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Published online 17 June 2008 Nucleic Acids Research, 2008, Vol. 36, No. 13 e77 doi:10.1093/nar/gkn358 Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx) Amy J. Baldwin, Kathy Busse, Alan M. Simm and D. Dafydd Jones* School of Biosciences, Cardiff University, UK Received March 19, 2008; Revised May 14, 2008; Accepted May 20, 2008 ABSTRACT site-directed mutagenesis but due to our limited under- standing of the complex and cooperative network of Trinucleotide exchange (TriNEx) is a method for gen- weak interactions that comprise a functional protein, it erating novel molecular diversity during directed is still difficult to predict the consequence of a mutation. evolution by random substitution of one contiguous To overcome these difficulties, a new approach was taken trinucleotide sequence for another. Single trinucleo- that mimicked the natural process of Darwinian evolution tide sequences were deleted at random positions in and has been termed directed evolution (2–4). Directed a target gene using the engineered transposon evolution involves generating molecular diversity by the introduction of random mutations within a gene followed MuDel that were subsequently replaced with a ran- by selection of desirable protein variants. Unlike site- domized trinucleotide sequence donated by the DNA NNN specific mutagenesis, directed evolution does not require cassette termed SubSeq . The bla gene encoding a detailed knowledge of protein structure or have any TEM-1 b-lactamase was used as a model to demon- preconceptions of the importance of certain residues to strate the effectiveness of TriNEx. Sequence analy- stability and function. This evolutionary protein engineer- sis revealed that the mutations were distributed ing strategy has been very successful in generating proteins throughout bla, with variants containing single, with new properties, with beneficial mutations observed at double and triple nucleotide changes. Many of the positions distant from those predicted to be crucial for resulting amino acid substitutions had significant generating novel activity (5,6). effects on the in vivo activity of TEM-1, including Although a variety of methods exist to introduce pre- up to a 64-fold increased activity toward ceftazidime dominantly single point mutations randomly throughout and up to an 8-fold increased resistance to the inhi- a whole gene, their limitations include: error, codon and bitor clavulanate. Many of the observed amino acid amplification biases, sampling of a restricted set of amino acids, the masking of beneficial mutations by deleterious substitutions were only accessible by exchanging at ones and the inability to sample many of the potential least two nucleotides per codon, including charge- variants due to large and undefined library sizes (7,8). switch (R164D) and aromatic substitution (W165Y) Some of these problems can be overcome by site-satura- mutations. TriNEx can therefore generate a diverse tion mutagenesis, in which diversity is incorporated into a range of protein variants with altered properties by synthetic oligonucleotide. However, this approach inherits combining the power of site-directed saturation the problems of site-directed mutagenesis and mutations mutagenesis with the capacity of whole-gene muta- are restricted to just a small portion of the protein. genesis to randomly introduce mutations through- A new method called trinucleotide exchange (TriNEx) is out a gene. proposed that will overcome the limitations of current methods for generating molecular diversity. TriNEx is a non-PCR-based method that combines the power of INTRODUCTION whole-gene random mutagenesis with the ability of oligo- nucleotide-directed mutagenesis to sample an expanded Protein engineering has provided crucial insights into pro- tein structure, function and folding, and has been pivotal range of amino acid substitutions. Based on a recently in adapting proteins for various biotechnological applica- developed transposon method for generating single tions (1). Initial protein engineering strategies focused on amino acid deletion variants (9), TriNEx involves first *To whom correspondence should be addressed. Tel: +44 0 29 2087 4290; Fax: +44 0 29 2087 4116; Email: jonesdd@cf.ac.uk Present addresses: Kathy Busse, Institut fu¨ r Biochemie der Med. Fakulta¨ t, Universita¨ t Leipzig, Leipzig, Germany Alan M. Simm, School of Engineering and Electronics, University of Edinburgh, Edinburgh, UK 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. e77 Nucleic Acids Research, 2008, Vol. 36, No. 13 PAGE 2 OF 9 the removal of a single contiguous trinucleotide sequence then its replacement with a randomized sequence (Figure 1). This potentially allows the sampling of every codon permutation throughout a gene and defines library diversity and sampling requirements. This method will overcome the error and codon bias inherent in methods such as error-prone PCR and will not be restricted to mutating predefined amino acid residues as is the case with oligonucleotide-directed mutagenesis. There are no PCR steps involved in diversity generation so avoiding any amplification bias. Also, as one transposon is inserted per gene, each variant will contain either a single amino acid or two adjacent substitutions per protein variant, preventing the masking of beneficial mutations by other, distant deleterious mutations. Furthermore, it allows directed evolution to be applied in a similar manner to the powerful site-directed single substitution scanning mutagenesis methods (e.g. alanine scanning mutagenesis) to assess the contribution of individual amino acid resi- dues to the structure, function and folding of proteins. Here, we demonstrate the TriNEx method using the bla gene encoding TEM-1 b-lactamase. TEM-1 is one of the main enzymes responsible for conferring bacterial resis- tance to b-lactam antibiotics such as ampicillin but has poor activity towards third generation cephalosporins such as ceftazidime and can be inhibited by clavulanate (10). We successfully generated libraries with triplet nucleotide exchanges present throughout the bla gene. The resulting amino acid substitutions had a range of effects on the in vivo activity of TEM-1, including increased activity towards ceftazidime (CAZ) and improved resistance to clavulanate (CLV). Many of the amino acid substitutions observed were only accessible by two or more adjacent nucleotide changes. MATERIALS AND METHODS Construction of libraries with MuDel inserted randomly within the bla gene The initial small-scale library (termed BLA ) that con- tained 175 variants with MuDel inserted randomly within Figure 1. Schematic outline of the TriNEx procedure. Stage 1. the bla gene was a subset of 391 variants generated pre- Trinucleotide deletion. The MuDel library is generated by in vitro trans- viously (11). Construction of the larger, extended library position of the engineered transposon MuDel (teal) into the target DNA. (termed BLA ) comprised of 1644 variants with Restriction endonuclease digestion removes MuDel from this library together with 3 bp of the original target DNA to generate a single break MuDel inserted randomly within bla was performed essen- per molecule. Stage 2. Trinucleotide replacement. The DNA cassette tially as described previously for the single amino acid NNN termed SubSeq (red) is then inserted between the break in the target deletion procedure (9,11). The details of this procedure NNN DNA to generate the SubSeq library. The last three nucleotides at one NNN are described in Supplementary Methods. end of SubSeq (red square) are randomized (NNN). Restriction endo- nuclease digestion removes all SubSeq, except for the NNN sequence that NNN Construction of library containing SubSeq is now incorporated into the target DNA, which replaces the 3 bp deleted in stage 1. Intramolecular ligation regenerates the complete gene contain- randomly placed within bla ing new and randomly placed trinucleotide segments. This is followed by NNN The SubSeq cassette was constructed using PCR as selection and/or screening to identify new variants with desired properties. described in Supplementary Methods. The library of NNN variants with SubSeq placed within bla was generated Madison, WI, USA) for 15 min at 378C. After inactivation by the excision of MuDel from the plasmid followed by NNN of the phosphatase by heating to 708C for 10 min, the the insertion of the SubSeq DNA cassette. The pooled 175 1644 linearized pNOM DNA was separated by agarose gel elec- DNA (1mg) from either the BLA or BLA libraries was digested with MlyI to remove MuDel. trophoresis and purified. Linear pNOM was ligated with TM NNN NNN The cleaved DNA was then treated with APex heat- SubSeq in 1:3 molar ratio (pNOM:SubSeq ) TM labile alkaline phosphatase (Epicentre Biotechnologies, using the Fast-Link DNA ligation kit (Epicentre PAGE 3 OF 9 Nucleic Acids Research, 2008, Vol. 36, No. 13 e77 Biotechnologies). A total of 200 ng of DNA was used in Characterization of the bla trinucleotide exchange variants the ligation. The bla gene of selected variants was isolated by PCR With regards to the BLA library, ca 15 ng of the using the GoTaq system (Promega) with primers flank- DNA from the ligation reaction was used to transform che- ing the gene (5 -TCCGCTCATGAGACAATAACCCTG- mically competent Escherichia coli DH5a cells. The trans- 0 0 0 3 and 5 -CTACGGGGTCTGACGCTCAGTG-3 ) and formed cells were grown on LB agar containing 15mg/ml the PCR product sequenced (DNA Sequencing Core, kanamycin (Kan) and incubated at 378C for 16 h. Colonies Molecular Biology Unit, Cardiff University) to determine were picked at random and used to inoculate 5 ml of LB the position and nature of the mutation. The in vivo activity broth containing 15mg/ml Kan. Plasmid DNA was isolated of the selected variants was estimated by measuring the from each culture using the QIAprep Spin Miniprep kit minimum inhibitory concentration (MIC) of Amp, CAZ (Qiagen Ltd, Crawley, UK). SubSeq was removed from or Amp and CLV that prevented cell growth. The Amp the plasmid (ca 2mg per 50ml reaction volume) by digestion MIC values for cells producing TEM-1 variants derived with MlyI. The plasmid (ca 20 ng) was recircularized from the BLA library was determined essentially as directly after digestion using T4 DNA ligase (Promega described previously (9,11) and detailed in the Supple- UK, Southampton, UK). An equivalent of ca 0.4 ng of mentary Methods. The CAZ or CLV+Amp MIC values DNA from the ligation reaction was used to transform of the selected TEM-1 variants derived from BLA chemically competent E. coli DH5a. Half of the cells were were estimated as follows. The glycerol stocks generated then spread on LB agar containing 32mg/ml Amp and incu- above were used to inoculate LB broth cultures that were bated at 378C for a minimum of 16 h. The bla gene sequence incubated at 378C for 4 h. Each culture was then trans- and the Amp MIC for clones producing active TEM-1 var- ferred, using a 96 pronged replicating fork, to LB agar iants were determined as described subsequently. The bla containing 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.6, 19.2 or 38.4mg/ gene prior to SubSeq removal was also sequenced for sev- ml CAZ, or 2, 3, 4, 6, 8, 12, 16, 32, 64mg/ml CLV with eral inactive variants to predict the mutation that resulted 100mg/ml Amp and the plates incubated at 378C for 16 h. in loss of b-lactamase activity. Four colonies containing pNOM were used as controls. With regards to the BLA library, the ligation reac- NNN The bla gene was isolated by PCR and sequenced, as tion to insert SubSeq within linearized pNOM was described earlier. then used to transform (ca 15 ng of DNA/transformation) high efficiency chemically competent NovaBlue E. coli cells (Merck KGaA). To calculate the number of transformed NNN RESULTS cells containing the pNOM-SubSeq plasmid, the equivalent of 1/30 of the transformation mix post-recovery Creation of trinucleotide substitution variants from BLA" was grown on LB agar plates containing 25mg/ml Kan and The replacement of one contiguous trinucleotide sequence the number of colony forming units (c.f.u.) observed after with another was achieved in a two-stage process, as out- incubation at 378C overnight was determined (routinely lined in Figure 1. The first stage involved the removal of a generated ca 4 10 c.f.u./mg DNA). The remainder of trinucleotide sequence from a target gene using a pre- the transformation culture was added to 50 ml of LB viously described method based on the use of the engi- broth containing 200mg/ml Kan and incubated at 378C neered transposon MuDel (9). The next stage involves for 16 h. Plasmid DNA was isolated from the culture the replacement of the deleted trinucleotide by the inser- using the QIAprep Spin Miniprep kit (Qiagen), and 2mg NNN tion of the DNA cassette termed SubSeq into the of this DNA was digested with MlyI to remove the SubSeq break within the target gene vacated by MuDel. The section. The DNA was desalted and 100 ng of the DNA TM mechanism by which trinucleotide deletion and replace- ligated using the Fast-Link DNA ligation kit ment is accomplished is described in Figure 2. (Epicentre Biotechnologies) to recircularize pNOM. The Initially, TriNEx was demonstrated using a subset of 175 ligation reaction (10 ng/transformation) was used to trans- variants, constructed previously (11), with MuDel inserted form high efficiency chemical competent NovaBlue E. coli at random positions within bla. This library was termed (Merck KGaA, Merck Chemicals Ltd, Nottingham, UK). BLA and MlyI digestion from a DNA pool comprised of the 175 variants removed MuDel. After insertion of Selection of TEM-1 variants with enhanced activity NNN SubSeq by ligation and transformation of E. coli, plas- towards ceftazidime and clavulanate mid DNA was isolated from a total of 106 different colonies Transformed cells were diluted 10-fold in SOC medium resistant to Kan and SubSeq removed by digestion with then spread on LB agar containing at least 0.3mg/ml MlyI. Recircularized plasmid was used to transform CAZ or 100mg/ml Amp and 4 mg/ml CLV. The plates E. coli, and bacterial resistance to Amp tested by plating were incubated at 378C for a minimum of 16 h. Cells trans- on LB agar containing 32mg/ml Amp. Of these 106, viable formed with pNOM containing wild-type bla did not grow cell growth (typically 10–500 colonies per plate) was under these CAZ and CLV selection conditions. Selected observed for 73 with no colony growth detected for the colonies were transferred to LB broth containing the equi- remaining 33. Of the 73 bla variants capable of conferring valent concentration of antibiotic used in the selection. Amp resistance, 34 were sequenced to determine the muta- The cultures were grown overnight at 378C and glycerol tions introduced and to assess their influence on the MIC of added to a final concentration of 25% (v/v) for storage Amp that prevented cell growth. To confirm that variants at 808C. unable to confer resistance were a consequence of e77 Nucleic Acids Research, 2008, Vol. 36, No. 13 PAGE 4 OF 9 Figure 2. Mechanism of TriNEx process. Step 1. Two MlyI recognition sites (5 GAGTC(N) #) were placed 1 bp from each end of MuDel, 1 bp away from the site of transposon insertion. MlyI cuts 5 bp outside its recognition sequence to generate a blunt end. Insertion of MuDel results in the duplication of 5 bp (N N N N N ) of the target gene at the insertion point (23,24). Step 2. Digestion with MlyI removes MuDel together with 8 bp of the target 1 2 3 4 5 NNN DNA (4 bp at each end). This equates to removal of a contiguous 3 bp sequence from the starting target gene (N N N ). Step 3. SubSeq is then ligated 2 3 4 NNN into the gap vacated by MuDel. SubSeq also contains two MlyI recognition sites strategically placed towards the ends of the cassette. One site is NNN NNN located so that MlyI will cut at the exact point where the target DNA joins SubSeq . The second site will cut 3 bp into SubSeq so donating 3 bp (N N N ) to the target DNA. Step 4. Digestion with MlyI removes SubSeq but with 3 bp of its sequence now replacing the 3 bp deleted from the target X Y Z gene. Step 5. Intramolecular ligation reforms the target gene but with one contiguous trinucleotide sequence replaced with another. observed twice and one three times. However, the tri- nucleotide replaced was not always the same (Table 1). For example, Q278W was generated by exchange of three nucleotides 1 bp upstream of those replaced to produce Q278C. At least 88% of the MuDel insertion positions predicted from the sequence data were judged to be Figure 3. Position and number of nucleotides changes introduced into the unique, close to that observed previously (11). Just over 39 bla TriNEx variants derived from BLA . The grey, orange and half (26 from 45) of the TEM-1 variants contained amino green triangles signify 1, 2 and 3 nucleotide replacements, respectively. acid substitutions only accessible by mutation of at least The black triangles represent frameshifts due to deletion of two nucleo- tides from bla. two of the three nucleotides. Exchange of contiguous trinucleotide segments can span two adjacent codons in theoretically 2/3 of cases, poten- potentially deleterious substitution mutations to TEM-1 tially leading to a double amino acid substitution. Due to and not gene frameshifts or trinucleotide deletions, 15 of the degeneracy in the genetic code and mutation of either the 33 bla variants that encoded a TEM-1 variant unable to NNN one or two nucleotides, the majority of variants (66%) confer Amp resistance were sequenced prior to SubSeq encoded a single amino acid substitution (Table 1), with removal and the TriNEx predicted. just under a half of these (15 from 31) containing a silent Sequence analysis revealed that the position of the muta- mutation. tions were distributed throughout the bla gene (Figure 3), The in vivo activity of TEM-1 variants also varied with 14, 45 and 41% of the variants containing single, depending on the mutation, with Amp MIC conferred double or triple nucleotide changes, respectively. No on E. coli ranging from 64mg/ml (e.g. E274P) to wild-type or trinucleotide deletion variants of bla were 4096mg/ml (e.g. Q205R and Q278W) observed for wild- observed, and no secondary mutations were seen. Two var- type TEM-1 (Table 1). Of the 15 variants that displayed iants were predicted to introduce frameshifts. Each of the no TEM-1 activity, three were predicted to contain sub- 49 variants was unique at the genetic level and only two stitutions that generated a stop codon (e.g. K192ter) and were identical at the protein level (Table 1), which both another disrupted the initiation codon (M1N-S2G). Many reconstituted a gene encoding wild-type TEM-1 but with of the other predicted mutations could be classed as poten- silent mutations within different codons (encoding R120 tially disruptive to TEM-1 structure and/or function. The and L193-L194). The nature of the replacement NNN M69R-S70C mutation knocks out the essential catalytic sequence could be determined exactly for 33 variants, and serine. Mutation of C77 in the core helix of TEM-1 revealed that 27 were unique with no NNN sequence repre- sented more than twice. Of the 45 variants that encoded will remove the sole disulphide bond in the protein. amino acid substitutions, mutation of six residues were Others may disrupt organized secondary structure PAGE 5 OF 9 Nucleic Acids Research, 2008, Vol. 36, No. 13 e77 Table 1. Sequence variation observed in bla variants derived from the Two variants were predicted to contain frameshifts due to BLA library and their influence on TEM-1 activity towards Amp the removal of two nucleotides from the bla gene (Table 1) in vivo but no trinucleotide deletions were predicted. However, it cannot be ruled out that errors introduced post SubSeq Amino acid Nucleotide Base Amp MIC a b c d removal and the subsequent self-ligation stages were substitutions substitutions changes (mg/ml) responsible for inactivation of some of the TEM-1 Wild type 4096 variants. M1N-S2G atg agt!aat ggt 3NG R9V cgt!gtt 2 512 Construction of an extended TriNEx library and D38E-Q39P gat cag!gaa ccg 2 256 L40ter ttg ggt!tga tgt 3NG the selection of enhanced TEM-1 variants G41S ggt!agt 1 4096 The main aim of directed evolution is the creation and N52K-S53A aac agc!aag gcc 3 1024 M69R-S70C atg agc!agg tgc 2NG sampling of a suitable range of molecular diversity to C77R cta tgt!cta cgt 1NG L76L alter or improve proteins. As a demonstration of the ability C77P tgt!cca 3NG of TriNEx to fulfil this aim, it was employed to improve the A79V gcg!gta 2 2048 activity of TEM-1 towards a third generation cephalos- E104G gtt gag!gtc ggg 2 2048 V103V E104L gtt gag!gtg ctg 3 1024 porin CAZ, and enhance resistance to the inhibitor CLV. V103V S106T tca!acc 2 256 First, a much larger transposon insertion library was S106C tca!tgt 2 256 required to sample all the potential insertion positions P107A cca!gct 2 256 within bla. A new library comprised of 1644 Amp sensitive D115Y acg gat!acc tat 2NG T114T M117I atg!ata 1 1024 colonies was constructed with MuDel inserted within bla T118M-V119I aca gta!atg ata 3 512 and was termed BLA . The size of this library was aga!agg 1 4096 R120R deemed sufficient to cover all potential insertion positions C123ter tgc agt!tga tgt 2NG within bla (coding region of 858 bp), even when redundancy A125G-A126S gct gcc!gga tcc 364 M129G acc atg!act ggg 3 512 in transposon insertion positions [10–15%; see above and T128T A135D-N136Y gcc aac!gat tac 3NG reference (11)] is factored in. The diversity of transposon M155S-G156W atg ggg!agt tgg 364 insertion was confirmed using a restriction endonuclease M155I-G156Q atg ggg!att cag 3 1024 procedure described previously (Figure 4a) (11). The diges- M155I-G156E atg ggg!atc gag 2 1024 E168L ccg gag!cct ttg 3 1024 tion products form a smear, as would be expected if MuDel P167P T181D acg!gac 3NG insertion within bla was essentially random (Figure 4b). K192ter cgc aaa!cgt taa 2NG R191R The BLA members were pooled and MuDel removed cta tta!ctc cta 2 4096 L193L-L194L NNN by digestion with MlyI and SubSeq ligated into the L194C-T195P tta act!tgt cct 3NG break. Transformation of E. coli with the ligation mixture Q205R cgg caa!cgc cga 2 4096 R204R W210 fs gac tgg atg NG – routinely generated ca 4 10 c.f.u./mg of DNA on agar !ga––ac atg plates containing Kan. This was deemed sufficiently high A213G gcg!ggt 2 4096 to sample the possible 54 912 different genetic variations of A227T ccg gct!cca act 2 4096 P226P bla that can be generated by TriNEx, if every possible con- S235 fs tct gga!tcg ––a –NG L250H-G251R ctg ggg!cac agg 3NG tiguous trinucleotide sequence is replaced (gene size of 858 G251R ctg ggg!cta cgg 2NG L250L multiplied by 64 different NNN combinations). The trans- R259I cgt!atc 3 256 formed cells were subsequently grown under a stringent T266K-G267S acg ggc!aag agc 2 512 Kan selection regime (200mg/ml) to ensure that only cells Q269L cag!ctg 1 4096 NNN M272R atg!agg 1 512 containing plasmid-borne SubSeq would survive. E274Q gat gaa!gac caa 2 2048 D273D DNA was then isolated from the culture to generate a plas- E274P gaa!cca 264 NNN mid pool of SubSeq inserted at random positions Q278W aga cag!agg tgg 3 4096 R277R within bla. Q278C cag!tgt 3 2048 E281S gct gag!gcg tcg 3 2048 The SubSeq portion was removed by MlyI digestion A280A I282T ata!acc 264 and the plasmid pool recircularized. This formed the K288N-H289V aag cat!aac gtt 3 512 library used for the selection of TEM-1 variants with improved properties. Minimum estimates of 41 000 var- Subscripted sequences represent silent nucleotide changes to the iants were tested under each condition to select those cap- codon. fs represent frameshift. Residues numbered according to stan- dard system (25). able of promoting cell growth on 0.3mg/ml CAZ or a Bold and underlined sequences represent the known nucleotides combination of 100mg/ml Amp and 4mg/ml CLV. NNN deleted from bla and replaced by SubSeq . However, this was probably an underestimate because in Actual number of nucleotide differences between the variant and wild- type bla. the absence of a second selectable marker, the calculation NG, no cell growth observed on 32mg/ml Amp LB agar upon removal was based on the number of colonies observed on LB agar of SubSeq and reconstitution of the full length bla. plates containing 100mg/ml Amp. While the majority of TEM-1 substitution variants were likely to retain even a (e.g. L194C-T195P) or introduce a charged residue into low level of activity towards Amp, the previous results of the hydrophobic core close to the active site (e.g. individual TEM-1 variants derived from BLA indi- A135D-N136Y). TEM-1 has also been shown previously cated that 31% would be inactive. Using this as a guide, not to tolerate mutations as positions T181 or G251 (12). the number of variants screened under each selection e77 Nucleic Acids Research, 2008, Vol. 36, No. 13 PAGE 6 OF 9 Figure 4. Restriction endonuclease analysis of the BLA library. (a) Rationale for determining MuDel (teal) insertion diversity within the bla gene (orange with black stripe) of pNOM. Digestion by XhoI (recognition site shown as blue triangle) of the pooled pNOM plasmid with MuDel inserted within the bla gene generates one major product of 3422 bp in size. Digestion with MlyI (recognition site shown as red triangle) generates two major products of 1310 bp (MuDel plus 8 bp from bla) and 2112 bp in size. Digestion with both XhoI and MlyI generates the 1310 bp MuDel fragment together with many different size fragments depending on the insertion position of the transposon. (b) Restriction analysis of the BLA library with XhoI and/or MlyI, as indicated in the figure. condition was likely to be closer to 52 000. Cells producing to improving resistance to CAZ (10) was also highlighted wild-type TEM-1 (via the parent pNOM plasmid) did not here as 30 of the analysed variants were mutated at this grow at these CAZ or Amp+CLV concentrations. A total residue, with six different substitutions observed of 408 or 93 colonies were observed to grow on LB agar (Supplementary Table 1). under either the CAZ or Amp+CLV selection conditions, Beneficial effects of two adjacent amino acid mutations respectively. Of these, 41 colonies selected on CAZ and were also observed. For example, there was up to a 4-fold 27 selected on Amp/CLV were arbitrarily chosen for improvement in the CAZ MIC values for variants contain- sequencing and in vivo activity analysis (Table 2 and full ing the W165G mutation when accompanied by R164P list in Supplementary Table 1). (Table 2 and Supplementary Table 1). The R164P mutation was only observed in combination with a substitution at Characterization of TEM-1variants with improved properties W165. This highlights the advantages of sampling such adjacent mutations. In other cases, the adjacent mutation Analysis of the TEM-1 variants with enhanced activity had little effect. For example, the CAZ MIC value for var- towards CAZ and increased resistance to CLV revealed a iants containing the P167H mutation was similar to those range of substitution mutations at different positions instil- that contained the accompanying E168K substitution ling various degrees of improvement over wild-type TEM-1 (Table 2). The use of different codons to specify mutations (Table 2 and Supplementary Table 1). Many of the best also appeared to influence the in vivo activities of TEM-1 in performing TEM-1 variants had amino acid substitutions some cases (Table 2 and Supplementary Table 1). For only accessible by at least two nucleotide changes per example, the R164P-W165R variants were encoded by codon. These included the charge-switch mutant R164D five different gene sequences but their CAZ MIC values with a 32-fold improvement in activity towards CAZ and varied 4-fold. It has been noted previously that silent muta- the semi-conservative aromatic substitution of tryptophan to tyrosine (W165Y), which increased CLV resistance tions to TEM-1 can also result in such differences (13,14). The majority of beneficial mutations occurred close to 3-fold (Table 2). The R275V mutation common to variants with the most improved resistance to CLV also required at the active site (Figure 5), of which several were to residues least two adjacent nucleotide changes to access the amino that had previously been identified as important in enhanc- acid substitution (Table 2). The importance of residue 164 ing activity to CAZ (e.g. R164S and R164H) and resistance PAGE 7 OF 9 Nucleic Acids Research, 2008, Vol. 36, No. 13 e77 Table 2. Examples of TEM-1 variants with enhanced properties in vivo included three independent variants with enhanced activity to CAZ that had the catalytically important E166 mutated. Amino acid Nucleotide changes MIC fold change One mutation, E197P, was relatively distance from the substitution active site (Figure 5) but slightly enhanced activity towards CAZ CLV CAZ and resistance to CLV (Table 2). This highlights the power of directed evolution to sample mutations M69I atg!atc 3 P145R ccg!cga 4 not predicted to be beneficial by structural analysis, and D163E-R164N gat cgt!gag aat 16 the ability of TriNEx to expand mutational sampling R164S gat cgt!gac tct 32 D163D (E!P requires substitution of at least two adjacent R164H gat cgt!gac cat 64 D163D nucleotides). R164D gat cgt!gac gat 32 D163D R164P-W165R cgt tgg!cct agg 32 cgt tgg!ccg cgg 4 R164P-W165G cgt tgg!cca ggg 8 DISCUSSION W165G tgg!ggg 2 W165P tgg!cct 2 TriNEx as a method for generating molecular diversity W165Y tgg!tac 3 tgg!tat 3 Directed evolution has been proven as an effective strategy E166G gaa!gga 8 for engineering proteins (2–6). Critical to directed evolu- P167H ccg!cac 8 tion is the ability to generate and sample molecular diver- E171P gaa!cca 8 sity. While whole-gene mutagenesis methods such as R178A cgt!gca 4 error-prone PCR (15) are relatively simple to perform E197P gaa!cca 2 1.5 E240R ggt gag!ggc agg 4 G238G and can introduce mutations throughout a given target R244T tct cgc!tcc acc 3 S243S DNA sequence, they can only sample a restricted muta- R275V cga!gta 8 tional range (with inherent biases) and generate large cga!gtg 8 libraries of which only a fraction can ever be sampled cga!gtt 8 R275T cga!acc 2 (8). Site-saturated mutagenesis using oligonucleotide- encoded molecular diversity overcomes the limited muta- The full list of sequenced TEM-1 variants are shown in Supplementary tional range of whole-gene mutagenesis but is restricted to Table 1. b sampling just a few residue positions in a protein so Numbering according to standard system (25). requires a detailed knowledge of the structure–function Fold change compared to MIC observed for cells producing wt TEM-1 encoded in the pNOM plasmid (CAZ MIC 0.3mg/ml, CLV relationship to predict which residues to target. MIC 4mg/ml). TriNEx attempts to overcome these problems by pro- viding a useful alternative that combines the benefits of both approaches. The random insertion and deletion (RID) method (16) was also developed to overcome simi- lar problems but this method is difficult to implement (including the requirement of single strand DNA) and uses PCR. Furthermore, many variants contained second- ary mutations and frameshifts that reduce the quality of the library. TriNEx does not use PCR at any stage during library construction and utilizes simple DNA manipula- tion and microbiological methods familiar to most mole- cular biologists. Only two frameshifts were predicted and no secondary point mutations or trinucleotide deletions were observed in the sequenced clones produced by TriNEx (Tables 1 and 2, Supplementary Table 1). TriNEx has been successfully demonstrated using the bla gene encoding TEM-1 b-lactamase as our test system. The observed mutations were distributed through- out bla, with the majority of the variants containing 2- or 3-bp changes with respect to wild-type (Figure 3 and Tables 1 and 2). Many of the resulting amino acid sub- Figure 5. Position of mutations with respect to the tertiary structure of stitutions introduced into TEM-1 could only be accessed TEM-1 [PDB code 1BTL (18)]. Mutations that enhance activity to by more than 1 bp change to a codon. Therefore, TriNEx CAZ or CLV or both are shown as cyan, orange or purple balls, can expand the molecular diversity sampled by whole-gene respectively. The active site serine (S70) is shown as space-fill and mutagenesis procedures to introduce novel amino acid labelled. The V-loop is coloured red. The residues are numbered using the recommended numbering system (25). substitutions that may enhance the properties of a protein. The first transposon step ultimately determines TriNEx to CLV (e.g. M69I) [see (10) and www.lahey.org/Studies/ library diversity. MuDel is based on the mini-Mu trans- and references therein]. Beneficial mutations included a poson, which has a reported 5-bp target site for insertion run of eight substitutions (six consecutively between 163 based on the consensus sequence 5 N-Py-G/C-Pu-N and 168) in the V-loop (Figure 5). This unexpectedly 3 (17). Such a sequence bias may result in hotspots for e77 Nucleic Acids Research, 2008, Vol. 36, No. 13 PAGE 8 OF 9 transposon insertion and therefore introduce a bias in the genetic code many of the mutations were silent at the pro- library. While some duplication of the trinucleotide tein level. The result is that 66% of the variants contained a sequences deleted in separate variants derived from the single amino acid substitution. Furthermore, sampling BLA library was observed (Table 1), the general such double substitutions may not be consider detrimental redundancy of transposon insertion positions was esti- but may enhance diversity. For example, a 4-fold increase mated to be 10–15%. This agrees with previously observed in TEM-1 activity towards CAZ was observed when redundancy but in a different mutational context (11). W165G was combined with R164P (Table 2). Restriction analysis of the BLA libraries reported both here (Figure 4) and previously (11) confirm the random Mutations introduced by TriNEx alter nature of MuDel insertion position. Therefore, the diver- the activity of TEM-1 sity of insertion positions sampled by MuDel allows muta- tions to be distributed throughout bla. A diverse range of TriNEx introduced a wide variety of amino acid substitu- NNN replacement NNN encoded as part of SubSeq was tions into TEM-1 with varying effects on the enzyme’s also observed (Table 1). in vivo activity (Tables 1 and 2). Many of the TEM-1 var- Insertion of MuDel outside of the target gene within the iants derived from BLA retained a high degree of activ- plasmid backbone may also be an issue. This was over- ity, with eight (including the two with wild-type amino acid come here by selection for clones resistant to Cam but sequence) of the 34 active variants conferring an Amp MIC sensitive to Amp due to the knockout of bla by MuDel value similar to that of wild-type. Interestingly, these insertion. Using linear DNA comprised solely of the target included mutations to residues generally conserved in gene can overcome this problem. It has been demonstrated class A b-lactamases (10) or not consider tolerant to a sub- previously that linear DNA acts as an efficient substrate stitution (12) (e.g. P107, A125, G156, M272 and I282). As for transposon insertion (17). While recloning the target 175 69% of the tested variants derived from BLA library gene back into pNOM after transposition may reduce the retained even a small degree of b-lactamase activity efficiency of the first step in the procedure, the subsequent towards Amp, this confirms previous work (12) that sug- selection for Cam resistance will result in theoretically gests TEM-1 is largely tolerant to amino acid substitutions. 100% of clones containing MuDel inserted within the TriNEx has also been successfully employed to improve target gene. This is particularly important when applying the activity of TEM-1 towards the third generation cepha- TriNEx to proteins in which there is no simple selection or losporin CAZ and increase resistance to the inhibitor CLV. screen available. We have recently demonstrated this strat- Most of these mutations lay within or close to regions egy, along with other approaches, in our laboratory to involved in the catalytic process and substrate binding implement TriNEx for non-antibiotic resistance proteins (Figure 5). Many of the beneficial mutations were observed (data not shown). in the V-loop (residues 163–178 (18)). This included sub- The number of variants that can be generated at each stitution of the catalytically important E166 (to glycine or stage is also critical to the ability of TriNEx to sample a lysine) observed in three independent clones that improved suitable range of diversity. The generation of 1644 variants TEM-1 activity towards CAZ by up to 8-fold (Table 2 and with MuDel inserted within the 858 bp bla gene was deemed Supplementary Table 1). A variety of substitutions to R164 more than sufficient even when taking into account redun- were observed in many of the variants with improved activ- dancy in transposon insertion. Blunt-end ligations akin to NNN ity towards CAZ, including those with highest observed that necessary for the insertion of SubSeq can be less activity (Table 2 and Supplementary Table 1). Mutations efficient than those performed with compatible overhangs, to R164 have been demonstrated previously to improve which may potentially reduce the number of subsequent NNN activity towards CAZ by removing a salt bridge that con- variants sampled. The blunt-end ligation of SubSeq strains conformation of the V-loop (10,19). To our knowl- into bla routinely generated 4 10 Kan resistant colonies edge, some of the specific mutations to R164 identified in per microgram of DNA after transformation, which was this study (to asparagine, aspartate and proline) have not considered high enough to sample the potentially 54 912 been observed previously. Mutations outside the V-loop different potential genetic variations of bla. The final recir- also contribute towards enhanced activity towards CAZ. cularization ligation to reform the trinucleotide replace- These include some novel mutations such as P145R and ment bla gene variants was extremely efficient, routinely P145R-K146E that lie close to both the catalytic centre producing ca 10 c.f.u./mg DNA. Therefore, TriNEx can and the V-loop (Figure 5). generate a suitable number of variants at each stage to TriNEx also introduced mutations to residues known to sample an extensive range of molecular diversity for a contribute to improved resistance to CLV (M69, W165, single and, if required, multiple iterative rounds where the R244 and R275) (10). Mutations at these positions are target gene may be comprised of several different variants thought to alter substrate binding, including for suicide selected from the previous round of TriNEx. substrates such as CLV. Mutations to R275 produced As TriNEx can span two codons in theoretically / of the largest improvements, with the R275V substitution cases, this can and does lead to substitution mutations in increasing resistance by 8-fold (Table 2). Natural variants two adjacent amino acid residues (Table 1). Of the 49 of TEM-1 resistant to CLV contain either a leucine or sequenced variants derived from BLA , 30 were known to contain trinucleotide exchanges that spanned glutamine at residue 275 and are found in combination two codons (Table 1), yet only 14 encoded a double with other beneficial mutations [(10) and www.lahey.org/ amino acid substitution. Due to the degeneracy in the Studies/]. The ability of TriNEx to expand the mutational PAGE 9 OF 9 Nucleic Acids Research, 2008, Vol. 36, No. 13 e77 5. Bloom,J.D., Meyer,M.M., Meinhold,P., Otey,C.R., MacMillan,D. range to incorporate R275V may explain why this muta- and Arnold,F.H. (2005) Evolving strategies for enzyme engineering. tion observed in our study had such a drastic effect alone. Curr. Opin. Struct. Biol., 15, 447–452. One TEM-1 variant containing the E197P mutation dis- 6. Tao,H. and Cornish,V.W. (2002) Milestones in directed enzyme played a slightly enhanced activity towards CAZ and evolution. Curr. Opin. Chem. Biol., 6, 858–864. 7. Lutz,S. and Patrick,W.M. (2004) Novel methods for directed resistance to CLV. Its potential mode of action is unclear evolution of enzymes: quality, not quantity. Curr. Opin. Biotechnol., as it is distant from the active site and to our knowledge 15, 291–297. has not been observed before. The fact this particular 8. Neylon,C. (2004) Chemical and biochemical strategies for the substitution required the exchange of 2 bp of the E197 randomization of protein encoding DNA sequences: library codon may explain why it has not been observed in nat- construction methods for directed evolution. Nucleic Acids Res., 32, 1448–1459. ural or engineered variants of TEM-1. However, the rela- 9. Jones,D.D. (2005) Triplet nucleotide removal at random positions tively small enhancements may not have been sufficient to in a target gene: the tolerance of TEM-1 b-lactamase to an amino overcome previous selection pressures. acid deletion. Nucleic Acids Res., 33, e80. 10. 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Nucleic Acids Research – Oxford University Press
Published: Aug 17, 2008
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