| Monascus species are widely used to produce natural colorants and functional food additives all over the world, especially in Asian countries. The Monascus pigments have been proved to possess beneficial functions such as anti-inflammation, anti-diabetes, anti-atherosclerosis and anti-cancer activities. Many other beneficial secondary metabolites, such as monacolins K, y-aminobutyric acid (GABA) have many beneficial properties including hypolipidemic effect, antihypertensive effect, anti-oxidation, anti-cancer and immunomodulation. Despites of the beneficial products mentioned above, Monascus species also have potential hazard owing to contain the mycotoxin-citrinin, which greatly limits the wide application of the Monascus-related products. So far, the molecular biology information associated with the secondary metabolites of Monascus and its regulation mechanism is lack, there is no good guidance for industrial fermentation production of pigment, so it is important to reveal the mechanism of metabolic regulation of Monascus at molecule level. In order to screen the genes that related to pigment synthesis, in this study, we chose the low pigment producing condition nd high pigment producing condition of a Monascus purpureus strain to perform a comparative proteomic experiment through two-dimensional gel electrophoresis (2-DE) and verify the genes through gene knocking out experiments.In this study, six kinds of carbon source (rice, fructose, sucrose, starch, glucose and maltose) were selected to reveal the capacity of pigment producing of a M. purpureus strain. This strain in rice medium generated highest pigments yields, and then followed by fructose, glucose, starch and maltose. In sucrose, this M. purpureus synthesized lowest amount of pigment. By comparison of mediums and pigment yields, starch and sucrose were selected as the comparative mediums. We selected the fermented products on eighth day to compare proteomes of this M. purpureus in two different medium. At the eighth day, the three kinds of pigments (red pigments, orange pigments and yellow pigments) yields in starch medium were up to 3442.25 U/g、3148.13 U/g、3703.43 U/g and in sucrose were up to 555.49 U/g、 396.66 U/g、1001.28 U/g, respectively. Two-dimensional gel electrophoresis (2-DE) were used to discriminate the different proteins of this M. purpureus in the two medium and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS) was performed to identify the proteins. Seventy two different protein spots could be observed clearly on the 2-DE gels. Among these spots,64 proteins were successfully identified and 11 proteins were speculated as potential pigment-related proteins.In order to verify the propertity of the protein in pigment biosynthesis, the genes c5.123 (salicylate hydroxylase) and c5.133 (Aflatoxin Bl aldehyde reductase) were selected to carry out gene knocking out experiments. Primers were designed according gene sequence and were used to amplify fragments upstream and downstream to the target genes. Then, recombinant DNA technology was employed to ligate the two fragments into the two hygromycin gene ends on pUCH2-8 vector which was used to obtain fragments containing long flanking regions that can be used to improve knocking efficiency. The length of upsteam fragment and downsteam fragment of c5.123 were 1091 bp and 1278 bp. The upstream region and downstream region of c5.133 were 1262 bp and 1229 bp. The recombinant pUCH2-8 vectors were transformed into the protoplast of M. purpureus through the chemical method. But unfortunately, the gene silenced mutants were not obtained.This study explores the influence of different carbon sources on the M. purpureus pigments yields. Eleven pigment-related proteins were identified through comparative proteomic approach between two fermented conditions.This study lays a good foundation on the research of pigment biosynthetic pathway in Monascus species. |
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