Phosphorothioate-base dDNA recombination method (PTRec) is a ligase-and restriction site-independent approach for recombining secondary structure elements, motifs or domains of proteins. PTRec comprises only four simple steps including a highly efficient chemical cleavage of phosphorothioated nucleotides to generate 12-nucleotide long overhangs in double-stranded DNA with subsequent hybridization in a one-pot assembly. As proof of principle, five domains of three phytases (sequence identity: 45%–53%; 15 fragments) were recombined in a combinatorial manner with four crossover points. PTRec is “almost” homology-independent, not limited by the number of genes to recombine, and enables immediate and efficient cloning of the resulting chimeras.
Rational guided generation of protein chimeras has developed into an attractive approach in protein engineering for tailoring catalytic and biophysical properties of enzymes. Combinatorial recombination of structural elements or whole protein domains is still technically challenging due to sequence dependent biases diminishing the overall quality of resulting chimeric libraries. Since methods for generating such libraries are often limited by a low frequency of crossover points and suffer from challenges in handling, we developed the phosphorothioate-based DNA recombination method (PTRec). PTRec is an enzyme-free method and only requires a short stretch of four amino acids that is identical among the proteins to be recombined in order to define a single crossover point. In a PTRec-generated chimeric library that shuffled five domains of phytase using genes from three different species, 88% of 42 randomly picked and sequenced genes were efficiently recombined. Furthermore, PTRec is a technically simple, fast, and reliable method that can be used for domain-and exon-shuffling or recombination of DNA fragments in general.
Directed evolution mimics natural evolution through screening or selection with the aim of generating genes that encode for proteins with new or improved functional traits (1). Experiments in directed evolution involve iterative cycles of diversification on the gene level (2) followed by screening or selection for the best variant on the protein level (3). Many different methods have been developed to generate rational guided or random mutant libraries (2, 4-7), usually targeting only a single gene. DNA recombination strategies differ from directed protein evolution of single genes by generating chimeras through rational or random recombination of at least two genes. On the protein level, secondary structure elements, motifs, or domains preselected for function are mixed and used for the generation of new proteins to screen or select from (8). Many methods for DNA recombination have been developed and applied in protein engineering campaigns (9). These methods can be grouped into homologous and nonhomologous strategies (10): homology-dependent recombination strategies such as DNA-shuffling (11), staggered extension process (StEP) (12), random priming (13), or random chimeragenesis of transient templates (RACHITT) (14) rely on the principle of hybridization and extension of homologous DNA fragments during PCRs. Hence, methods for homologous recombination require at least 70% sequence identity on the gene level. Homology-independent recombination strategies generate hybrid proteins of more distant related genes by using truncation-based methods such as incremental truncation for the creation of hybrid enzymes [ITCHY (15), SCRATCHY (16)] and sequence homology-independent protein recombination (SHIPREC) (17). Two genes were recombined in a single experiment as proof of concept in the latter methods. The sequence-independent site-directed chimeragenesis (SISDC) (18) method was reported to be able to recombine more than two genes, relying on the site-directed incorporation of marker tags that later have to be removed by BaeI-restriction. Degenerate oligonucleotide gene shuffling (DOGS) (19) makes use of degenerate primers, which allow control over relative levels of recombination between the genes that are recombined, but requires endonucleases for cloning of chimeric libraries. Due to its simplicity, overlap extension PCR (OE-PCR) (20, 21) is widely used and requires at least 15 overlapping nucleotides between individual DNA-fragments that have to be assembled in a final PCR step. Recently, USER friendly DNA recombination (USERec) (10) has been introduced claiming “unique advantages in comparison to alternative recombination strategies,” for instance by overcoming sequence constraints with the usage of a 5'-AN4–8T-3' motif at crossover sites. USERec relies on the activity of multiple enzymes: uracyl DNA glycosylase, endonuclease VIII (together termed “USER enzyme”) (22), T4 DNA ligase, and requires a final PCR amplification step to generate large quantities of the recombined library. Homology-independent methods for DNA recombination have in common that they require enzymatic activities after DNA amplification/purification to generate the desired libraries. Additionally, a final PCR amplification step is often needed to amplify the chimeric library before transformation.
Here we report the phosphorothioate-based DNA recombination method (PTRec) as a ligase-and restriction site-independent method for recombining secondary structure elements, motifs or domains of proteins. PTRec is based on phosphorothioate chemistry, which allows site-specific cleavage of phosphorothiodiester bonds in phosphorothioate oligonucleotides in the presence of ethanol and iodine in an alkaline solution (23). PTRec starts with PCR amplification of the target DNA fragments and vector backbone by PCR using primers with complementary phosphorothioate nucleotides at their 5'-end. The PCR products are cleaved in an iodine/ethanol solution at elevated temperatures producing single-stranded overhangs. Subsequently, these complementary ends hybridize at room temperature, and the resulting DNA constructs can be directly transformed into competent host cells. As proof of principle, PTRec was used to recombine five domains of three different phytases (amino acid sequence identity: 45%–53%) by using four crossover points in the targeted proteins. Only a stretch of four amino acids that is identical among the proteins is required to define a crossover point, making PTRec almost independent from sequence constraints. PTRec is not limited by the number of genes to be recombined and enables immediate and efficient cloning of the resulting chimeric library. PTRec was developed after extensive optimization of DNA cleavage and hybridization conditions using the recently published phosphorothioate-based ligase-independent gene cloning (PLICing) method (24) and the OmniChange method (25), which were developed as a DNA fusion technology for cloning random mutant libraries of single genes and a method for the simultaneous site saturation mutagenesis of five codons in a single protein, respectively.