Selection of Error-Less Synthetic Genes in Yeast

Conventional gene synthesis is usually accompanied by sequence errors, which are often deletions derived from chemically synthesized oligonucleotides. Such deletions lead to frame shifts and mostly result in premature translational terminations. Therefore, in-frame fusion of a marker gene to the downstream of a synthetic gene is an effective strategy to select for frame-shift-free synthetic genes. Functional expression of fused marker genes indicates that synthetic genes are translated without premature termination, i.e., error-less synthetic genes. A recently developed nonhomologous end joining (NHEJ)-mediated DNA cloning method in the yeast Kluyveromyces marxianus is suitable for the selection of frame-shift-free synthetic genes. Transformation and NHEJ-mediated in-frame joining of a synthetic gene with a selection marker gene enables colony formation of only the yeast cells containing synthetic genes without premature termination. This method increased selection frequency of error-less synthetic genes by 3- to 12-fold.