High‐level production of yeast ( Schwanniomyces occidentalis ) phytase in transgenic rice plants by a combination of signal sequence and codon modification of the phytase gene

Summary This study was designed to produce yeast ( Schwanniomyces occidentalis ) phytase in rice with a view to future applications in the animal feed industry. To achieve high‐level production, chimeric genes with the secretory signal sequence of the rice chitinase‐3 gene were constructed using either the original full‐length or N‐truncated yeast phytase gene, or a modified gene whose codon usage was changed to be more similar to that of rice, and then introduced into rice ( Oryza sativa L.). When the original phytase genes were used, the phytase activity in the leaves of transgenic rice was of the same level as in wild‐type plants, whose mean value was 0.039 U/g fresh weight (g‐FW) (1 U of activity was defined as 1 µmol P released per min at 37 °C). In contrast, the enzyme activity was increased markedly when codon‐modified phytase genes were introduced: up to 4.6 U/g‐FW of leaves for full‐length codon‐modified phytase, and 10.6 U/g‐FW for truncated codon‐modified phytase. A decrease in the optimum temperature and thermal stability was observed in the truncated heterologous enzyme, suggesting that the N‐terminal region plays an important role in enzymatic properties. In contrast, the optimum temperature and pH of full‐length heterologous phytase were indistinguishable from those of the benchmark yeast phytase, although the heterologous enzyme was less glycosylated. Full‐length heterologous phytase in leaf extract showed extreme stability. These results indicate that codon modification, combined with the use of a secretory signal sequence, can be used to produce substantial amounts of yeast phytase, and possibly any phytases from various organisms, in an active and stable form.