Codon optimization enables the Zeocin resistance marker's use in the ascomycete yeast Debaryomyces occidentalis.

applicability of Zeocin resistance means that a single selectable marker can be used for several different cell types.Native and commercial Sh ble genes (Zeocin resistance genes) have high G+C compositions (69.9%), but our study showed that the native Sh ble gene was not functionally expressed in D. occidentalis.We speculated that the codon usage in the Zeocin resistance gene was not appropriate for D. occidentalis.Codon optimization is known to improve the expression of heterologous proteins (Gustafsson et al., 2004).A gene's sequence can influence protein expression in a number of ways, including affecting messenger RNA (mRNA) levels, translational velocities, initiation efficiencies, and consumption rates of charged tRNA (Gustafsson et al., 2012).In this study, we optimized the codon use in the Zeocin marker for expression in D. occidentalis as part of a useful transformation system.A codon-optimized Zeocin resistance gene (Zeo/opt) was synthesized and subcloned in pUC57 using GenScript (GenScript, Piscataway, NJ, USA).The resulting plasmid was pUC57-Zeo/opt.The codons were designed based on the D. occidentalis codon usage database (http:// www.kazusa.or.jp/codon/).The GC content of the coding region of the native Zeocin was 69.9% GC, while the GC content of D. occidentalis was 35.8%.Using codons optimized for D. occidentalis, the GC content of the modified Zeocin resistance gene (accession number LC164249) was 41.9%.The alignments of nucleotide sequences and codon usage rates of amino acids are shown in Fig. S1 and Table S1, respectively.The CTG codon was not found in the modified Zeocin resistance gene for D. occidentalis.However, some CTG codons were found in the native Zeocin resistance gene (Table S1).DebaryomycesCodon optimization enables the Zeocin resistance marker's use in the ascomycete yeast Debaryomyces occidentalis