Cloning And Functional Analysis Of The PigE Gene In Monascus Purpureus

Monascus pigments,a kind of natural pigments found in the secondary metabolites of Monascus fermentation,had diverse biological activities and were widely used as colorants in food,cosmetics and health-care products.Researchers were generally focused on improving the yield of Monascus pigmnents while reducing citrinin content in the industrial production recently.This study on pigment synthesis related gene will provide a reference for the construction of genetic engineering strain with high yield of Monascus pigments and low production of citrinin.In this paper,The pigE gene related to pigment metabolism was cloned from the genome DNA of Monascus purpurse M-21 strain.We successfully knocked out the PigE gene and obtained the PigE deletion strain(?PigE)based on the homologous recombination principle.The changes of growth,metabolism and PigE gene expression in ?PigE strain were analyzed.Finally,Monascus pigments were also extracted to investigate the stability and antimicrobial activity.The main contents were illustrated as following:1 Screening and identification of Monascus with high yield of piggments and low production of citrininThe wild-type strain library of Monascus spp.,including 238 Monascus strains which were isolated and purified from different kinds of Monascus products,was constructed.The yield of pigments and citrinin was measured by spectrophotometry and HPLC respectively.Through comparative analysis,The Monascus strain M-21 which the Monascus pigments production up to 284.71 U/g while the citrinin concentration was only 1.1 mg/L was finally obtained.The identification results based on ITS gene sequences showed that it was Monascus purpureus.2 Cloning and deletion of PigEPigE gene,which was about 1200 bp full-length and encoded a protein of 342 amino acids,was cloned from the genome DNA of M.purpurse M-21 strain.According to the homologous recombination principle,We constructed a gene knockout vector and transformed it into Monascus strain by Agrobacterium tumefaciens-mediated transformation for the Monascus transformants,in which the hygromycin resistance gene(hph)was used as screening marker gene.Finally,The?PigE strain was identified from the transformant library which contained 116 Monascus transformants by hph resistance plate,PCR and Southern blot methods.3 Growth and metabolism analysis of ?PigE strainCompared with the wild-type strain,The ?PigE strain had little changes in the microstructure characteristics and growth rates,while colony morphologies changed significantly.The deletion of PigE gene mainly caused the changes of Monascus pigments in variety and yield.The wild-type strain produced two kinds of Monascus pigments,and the red pigment was dominant.However,the PigE-deleted mutant only produced yellow pigment which was up to 1050 U/g and about 1.84-fold production as that in wild type strain after fermentation for 13 d in rice medium.The production period of citrinin was also delayed and the yield was reduced to 1.57 mg/L after fermentation for 13 d in YES liquid medium.The expression level of PigE in ?PigE strain could not be detected by RT-PCR on account of the deletion of PigE gene.4 Analysis of Monascus pigments in stability and antimicrobial activityThe pigments in M.purpurse M-21 and ?PigE strain were extracted for the analysis of stability and antimicrobial activity.The analysis results showed that Monascus pigments were stability when the pH is 7-9 at 60?.The oxidizing agent NaClO4 and the reducing agent Na2SO3 had little effect on the stability of Monascus pigments.Compared with Mg2+,Ca2+,K+ and Cu2+,Monascus pigments were more stable in extract liquid containing Na+.The antimicrobial activity of Monascus pigments in four types of bacteria and six kinds of fungi was also detected.As for antibacterial activity,Red pigment had week antibacterial activity in Bacillus subtilis,while yellow pigment in Staphylococcus aureus and Bacillus subtilis.As for antifungal activity,Red and yellow pigments all had different levels of antifungal activity in Fusarium oxysporum f.sp.cucumerinum,Alternaria solani,Fusarium graminearum,Fusarium solani and Penicillium oxalicum and it was especially obvious in Alternaria solani strain.