[Construction of high-quality gene mutant pool in Pichia pastoris by a PCR dependent method]

We developed a method to construct a gene mutant pool in Pichia pastoris based on in vivo homologous recombination. It was an absolute PCR-dependent method (PDM) and could avoid the disadvantages of traditional mutant pool construction process such as long-experimental period, low pool capacity and inadequate abundance. The method consisted of four steps: (1) construction of recombinant expression plasmid of target gene; (2) design of long primers that have 40-70 bp of homology to expression vector fragments at both ends and amplification of target gene by error-prone PCR, DNA Shuffling or other methods; (3) PCR amplification of expression vectors fragments; (4) mixture of gene and vectors by appropriate mole ratio, electroporation, formation of expression cassette in vivo, homologous recombination with host genome and achievement of mutant pool. Screening from this library, we obtained mutants with improved enzyme activity, protein expression level and thermostability. In conclusion, PDM was very efficient and convenient with advantages of shortened pool construction cycle from 2 weeks to 3 days, enlarged pool capacity from the original 10(3)-10(4) to more than 10(5), with a positive rate of more than 95%.