Breeding Of Monascus Spp With High Monacolin K Production Via Agrobacterium-mediated Transformation And Protoplast Fusion

One of cholesterol synthesis inhibitor, Monacolin K, was found in Monascus fermented products. It is the same substance as lovastatin in Aspergillus terreus. It inhibits the activity of hydroxymethyglutaryl coenzyme A reductase, which catalyzes the rate-limiting step in cholesterol biosynthesis and regulate abnormal blood liqid in organism. Over the last 30 years, many Monascus spp such as M.purpureus, M.albidus and M.pilosus were found to produce Monacolin K. Although the ability of synthesizing Monacolin K by Monascus spp degenerated with the generation passing on, and yield of Monacolin K from Monascus spp is less than A. terreus, but with its edibility, Monacolin K produced from Monascus spp seems more acceptable than from A. terreus. Constant selection and breeding of Monascus spp are necessary. In order to investigate the related gene in Monacolin K synthesis pathway and find out a simple and effective method to screen strains of Monascus spp with high Monacolin K production, the approach of combinated techniques with gene cloning and protoplast fusion may be a feasible choice.The aim of this work was to develop the Agrobacterium tumefaciens-mediated transformation (ATMT) method in Monascus spp, and transfer the T-DNA with genetic marker into Monascus spp, expect the insertion of T-DNA would change the host DNA sequence arrangement and affect the growth and metabolism of Monascus transformants, and then the Monascus transformants with high Monanolin K could be selected and analysed. The flanking sequence of T-DNA from these transformants will be amplified to find out the related Monacolin K biosynthetic gene. Monascus fusants with enhanced Monacolin K production could be screened via proroplast fusion between parental strains with gene hph and the ability of Monacolin K production respectively, the fermentation conditions by Monascus fusants during solid-state fermentation will be optimized, and provide finally some data for industrialization of Monacolin K production.The related results are as following:(1) The binary vector pCAMBIA 3300-gpdA-hph-trpC was constructed and transformed into Agrobacterium tumefaciens GV3101. The exogenous gene gpdA-hph-trpC was transformed into M. albidus by ATMT. Various influencing factors on transformation efficiency were optimized including the concentration of antibiotics, concentration of M. albidus spores, cell density of A. tumefaciens, co-cultivation time, temperature and the concentration of acetosyringone. The highest transformation frequency was about 5.2×104 . Some of the transformants were selected for PCR analysis, and the results further confirmed that the transformants tested were arbitrarily integrated with exogenous T-DNA in genome. Among many transformants, one transformant H1 with high Monacolin K production was selected, from which the flanking sequence mkWL (0.88 kb) of T-DNA by inverse PCR was obtained. The result of homology between the gene mkWL and Monacolin K biosynthetic gene cluster from M. albidus in the Genbank was analyzed, which revealed that there was identity of 97% at DNA sequence homology. The gene mkWL was deduced to be the related gene in Monacolin K biosynthetic pathway of M. albidus. (2) Main factors such as mycelium age, osmotic stabilizer, different enzymatic combinations, duration of enzyme treatment and regeneration medium for efficient protoplast formation and regeneration from transformant H1 and M. fumeus 9908 were investigated. Monascus fusants HH1 with high Monacolin K production were selected via protoplast fusion between tansformant H1 and M. fumeus 9908, and the concentration of Monacolin K in fermented millet by fusant HH1 increased by 404% and 27% compared with that produced by parental Monascus spp.(3) The fermentation conditions for promotion Monacolin K production by solid-state fermentation were optimized, and the Monacolin K yield by fusant HH1 reached to 17.50 mg/g after fermentation for 20 d. The response surface methodology of Box-Behnken was applied to further optimize the fermentation conditions including water volume, pH of water and inoculant volume. A model was constructed, and further experiments were carried out to verify the accuracy and reliability of the model, the results showed that the predicted value was agreed well to the experimental value.The gene mkWL related to Monacolin K biosynthesis in M. albidus was amplified, and Monascus fusants with high Monacolin K production were bred via Agrobacterium -mediated transformation and protoplast fusion, these works laid a good foundation for genetic modification and industrialization of Monacolin K fermentation of M. albidus.