Effects Of RNA Interference On Metabolic Repressor CreA On Aspergillus Terreus Producing Lovastatin

Lovastatin was formally applied to cardiovascular and cerebrovascular diseases in clinical in 1987.Lovastatin is become one of the most commonly used drugs for the treatment of cardiovascular and cerebrovascular diseases.By the methods of optimizing the basic parameters of fermentation,regulating the metabolism,screening high-yield strains and developing new fermentation methods etc.The production of lovastatin in Aspergillus terreened has increased 75 times.In order to improve the yield of lovastatin of Aspergillus terreus and reduce the effect of Cre A on lovastatin biosynthesis,the si RNA interference system was successfully established in Aspergillus terreus.The effect of Cre A on lovastatin synthesis was studied by RNAi technique.In this work,an effective expression system was first constructed in A.terreus ATCC20542.Using the p UC19 plasmid as the skeleton,the expression cassette p UCACE was constructed by the promoter and terminator of the constitutive expression gene pdc of A.terreus.The red fluorescent protein gene Ds Red was inserted into p UCACE and the expression cassette p UC-ACE-Ds Red was transformed into A.terreus,in which the expression cassette would integrated into the genome.After cultivation of the recombinant strain,the morphology of the mycelium was clearly observed and the red fluorescence was distributed on the whole mycelium,indicating that the transformant could stably express the red fluorescence.This result proved that the expression vector p UC-ACE could be used for the gene expression in A.terreus.The interference fragment of creA gene in A.terreus was inserted into p UC-ACE,resulting in the interfering expression cassette of creA gene,p UC-ACE-si RNA,and this cassette was transformed into A.terreus,resulting in the recombinant strain A.terreusi Cre A,in which the cassttee p UC-ACE-si RNA integrated into the genome.The transformant was cultivated in lovastatin fermentation medium,and the gene expression levels and the productivity of lovastatin were analyzed.The results of realtime quantitative PCR revealed that the expression level of creA was decreased gradually with cultivation time,and the yield of lovastatin increased,as revealed by the result analyzed with HPLC.In the recombinant strain,the expression level of creA was significantly lower,and the productivity of lovastatin was significantly higher than those of the control strain,the expression level of creA gene decreased 63% and the production of lovastatin increased 22.59% after 7 days of fermentation,compared with the control strain.These showed the method for knock-down of creA gene expression through RNA interference,proved the role creA gene of negative regulation of Cre A on lovastatin production,and proposed a new approach to improve lovastatin productivity through lowering the expression level of creA.