Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation prior to shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G+C contents.     

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 ± 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure–function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.     

Improved production and function of llama heavy chain antibody fragments by molecular evolution

The aim of this study was to improve production level of llama heavy chain antibody fragments (V_HH) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V_HH fragments specific for the azo-dye reactive red-6. In the DNA shuffling process, three parental llama V_HH with high amino acid sequence identity with significant differences in production and functional characteristics were used. From these parental sequences, a S. cerevisiae library was created and 16 antigen specific shuffled V_HH fragments were selected. We found that these shuffled V_HH fragments were, (i) unique in sequence; (ii) composed of two or three parental sequences; (iii) in three V_HHs point mutations occurred; and (iv) antigen specificity was not changed. The four highest producers in the yeast S. cerevisiae were selected and production, affinity, and antigen binding at 90°C were compared with parental V_HHs. One shuffled V_HH was enhanced both in production (3.4-fold) and affinity (four-fold). A second shuffled V_HH displayed increased production (1.9-fold), and improved stability (2.4-fold) in antigen binding at 90°C. Structural analysis suggested that improved antigen binding is associated with the A24→V24 substitution, which reduces the size of the hydrophobic pit at the llama V_HH surface. We demonstrate that it is possible to improve desired characteristics of the same V_HH fragment simultaneously using DNA shuffling. Finally, this is one of the first examples of DNA shuffling improving temperature stability of an antibody fragment.     

Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): two complementary techniques for enzyme evolution

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled, and reduces the regeneration of unshuffled parental genes. This shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G+C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space.     

Digestion-after-shuffling: a method for combinatorial library construction

We have developed a convenient method for family shuffling of amino acid sequences, termed digestion-after-shuffling. After DNA shuffling of homologous genes, plasmid mixture is extracted from a library and used for several double digestions with restriction enzymes. For each double digestion, two restriction enzymes are selected, corresponding to the single restriction sites of different parental genes. After digestions, fragments with expected sizes are obtained by gel purification and religated to construct recombinant plasmids. Thus, the obtained genes should be chimeras and have at least two restriction sites originating from different parental sequences.     

A shuffled CYP2C library with a high degree of structural integrity and functional versatility

Cytochrome P450 (CYP) enzymes involved in mammalian xenobiotic metabolism are attractive targets for the engineering of biocatalysts since they have broad and overlapping substrate and reaction substrate specificities. In this report, a library of chimeric mutants was prepared from CYP2C8, CYP2C9, CYP2C18 and CYP2C19 by DNA family shuffling. Twelve randomly selected clones were fully sequenced and showed 9 ± 2 crossovers and 1.5 ± 0.5 spontaneous mutations per ∼1.5 kbp open reading frame. CYP hemoprotein expression was observed in 50% (microaerobic culture) to 54% (aerobic culture) of clones. The functional diversity of the library was assessed using three luminogenic substrates, diclofenac and indole as probe substrates. A random sample of 26 clones revealed two clones with activity towards luciferin ME, one towards luciferin H and five towards diclofenac 4′-hydroxylation. One mutant showed activity towards all three substrates. Of 96 clones screened on solid media, one showed elevated indigo production compared to the parental forms. Turnover rates for luciferin ME and H metabolism by CYP2C9 and mutants were at least one order of magnitude higher in experiments with membranes compared to whole cells, consistent with impaired product egress from cells. Apparent K_m values were increased in whole cell incubations with luciferin H suggesting impaired access of the substrate to the active site of the enzymes in whole cells. Finally screening with a panel of CYP2C ligands using CYP2C9 or active mutants revealed different patterns of inhibition and heteroactivation of metabolism of luciferin analogs.     

Versatile capacity of shuffled cytochrome P450s for dye production

DNA family shuffling is a relatively new method of directed evolution used to create novel enzymes in order to improve their existing properties or to develop new features. This method of evolution in vitro has one basic requirement: a high similarity of initial parental sequences. Cytochrome P450 enzymes are relatively well conserved in their amino acid sequences. Members of the same family can have more than 40% of sequence identity at the protein level and are therefore good candidates for DNA family shuffling. These xenobiotic-metabolising enzymes have an ability to metabolise a wide range of chemicals and produce a variety of products including blue pigments such as indigo. By applying the specifically designed DNA family shuffling approach, catalytic properties of cytochrome P450 enzymes were further extended in the chimeric progeny to include a new range of blue colour formations. This mini-review evokes the possibility of exploiting directed evolution of cytochrome P450s and the novel enzymes created by DNA family shuffling for the production of new dyes.     

Aflatoxin B1-4-hydroxylase is associated with cytochrome P3-450 in C57BL/6 mouse liver

Messenger RNA from the livers of Aroclor 1254 treated mice was used to produce a cDNA library. cDNA clones corresponding to cytochromes P1-450 and P3-450 were isolated from this library by screening with a probe for the rat cytochrome P-450c gene. Specific non-cross hybridizing probes for P1-450 and P3-450 were prepared from unique restriction fragments. The radiolabeled probes were hybridized to RNA from mice treated with a low (15 mg/kg) and high (150 mg/kg, 400 mg/kg) doses of beta-naphthoflavone. The low dose of beta-naphthoflavone was found to induce only P3-450 mRNA, whereas higher doses induced both P1-450 and P3-450 mRNA. Similarly, a low dose of beta-naphthoflavone induced aflatoxin B1-4-hydroxylase, whereas higher doses induced both aflatoxin B1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. These results suggest that P3-450 mRNA codes for the cytochrome that is associated with aflatoxin B1-4-hydroxylase activity.     

Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling

Evolutionary protein engineering is now proceeding to a new stage in which novel technologies, besides the conventional point mutations, to generate a library of proteins, are required. In this context, a novel method for shuffling and rearranging DNA blocks (leading to protein libraries) is reported. A cycle of processes for producing combinatorial diversity was devised and designated Y-ligation-based block shuffling (YLBS). Methodological refinement was made by applying it to the shuffling of module-sized and amino acid-sized blocks. Running three cycles of YLBS with module-sized GFP blocks resulted in a high diversity of an eight-block shuffled library. Partial shuffling of the central four blocks of GFP was performed to obtain in-effect shuffled protein, resulting in an intact arrangement. Shuffling of amino acid monomer-sized blocks by YLBS was also performed and a diversity of more than 10(10) shuffled molecules was attained. The deletion problems encountered during these experiments were shown to be solved by additional measures which tame type IIS restriction enzymes. The frequency of appearance of each block was skewed but was within a permissible range. Therefore, YLBS is the first general method for generating a huge diversity of shuffled proteins, recombining domains, exons and modules with ease.     

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.     

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.     

The use of random chimeragenesis to study structure/function properties of rat and human P450c17

The microsomal 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) catalyzes the 17alpha-hydroxylase reaction required to produce cortisol, the major glucocorticoid in many species and the 17,20-lyase activity required for the production of androgens in all species. Utilizing the technique of random chimeragenesis we have attempted to map regions of primary sequence that contribute to the species-specific biochemical differences between rat and human P450c17. We have previously reported significant differences between rat and human P450c17 in their activities, stability and substrate-dependent coupling efficiencies even though they share 68% amino acid identity. Identification of the regions of primary sequence that contribute to each of these properties would be helpful in understanding the structure/function relationships in this enzyme. A single plasmid containing the cDNAs encoding both enzymes in a tandem orientation was constructed. This plasmid was linearized at unique restriction sites and used to transform Escherichia coli. A three-step screening protocol identified five chimeras with a uniform distribution of 5' rat and 3' human sequence. All chimeric proteins yield the characteristic reduced-CO difference spectra, indicating proper folding. The chimeras exhibit a range of stability and activities that are not consistent with the degree of parental primary sequence. A chimera containing 301 N-terminal rat P450c17 amino acids and lacking the rat P450c17 phenylalanine 343, had the highest lyase activity. Generation of these functional rat/human chimeras suggests that the tertiary structures of rat and human P450c17 are sufficiently conserved to allow proper folding of chimeric enzymes. However, the properties of these chimeras did not permit identification of a region of primary sequence that contributes to a species-specific property of rat and human P450c17. Stability of these chimeras and insight into the presence of secondary structural elements is discussed.     

Assignment of the functional gene for human adrenodoxin to chromosome 11q13----qter and of adrenodoxin pseudogenes to chromosome 20cen----q13.1.

Adrenodoxin is a small iron/sulfur protein serving as an electron-transport intermediate for all mitochondrial forms of cytochrome P450. Southern blots of normal genomic DNA cleaved with six restriction endonucleases probed with full-length human adrenodoxin cDNA revealed complex patterns indicating the presence of multiple adrenodoxin genes. Southern blots of DNA from a panel of mouse/human somatic cell hybrids identified cross-hybridizing adrenodoxin DNA in two loci, chromosome 11q13----qter and chromosome 20cen----q13.1. Examination of adrenodoxin clones from a genomic DNA library in phage lambda revealed some clones bearing gene fragments interrupted by introns and other clones bearing processed pseudogenes. By probing the mouse/human hybrids with unique intronic DNA and by correlating restriction maps of the phage clones with that of uncloned genomic DNA, we show that the authentic transcribed adrenodoxin gene lies on chromosome 11, while pseudogenes lie on chromosome 20.     

Breeding of retroviruses by DNA shuffling for improved stability and processing yields

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.     

Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification

A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling.     

Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions

We have constructed, in the yeast Saccharomyces cerevisiae, a mosaic assembly of genes by in vivo recombination of partially homologous sequences. The approach was tested on cDNAs encoding functionally distinct mammalian cytochromes P-450 (P-450). The selection for recombinant cDNAs used the transformation of yeast cells, which required the recircularization of a linearized plasmid by recombination of two partially homologous cDNAs. Libraries of mosaic genes with bipartite or tripartite structures were generated by intramolecular and intermolecular recombination events. The presence of yeast promoter and terminator sequences on the flanking sides of the recombined cDNAs has allowed the synthesis of encoded mosaic proteins. A library of yeast clones producing recombinant mouse P-450 P1 and rabbit P-450 LM4 was screened using functional criteria to identify chimeras with shuffled substrate specificity. Restriction mapping of mosaic genes, biochemical analysis of the synthesized proteins, comparison of chimeric enzymes, and the alignment of sequences with bacterial P-450 camphor hydroxylase of known three-dimensional structure, all suggest that the P-450 P1 amino acid residues 203-238 play a major role in the control of cytochrome activity toward carcinogenic polycyclic aromatic hydrocarbons. Similar approaches to structure-function analysis are believed to be applicable to other protein families.     

DNA shuffling of subgenomic sequences of subtilisin.

DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.