Breeding Of Monascus Spp With High Monacolin K Production By Interspecific Protoplast Fusion Of Inactivated Parental Strains

As fermentated food, the application of Monascus has a very long history in China. The study found that, Monacolin K found in Monascus fermented products and lovastatin in Aspergillus terreus are the same substance.It inhibits the activity of hydroxymethyglutaryl coenzyme A reductase,which catalyzes the rate-limiting step in cholesterol biosynthesis.Yield of Monacolin K by Monascus spp is less than Aspergillus terreus,but with its safity on food , there is a realistic significance to improve the level of Monacolin K production. the level of Monacolin K production can be raised by breeding of Monascus spp with high Monacolin K production and improving fermentation conditions.The aim of this work was to breeding of Monascus spp with high Monacolin K production by interspecific protoplast fusion of inactivated parental strains. Monascus albidus H2 and Monascus fumeus 9908 were used as Parents. M.albidus H2 with hygromycin resistance gene was acquired by Agrobacterium-mediated transformation and had high Monacolin K production by liquid state fermentation with glycerol as main carbon source, but had low pigment production; M. fumeus 9908 with high pigment production can convert rice into Monacolin K in solid state fermentation. The protoplasts of parents were inactivated by heat and ultraviolet respectively, and fused with PEG as fusion agent. Fusants with hygromycin resistance gene were selected for further fermentation and compared with parental strains,fusant with high production of Monacolin K and pigment was obtained. The related results:(1) The improved condition of formaiton and regeneration of protoplasts from Monascus fumeus 9908 and Monascus albidus H2: 1.0 mol/L MgS04 was used as osmotic stabilizer; the optimal mycelium age were 26 h and 24 h respectively; the best cellulase concentration was 1%;the duration of enzyme treatment were 3.5 h and 4 h; the regeneration medium was PDA with 0.6 mol/L sucrose . The protoplasts could reach 106个/mL,and the regeneration rate were 80%and 25% respectively.The protoplasts of M. albidus H2 were inactivated by heat, treated 25 min in 65℃water bath; the protoplasts of M. fumeus 9908 were inactivated by ultraviolet,treated 15 min below the UV light(15 W) with 10 cm.The fusion rate reached 5.63%.(2) Fusants with hygromycin resistance gene were selected for further fermentation, Monascus fusant13-2 with high Monacolin K production was selected by screening, the Monacolin K yield was 1752.46 mg/L, increased by 32.98%, compared with M. albidus H2,its pigment production was between two parental strains'. the stability testing found that, hygromycin resistance gene could be inherited stably,and the level of Monacolin K production in liquid state fermentation was stable. (3)Main characters of fusant such as colony,color of fermental liquid and morphology of mycelia and spore were investigated,and found that M. albidus H2 was the main parent of fusant13-2,and fusant13-2 obtained the good character of high pigment production from M. fumeus 9908.PCR test showed that the hygromycin resistance gene was successfully and stably integrated on the chromosome DNA of fusant13-2.(4) As a main carbon source,glycerol played an important role in the production of Monacolin K,when the initial glycerol concentration was 10%,the level of Monacolin K production was high.The glycerol utilization ratio of fusant13-2 was 93.8%,which was higher than parental strains.