Rice (Oryza sativa L.) is one of the major grains of the world and an important energy source for humans. Rice grains contain functional ingredients such as γ-aminobutyric acid, tocophenols, tocotrienols, and γ-oryzanol that are beneficial to human health (Ng et al. 2013, Goufo et al. 2017). Rice breeding and mutation studies are ongoing to improve the quality and yield of rice.

Oryzamutaic acids were isolated and identified as novel compounds from the mutant cultivar Hatsuyamabuki with yellow endosperm. This mutant was obtained by gamma irradiation of the rice cultivar Kinuhikari (Kaji et al. 2010). Oryzamutaic acids A–K, and the related alkaloids oryzadiamine A–C, have been isolated and structurally determined (Nakano et al., 2009, Nakano et al., 2010a, Nakano et al., 2010b, Nakano et al., 2020a, Nakano et al., 2020b). Oryzamutaic acids A–C are yellow in color, and oryzamutaic acids A and D are mainly present in Hatsuyamabuki (Nakano et al. 2011). However, because oryzamutaic acids comprise many molecular species, present at trace amounts, they are difficult to purify, and their functionality has not been evaluated.

In the jmw rice mutant, which accumulates oryzamutaic acid A, an aldehyde dehydrogenase (ALDH7) was reported to be responsible for synthesis (Shen et al. 2012). ALDHs are NAD(P)+-dependent enzymes that oxidize aldehydes to acetate. At least 22 families of ALDHs have been reported depending on protein sequence. In plants, 12 families have been reported (Kirch et al., 2004, Gao and Han, 2009). The ALDH7 family is highly conserved in animals and plants, and some members contribute to lysine metabolism (Brocker et al. 2010). Rice ALDH7 was reportedly involved in seed maturation and viability (Shin et al. 2009). The accumulation of oryzamutaic acid A is believed to be due to a functional defect in ALDH7, preventing the conversion of α-aminoadipate-δ-semialdehyde—an intermediate in L-lysine metabolism—to adipic acid. However, the mechanism of oryzamutaic acid biosynthesis in vivo is unclear.

In this study, we focused on Agrobacterium tumefaciens-derived L-lysine ε-dehydrogenase (LysDH) to develop a simple method for the enzymatic synthesis of oryzamutaic acids in vitro. LysDH catalyzes the oxidative deamination of L-lysine in the presence of NAD+ to generate α-aminoadipate-δ-semialdehyde. We cloned and expressed a large amount of LysDH in Escherichia coli to examine its activity. Furthermore, by evaluating the reaction conditions using this enzyme, we synthesized oryzamutaic acid H in vitro for the first time.