The enzymatic digestibility of rice grains is a crucial property affecting the efficiency of the starch-to-alcohol conversion during shochu and sake brewing. However, the effects of the genetic background of rice on its enzymatic digestibility have not been clarified. To bridge this gap, we probed the relationships among starch biosynthesis genes, starch structure, and the enzymatic digestibility of steamed rice during brewing. In shochu and sake koji digestion tests, the digestibility of the Alk/Wx^a genotype was lower than that of the alk/Wx^b genotype. The four examined genotypes (Alk/Wx^a, alk/Wx^a, alk/Wx^b, and alk/wx) differed in terms of the digestibility of steamed rice under commercial-enzyme, shochu koji, and sake koji conditions, with digestibility following the order of alk/wx > alk/Wx^b > alk/Wx^a > Alk/Wx^a. The retrogradation rate of steamed rice under sake conditions followed the order of alk/wx < alk/Wx^b < alk/Wx^a < Alk/Wx^a. Digestibility showed positive and negative correlations with the amylopectin short-chain ratio and amylose content, respectively. Differential scanning calorimetry analysis indicated that crystalline amylopectin in the higher-crystallinity and higher-gelatinization-temperature Alk variety was not completely gelatinized by steaming, which resulted in reduced digestibility. Starch biosynthesis genotype (Alk/Wx) influenced the amylopectin short-chain ratio and amylose content, which resulted in a diversity of the enzymatic digestibility of steamed rice under shochu and sake production conditions. This study provides fundamental insights for selecting or breeding rice cultivars for shochu and sake brewing.

Lytic polysaccharide monooxygenases (LPMOs) are activated by electrons supplied from external redox enzymes. Cellobiose dehydrogenase (CDH) and pyrroloquinoline quinone (PQQ)-dependent pyranose dehydrogenase (PDH) are electron donors to LPMOs. CDH and PDH each consist of two domains: a catalytic domain that oxidizes substrates to extract electrons, and an AA8 domain that receives and transfers these electrons to LPMOs. Although non-catalytic AA8 proteins comprising only an AA8 domain and a cellulose-binding module have been reported, their electron donors and physiological roles remain largely unclear. In this study, we characterized a protein from Armillaria tabescens (syn. Desarmillaria tabescens) that lacks a catalytic domain but includes both an AA8 domain and a cellulose-specific binding module, CBM104, which we designated AtAA8-CBM104. Electron-transfer assays demonstrated that the AA8 domain can accept electrons from an external AA12 catalytic domain, strongly suggesting that AtAA8-CBM104 act as an electron mediator transferring electrons from redox partners such as AA12 to adjacent LPMOs. Further, adsorption experiments showed that the CBM104 domain binds to crystalline cellulose but with lower adsorption efficiency than a previously characterized CBM104 appended to LPMO9. This observation suggests that despite AtAA8-CBM104 localization on cellulose being necessary for electron transfer to cellulose-bound LPMO9, excessive retention on the cellulose surface may interfere with LPMO9 catalysis. Furthermore, phylogenetic analysis indicated that AA8 appended to CBM104 lacking catalytic domains share similar electrostatic properties. These findings advance our understanding of non-catalytic AA8 proteins, underscoring their potential involvement in fungal redox networks linked to cellulose decomposition.