Effect Of Mphy Gene Overexpression On Monascus Pigment Metabolism And Its Application

Light is an important signal factor and natural energy in the environment,which can affect the growth and development,physiological cycle,reproduction and various metabolic activities of fungi.The photochromophores and other luminescent chromophores are the core substances of fungi for light signal sensing.Photochromes are a class of photoreceptor proteins based on pyrrole derivatives and responsive to red and far-red light.In this paper,the gene sequence homologous to plant phytochrome was found from Monascus ruber C100 by comparing with the genome.In this paper,M.ruber C100 was taken as the research object to systematically study the effects of phytochrome genes on the growth and development of Monascus and Monascus production.RT-q PCR was used to explore the relative expression levels of key genes related to pigment synthesis and further analyze the function of photosensitive genes.By studying the overexpression of phytochrome,we found that phytochrome,as a red light sensor,inhibited its development under red light.At the same time,liquid and solid fermentation of Monascus showed that the color value of the overexpressed strain increased under red lightThe main results are as follows:(1)Cloning of phytochrome geneThe photochrome gene was cloned from M.ruber C100 genome by using the designed specific primers.The sequencing results showed that the gene was 3963 bp in length,and the comparison analysis predicted that the gene encoded 1292 amino acids,including six functional domains: PAS,GAF,PHY,His KA,Hatppase-C and RRD.The photochrome overexpression vector was constructed and introduced into Monascus genome through agrobacterium-mediated transformation.A photochrome overexpression strain OE-Mphy was screened and verified.(2)Growth and development analysis of overexpressed strain OE-MphyOE-Mphy overexpressed strain was cultured on different media under different culture conditions and characterized and analyzed.Microscopic analysis of OE-Mphy overexpressed strain was conducted by fluorescence microscope.The results showed that:The mycelium morphology of OE-Mphy overexpressed strain cultured under different conditions was different,and the number of spores in its obturator sheath was lower than that of the original strain M.ruber C100.Meanwhile,the mycelium were filled with more metabolites,and the overexpressed strain OE-Mphy promoted the formation of metabolites.The results showed that the gene of photochrome was related to the growth,development and reproduction of Monascus.(3)Analysis of Monascus pigment produced by fermentation of overexpressed strain OE-MphyThe production of Monascus pigment of overexpressed strain OE-Mphy under different conditions of solid and liquid fermentation was determined by UV-VIS.The results showed that the production of red,orange and yellow pigment of overexpressed strain OE-Mphy reached the highest under dark conditions and natural light on the 9th day of liquid fermentation.In solid state fermentation,the metabolism of Monascus pigment,the main secondary product of Monascus,was accelerated under red light,and the pigment yield reached the maximum on the 13 th day of fermentation,and the value of red,orange and yellow pigment was significantly increased compared with that of M.ruber C100,in which R2,Y1,O1 and O2 increased by more than 60%.The total color value of pigment reached 6868U/g,33.3% higher than that of M.ruber C100,86.2% higher than that of OE-Mphy under natural light.HPLC was used to determine the Monascus pigments components of overexpressed strain OE-Mphy in PDB liquid fermentation and rice solid fermentation,and six main Monascus pigments were found in overexpressed strain OE-Mphy,knockout strain W7 and original strain M.ruber C100: Monascorubramine(R-1),Rubropunctamine(R-2),Monascin(Y-1),Ankaflavin(Y-2),Monascorubrin(Monascorubrin),Rubropunctamine(R-2),Monascin(Y-1),Ankaflavin(Y-2).O-1),Rubropunctatin(O-2),and two other red pigments R-3 and R-4,and the molecular weights of R-3 and R-4 were 397 D and 425 D respectively by mass spectrometry.In conclusion,OE-Mphy overexpressing strain significantly improved the total yield of Monascus pigment under the condition of 100 Lux red light intensity,12 h light time and 12 h dark culture.(4)Analysis of OE-Mphy gene expression of overexpressed strainThe phytochrome gene has a significant effect on pigment metabolism,so the influence of phytochrome gene on pigment synthesis and metabolism was studied,and four key Monascus pigment genes were analyzed by RT-q PCR.The results showed that under the condition of 12 h red light irradiation and 100 Lux per day,the dark culture for 12 h could promote the secondary metabolism of Monascus.Under the condition of red light,Monascus pigment metabolism could be accelerated,while under the same condition,blue light had an inhibitory effect.