Abstract
In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE) was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200–500 bp fragments with 20–25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp) and Aspergillus niger phytase gene phyA (1404 bp). Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.
Highlights
In vitro chemical synthesis of long DNA sequences is the foundation of synthetic biology
Gene design Because of the significant difference in codon usage bias between R. oryzae, A. niger and P. pastoris, the usage frequency of most of the codon what R. oryzae lipase gene (ROL) and PhyA genes encoded are less frequently used in P. pastoris (Fig S1 and S2)
This step was similar to the general one-step assembly PCR gene synthesis method described by Stemmer et al [5]
Summary
In vitro chemical synthesis of long DNA sequences is the foundation of synthetic biology It was widely used in diverse fields, including codon optimization and in vitro functional evaluation of gene, nucleic acid immunity and gene chip preparation, etc. The method for synthesis and assembly of DNA sequences based on oligonucleotides was first described by Agarwal and co-workers [4]. According to their description, the gene synthesis was a typical enzymatic ligation which included 1) chemical synthesis of oligonucleotides, 2) 5’-end phosphorylating the oligonucleotides by T4 polynucleotide kinase, and 3) ligating the oligonucleotides into the full length gene by T4 ligase. To facilitate the design and assemble of the oligonucleotides, softwares such as DNAWorks [7], Gene2Oligo [8], and GeMS [9] were developed to make all the oligonucleotides thermodynamically unity
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