Construction Of Monascus Transformant Library,Screening High-Yielding Pigment And Low Citrinin Mutants And Analysis Of Fermented Products

Monascus spp. has been widely applied for a long time in China. In recent years, the effective physiological active substances produced by Monascus been extensively investigated. Such as:Monascus pigment, Monacolins, gamma-aminobutyric Acid (GABA) and so on have been repeatedly studied, Therefore increasingly more health value and medicinal function about Monascus was found, widely applied in pharmaceutical and food industry. But in molecular biology, the study of the Monascus was not so much.This topic from red yeast rice by screening gained a Monascus strain of Mp-41 as the basis of the experimental materials, and due to our laboratory has already set up the effective transformation system, building agrobacterium mediated transformation system, establishing the transformation library, molecular verification at the same time. Subsequently, under deep observation into the biological characteristics of transformants, gaining some transformants different from the original strain. Through the research on the Monascus pigment and citrinin, selecting a high-yield pigment strain but lower citrinin. And HPLC used for the analysis of products of original strains and this strain. The main results of research are as follows:1. Using the method of Agrobacterium tumefaciens building Monascus Mp-41 mutant libraryReferring to the available agrobacterium mediated transformation system: Monascus was cultured on PDA medium cultivation for 20 d, concentration of Monascus spores was 106/mL, optical density value at 600 nm of Agrobacterium was 0.5, concentration of inducer AS was 100 μmol/L, agrobacterium and Monascus spores on the NC membrane were co-cultured for 3 d at 25 ℃. More than 800 transformants were amplified under this condition in the transformation library,2. Genetic stability analysis, PCR identification and morphological observation of Monascus Mp-41 transformants60 transformants were cultivated on the PDA medium containing 200 μg/mL hygromycin B, to study the genetic stability of the transformation. As control, the original strain Mp-41 with the same. The results revealed that the genetic stability reached 98.6%, only one transformant cannot grow. Meanwhile, detection of gene hph in transformants was performed through PCR analysis. The results show that almost all the examined transformants kept the resistance, and what’s more, the PCR identification confirmed that the T-DNA has successfully integrated into transformants genome. The transformants diversified markedly from the parental strain were screened from more than 800 transformants, which cultured for 7d on PDA medium at 30 ℃ (original strains Monascus Mp-41 as a control). The results showed that most transformants were the same with the original, however, there were some transformants’cleistothecia, hypha and conidia had significantly changed.3. Selection of high yield pigment and lower citrinin from transformantsCitrinin and color value of pigment in liquid and solid produced by 9 tranformants were measured by thin-layer chromatography (TLC), ultraviolet-visible scanning method. The results were as follows. Color value of pigment of 9 mutants were higher than Mp-41; Concentration of citrinin of 3 belonging to 9 mutants were relatively lower; So we finally chose Mp-41-347 as our candidate strain.347# was the higest color value up to 51.024 U/mL in liquid state and was 31.712 times that of Mp-41; In solid state, the color value up to 1435.0 U/g and was 2 times that of Mp-41.4. Analyzing the fermentation products of original strain and strain Mp-41-347HPLC were used on the mensuration of citrinin and Monacolin K produced by 347# in liquid fermentation.The results showed that the original strains and 347# had the similar growth period,347# relatively grew faster, the dry weight of the maximum was 20.650 g/L, comparatively, original strains’maximum dry weight was 15.260 g/L. In the fermentation process, color value of pigment of 347# was up to 51.423 U/mL, was 183.96% times than the original strain (Coloe value of pigment was 27.962 U/mL). Maximum citrinin of 347# was 0.743 mg/L, only 50.70% of the maximum citrinin of original strains. Monacolin K. of 347# was 17.675 mg/L, Monacolin K content of the original in liquid fermentation was 32.716 mg/L, a little higher than the original strain. Thus in can be seen that the 347# was superior to the original in quality.