Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification

Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IFS) method consists of two steps. The first one is the synthesis of 300-400 bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15 bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256 bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR). (C) 2010 Elsevier B.V. All rights reserved.