open table in a new tab Molecular Modeling—We visualized crystal structures, made sitedirected mutants in silico and performed energy minimization calculations using SYBYL (Tripos, St. Louis, MO) at the Biomolecular Computing Resource (BimCore) at Emory University. Saturation Mutagenesis—Our objective was to direct the evolution of GUS variants with XYL activity. The ancestral plasmid for these experiments was a constitutive gusA expression vector constructed for a previous study (13Rowe L.A. Geddie M.L. Alexander O.B. Matsumura I. J. Mol. Biol. 2003; 332: 851-860Crossref PubMed Scopus (57) Google Scholar). We first employed gene site saturation mutagenesis to randomize the sequences of three residues: 557, 566, and 568, which were identified in a different directed evolution experiment (1Matsumura I. Ellington A.D. J. Mol. Biol. 2001; 305: 331-339Crossref PubMed Scopus (156) Google Scholar). We encoded all twenty amino acids in a library containing 323 (or 32,768) different clones, which is about three times the throughput of our screen. The library was generated by whole plasmid PCR (28Eisinger D.P. Trumpower B.L. BioTechniques. 1997; 22: 250-252, 254Crossref PubMed Scopus (7) Google Scholar, 37Stemmer W.P. Morris S.K. Kautzer C.R. Wilson B.S. Gene (Amst.). 1993; 123: 1-7Crossref PubMed Scopus (21) Google Scholar, 38Parikh A. Guengerich F.P. BioTechniques. 1998; 24: 428-431Crossref PubMed Scopus (31) Google Scholar) 2L. A. Rowe and I. Matsumura, submitted manuscript. using primers containing the degenerate NNK (where K is T or G) sequence. The PCR product was purified, self-ligated, and transformed into E. coli strain InvαF′. E. coli K-12 and its derivatives, including InvαF′, do not contain endogenous XYL activity (39Whitehead T.R. Curr. Microbiol. 1997; 35: 282-286Crossref PubMed Scopus (16) Google Scholar). We evaluated ∼10,000 GUS mutants in a semi-automated, high throughput microplate screen (32Geddie M.L. Rowe L.A. Alexander O.B. I. M. Methods Enzymol. 2004; 388: 134-145Crossref PubMed Scopus (11) Google Scholar). We used a microplate dispensor to distribute transformed cells into 77 × 384 well microtiter plates such that each of the 29,568 wells received an average of one cell in 5 μl of LB-ampicillin medium. As a control, 3 × 384 microcultures were seeded with cells transformed with the ancestral gusA expression vector. The 80 microplates were manually sealed with autoclaved silicone seals and inverted end-over-end in an environmental rotator for 16 h at 37 °C. The microcultures grew to saturation under these conditions, and the GUS proteins were constitutively expressed at high levels. The seals were removed, and 75 μl of 1.0 mm pNP-xyl in 50 mm Tris, pH 7.6, were added to each well. The microplates were incubated at a 45° angle (so that the cells settled into the corner of the wells) at 37 °C for 16 h. The XYL activity associated with each of the 29,568 microcultures was measured in a microplate spectrophotometer. Reconstruction experiments showed that all XYL activity was associated with the cells, rather than the supernatant, suggesting that pNP-xyl was somehow entering the cytoplasm. A Microsoft Excel macro facilitated the identification of the microcultures containing the most XYL activity. The vast majority of the GUS mutants exhibited less XYL activity than the ancestral controls (Fig. 3). We were not surprised because nearly all of the clones were supposed to contain random mutations in three active site residues. The 24 fittest clones were characterized in a more quantitative assay to demonstrate that the increases in xylosidase activity were reproducible (secondary screen). 16 replicates of each clone were propagated in a 384-well plate, in parallel with 48 replicates of the ancestral clone. Each of the cultures was reacted with 1.0 mm pNP-xyl for 16 h at 37 °C. The activity of each clone was calculated by averaging the ΔA405 of 16 replicates. The improvement initially exhibited by 10 of 24 selected clones was not reproducible, perhaps because the corresponding plasmids contained mutations that caused GUS expression to become genetically unstable. These ten clones were discarded. The average XYL activity of the remaining 14 clones was 30-fold higher than that of the ancestor. The best clone evinced 70-fold improvement in fitness (clone 1.11, Table II and Fig. 4). In our previous study, we found that random mutagenesis of the whole gene generated mutants with only 2–4-fold increases in β-galactosidase activity (40Matsumura I. Ellington A.D. Braman J. In Vitro Mutagenesis Protocols. 182. Humana Press, Totowa, NJ2001: 259-267Google Scholar). The higher values in the current study support the validity of our saturation mutagenesis strategy.Table IIAnalysis of mutants isolated in first round of evolutionVariant557566568Other mutationsaThe 3′-end of each gusA allele (encoding codons 440-604) isolated in the first round of screening was sequenced. Clone 1.15 was sequenced in its entirety. It is identical to a variant that was evolved to utilize β-galactosides (1), and is almost certainly a result of cross-contaminationRelative fitnessbThe selection coefficient (relative fitness) was determined by reacting clonal cultures with p-nitrophenyl-β-d-xylopyranoside, measuring the rate of product formation by monitoring A405 in a microplate spectrophotometer and dividing by the ancestral rateWTcWT, wild typeSerAsnLys11.1LysAlaPhe23 ± 31.2GlnLysSerQ598stop3.3 ± 11.3ProSerVal30 ± 21.4TyrGluGly20 ± 51.5AspAsnPhe48 ± 41.7LeuCysMet33 ± 21.8AspAsnLeu46 ± 21.10SerSerLys21 ± 31.11CysSerLys72 ± 81.12CysSerLys30 ± 101.13ValCysMetD531E26 ± 21.15aThe 3′-end of each gusA allele (encoding codons 440-604) isolated in the first round of screening was sequenced. Clone 1.15 was sequenced in its entirety. It is identical to a variant that was evolved to utilize β-galactosides (1), and is almost certainly a result of cross-contaminationProSerGlnS22N, G81S, K257E, T509A, Q598R, stop604W25 ± 41.16IleAlaArgA580V11 ± 71.17ProCysPhe32 ± 5a The 3′-end of each gusA allele (encoding codons 440-604) isolated in the first round of screening was sequenced. Clone 1.15 was sequenced in its entirety. It is identical to a variant that was evolved to utilize β