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To investigate the relationship between starch hydrolysis and Monascuspigments (MPs) production, the α-amylase gene (AOamyA) from Aspergillus oryzaewas heterologously expressed in Monascus ruberCICC41233, and we obtained a positive transformant named Monascus ruberAmy9. In M. ruberAmy9, the α-amylase activities were 6.65- and 4.26-fold higher at 72 h and 144 h, respectively, than those in the parent strain with the glucose as solo carbon medium. Surprisingly, in the MPs fermentation medium with rice powder as solo material, M. ruberAmy9 completely degraded starch at 48 h, while 43.93 and 7.29 mg/mL starch remained at 48 and 144 h, respectively, in the parent strain. Monascus ruberAmy9 accelerated starch hydrolysis, which enhanced biomass and also increased total MPs by 132% after 144 h. Compared with M. ruberCICC41233, the relative gene expression levels, as determined by a quantitative real-time polymerase chain reaction analysis, of acl2encoding ATP-citrate lyase subunit 2, pksencoding polyketide synthase, and fasBencoding the fatty acid synthase beta subunit increased by 33.14, 145.18, and 32.15%, respectively, after 144 h in M. ruberAmy9. The up-regulated expression of these key genes in MPs synthesis contributed to the large increase in MPs production. This interesting work provided us with a new idea and a new target for the study of the MPs production.

Previously, AoamyA,the alpha amylase-encoding gene from Aspergillus oryzae,was heterologously expressed in Monascus ruberCICC41233 to promote starch hydrolysis and increase the production of Monascuspigments. The target of this study is to screen the effective alpha-amylases from M. ruberfor starch fast degradation and investigated for Monascuspigments production. The 13 types of predicted alpha-amylases in the M. ruberNRRL1597 genome were divided into four classes based on EC number and into five groups based on the glycoside hydrolase sub-family. The predicted alpha-amylases MrAMY1 (protein ID 440333) and MrAMY2 (protein ID 324551) showed the closest match with AOamyA by phylogenetic analysis. The genes encoding alpha-amylase, Mramy1and Mramy2,were cloned from M. ruberCICC41233. However, the gene sequence of Mramy1from M. ruberCICC41233 differed from that of M.ruberNRRL1597 in the length of the intron sequence. Furthermore, the Mramy1-overexpressed strain M.ruber440333-6A completely degraded the starch of rice grain in 2 d; in contrast, starch (40.32 mg/mL) remained when rice grain was incubated with the Mramy2-overexpressed strain, M. ruber324551-D even after 2 d, while 45.43 mg/mL and 10.48 mg/mL starch remained after 2 d and 6 d, respectively, in wild type M. ruberCICC41233. Compared to that of M. ruberCICC41233, the total Monascuspigments and ethanol-soluble pigments in M.ruber440333-6A increased by 71.69% and 119.33% after 6d, respectively; however, it decreased by 21.40%and 26.58% after 6d, respectively, in M. ruber324551-D. This study demonstrated that alpha-amylase MrAMY1 was superior to MrAMY2, as it effectively degraded the starch of rice grain and enhanced Monascuspigments production.