Revealing biases inherent in recombination protocols.

BACKGROUND: The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing. RESULTS: We developed different test systems to compare and reveal biases from DNA shuffling and recombination-dependent PCR (RD-PCR), a StEP-like recombination protocol. An assay based on the reactivation of beta-lactamase was developed to simulate the recombination of point mutations. Both protocols performed similarly here, with slight advantages for RD-PCR. However, clear differences in the performance of the recombination protocols were observed when applied to homologous genes of varying DNA identities. Most importantly, the recombination-dependent PCR showed a less pronounced bias of the crossovers in regions with high sequence identity. We discovered that template variations, including engineered terminal truncations, have significant influence on the position of the crossovers in the recombination-dependent PCR. In comparison, DNA shuffling can produce higher crossover numbers, while the recombination-dependent PCR frequently results in one crossover. Lastly, DNA shuffling and recombination-dependent PCR both produce counter-productive variants such as parental sequences and have chimeras that are over-represented in a library, respectively. Lastly, only RD-PCR yielded chimeras in the low homology situation of GFP/mRFP (45% DNA identity level). CONCLUSION: By comparing different recombination scenarios, this study expands on existing recombination knowledge and sheds new light on known biases, which should improve library-creation efforts. It could be shown that the recombination-dependent PCR is an easy to perform alternative to DNA shuffling.

Improvement of a CrtO-type of beta-carotene ketolase for canthaxanthin production in Methylomonas sp.

Two types of non-homologous beta-carotene ketolases (CrtW and CrtO) were previously described. We report improvement of a CrtO-type of beta-carotene ketolase for canthaxanthin production in a methylotrophic bacterium, Methylomonas sp. 16a, which could use the C1 substrate (methane or methanol) as sole carbon and energy source. The crtO gene from Rhodococcus erythropolis was improved for canthaxanthin production in an E. coli strain engineered to produce high titer carotenoids by error-prone PCR mutagenesis followed by in vitro recombination. The best mutants from protein engineering could produce approximately 90% of total carotenoids as canthaxanthin in the high titer E. coli strain compared to approximately 20% canthaxanthin produced by the starting gene. Canthaxanthin production in Methylomonas was also significantly improved to approximately 50% of total carotenoids by the mutant genes. Further improvement of canthaxanthin production to approximately 93% in Methylomonas was achieved by increased expression of the best mutant gene. Some mutations were found in many of the improved genes, suggesting that these sites, and possibly the regions around these sites, were important for improving the crtO's activity for canthaxanthin production.