Construction And Functional Analysis Of The ACS And GMC Gene Disruption Mutants In Monascus Aurantiacus AS3.4384

The filamentous fungus Monascus, one of the important microorganism resources of China, has a versatile ability to produce different kinds of secondary metabolites including antibiotics, anticancer drugs, and pigments. However, due to citrinin contamination during the fermentation process, it is called in question about food safety evaluation of Monascus fermentation.In this paper, the functions of two genes were preliminary researched by molecular biotechnology, one was coding Acyl-CoA synthetase, the other was glucose-methanol-choline oxidoreductase coding gene, in order to provide basic theory support on elucidating the citrinin biosynthetic pathway. The major findings were expounded as follows:1 Two gene sequences were analyzed by blast, ACS gene were putatively deduced to coding Acyl-CoA synthetase, and GMC gene were deduced to coding glucose-methanol-choline oxidoreductase. They might be relative with the citrinin biosynthesis.2 Two replacement vectors, pACS-HPH and pGMC-HPH contained hygromycin B resistance selected marker, were constructed by the DNA recombinant technique such as: restriction enzyme cutting and ligase ligation and transformation.3 Two replacement vectors, pACS-HPH and pGMC-HPH, were respectively transformed into protoplast of M. aurantiacus AS3.4384 to disrupt ACS gene and GMC gene. Then, methods of hygromycin resistance and genomic-PCR were used to screen disruptants. Two Monascus mutants, ACS-6 and GMC-8 were gained.4 The production, citrinin and red pigment of Monascus mutants ACS-6 and GMC-8, were detected by HPLC to research the biological function of ACS gene and GMC gene. According to the results,ACS gene might be responsible for decenoyl-CoA and tetraketide synthesis. GMC gene was took part in the PKS-modifying of citrinin and red pigment biosynthetic pathways.