Genetic Transformation System From Monascus, Cloning And Identification Of PKS Gene For The Biosynthesis Of Citrinin And Monacolin K

Monascus, the important industrial filamentous fungi, is considered to be a mine for exploitation pharmaceutical because of abundant polyketide in its fermentation products. To investigate polyketide synthetic pathway of Monascus and regulation of relative pathway, research works below were fulfilled.To establish protoplast-mediated genetic transformation system of Monascus sp., conditions for the protoplast isolation and regeneration of the mycelia of various enzymes and osmotic stabilizers were examined. To investigate suitable cell age for the protoplast preparation of mycelia of M. purpureus, the mycelia were cultured in different ways at 30oC. Mycelia obtained through cellophane-mediated culture for 30-40 h were adequate to for protoplast preparation. When lysing enzyme, cellulase and snailase were added to the mycelia in combination or alone, combination of lysing enzyme, cellulase and snailase at the concentration of 0.3% lysing enzyme, 0.1% cellulose and 1% snailase respectively was most benefit for protoplast yield. When we applied various osmotic stabilizers at different concentration on the protoplasts preparation, 1 mol/L MgSO4 was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5-hr. When we investigate various osmotic stabilizers for the regeneration of the protoplasts of mycelia of strain M34 and N18, the complete medium containing 0.6 mol/L sucrose induced highest hyphal growth with regeneration frequency of 8.5% and 36.4%, respectively.In order to facilitate the producer of polyketide pathway, four different transformation methods were tested and compared in an attempt to develop the genetic transformation system of Monascus sp. Using vector pBC-Hygro, the fungus was transformed to be hygromycin B-resistant, by conventional transformation as well as electroporation based on protoplast, electroporation based on germinated conidia, and restriction enzyme-mediated integration (REMI). Electroporation based on germinated conidia was found to be inappropriate for transforming Monascus sp. due to a low transformation frequency. The conventional transformation and electroporation technique based on protoplasts were thought not to be fit for transforming Monascus sp., due to a low stability of transformants though they yielded up to 135 transformants and 125 transformants per microgramme DNA, respectively. Transformant number was increased by 20-fold by REMI (2500 transformants per microgramme DNA) and 70%~75%of them were stable. REMItechnique would be very beneficial to the establishment of the genetic transformation system of Monascus sp..We isolated and cloned pksCTl andpksCT2 gene. Then they were sequenced and analysed. The pksCTl was identified to be the gene coding for a key ketidesynthase of citrinin synthetic pathway by REMI-mediated disrupting pksCTl gene of Monascus rubber. While not producing citrinin genetically engineered Monascus was constructed.We Isolated and cloned monKl gene. Then they were sequenced and analysed. The monKl was identified to be the gene coding for nonaketidesynthase by REMI-mediated disrupting monkl gene of Monascus rubber.