As a first screen, 25 clones were randomly picked, and colony PCRs were performed. In all 25 cases, full-length genes in the expected size range from 1250–1300 bp were obtained as PCR products. The chimeric genes differed slightly in length, since individual domains, depending on their respective origin, also varied in size. Prior to the development of PTRec, we expected that the repair of 10 DNA nicks by E. coli after transformation might become a critical step for PTRec, since hybridization and subsequent repair of 12 nicks proved to be challenging in the past (29). We assume that enzymatic fragment generation might not have been complete prior to hybridization and transformation, thereby causing reduced efficiencies in the past. In PTRec, the chemically generated 12-nucleotide overhangs were stable to transformation conditions, and the E. coli host BL21(DE3) closed the 10 nicked DNA positions without causing frameshifts or mutations at the crossover points. Encouraged by these results, 42 clones were randomly picked and fully sequenced. We consider this number of clones to be large enough to determine the efficacy of PTRec for DNA recombination. Figure 2 shows the diverse pattern of chimeric phytase genes. All 42 genes contained the five expected domains in the correct order, demonstrating an efficient assembly through oriented hybridization. No attempt was made to screen the generated chimeric library for activity, because the study was intended to develop PTRec and to evaluate its performance as an universal method for recombining DNA fragments of distantly related genes. In order to benchmark PTRec, the number of fragments that can be assembled, the number of crossover points, and number of genes successfully recombined were determined. The distribution of individual fragments in the 42 clones was analyzed in detail. In none of the sequenced clones was a domain missing or occurring more than once. No preferential occurrence of any DNA fragment of the three phytase genes could be detected during analyses of the domains 1, 2, 4, and 5 (domain 1: appA, 18; ympH, 12; phyX, 12; domain 2: appA, 19; ympH, 13; phyX, 10; domain 4: appA, 14; ympH, 14; phyX, 14; domain 5: appA, 14; ympH, 15; phyX, 13). Only phyX domain 3 can be regarded as being underrepresented (domain 3: appA, 21; ympH, 14; phyX, 7) in the library, since this domain was identified only seven times. From 42 phytase genes sequenced, only five were not recombined (3× appA and 2× ympH). This can likely be attributed to incomplete DpnI digestion of template DNA prior to chemical cleavage. In addition, two identical chimeric phytase genes occurred twice. The theoretical size of the chimeric phytase library comprises 243 combinations (5 domains of 3 genes: 35), and thus screening of 729 clones (three times oversampling) is already sufficient to explore 95% of all possible combinations (30). Interestingly, no mutations were observed around the five crossover sites (+/-50 nucleotides), which would reduce the overall quality of the chimeric library. Altogether three transition mutations could be identified within the chimeric genes, which corresponds to the error-rate of 2.2 × 10-5 of the used Taq DNA polymerase.
DNA recombination is a powerful tool for the laboratory evolution of proteins, and various methods were developed for generating such genetically diverse libraries (9). Table 1 summarizes the most advanced methods for nonhomologous gene recombination. Approximately one decade ago, the methods ITCHY (15), SCRATCHY (16), and SHIPREC (17) allowing random recombination of two genes with one to three crossover points were developed. The limitation regarding the number of crossover points shifted the attention to the development of methods with rationally guided selection of crossover points. Application of SISDC (18), OE-PCR (20, 21), or USERec (10) allows recombination with up to nine crossover points (Table 1). However, enzymatic steps involved in fragment generation, ligation, and final PCR assembly limit the efficiency of nonhomologous recombination methods.
Compared with the above mentioned nonhomologous recombination methods, PTRec is unique, since enzymatic steps during fragment generation are avoided, and neither a ligation step nor a final assembly PCR is required. Key to DNA fragment assembly in PTRec is the efficient and specific chemical cleavage of multiple phosphorothioate diester bonds generating single-stranded 12-nucleotide overhangs for hybridization. Any DNA purification prior to hybridization and transformation can be skipped, since iodine and ethanol do not interfere with hybridization of complementary single-stranded DNA (24). Therefore, the time requirement for recombination experiments with PTRec is significantly reduced compared with all other nonhomologous recombination methods. In addition, avoiding inefficient enzymatic steps during fragment generation and eliminating ligation and PCR assembly steps reduces the possibility of problems during library generation (24). A further advantage of PTRec is the minimal sequence requirement at each crossover point; a stretch of four amino acids that is identical among the proteins is sufficient to define a crossover point (Supplementary Figure S1). Differences in gene sequences can therefore be compensated by incorporation of silent mutations in the primers used in step 1, which lowers sequence requirements at individual crossover points on the nucleotide level even more. In 19 cases, incorporation of silent mutations in the PCR primers was necessary to ensure complementary single-stranded 5'-overhangs for efficient hybridization (Supplementary Table S1). Due to these minimal requirements, PTRec should enable researchers to use this method to recombine even more distantly related or even unrelated proteins, possibly leading to proteins with new functions. Moreover, due to the modular nature of the PCR-based fragment generation in step 1, PTRec allows generation of chimeric sublibraries; the number of crossover points can be adjusted by simply changing primer combinations. For example, if it turned out that it was better for domain 2 and domain 3 to originate from the same gene in order to yield functional proteins in the previous recombination experiments, the forward primer of domain 2 and the reverse primer of domain 3 are used during fragment generation in step 1 to omit the crossover point between these two domains.
In summary, due to its simplicity in handling, high recombination efficiency, and flexibility, the PTRec method represents an innovative and powerful technique to generate high-quality chimeric libraries in a modular manner.