The Effect Of Mycelial Morphology And Histone Modification On Lovastatin Biosynthsis In Aspergillus Terreus

Aspergillus terreus is the main producer of lovastatin,a cholesterol-lowering drug.The efficiency of using this fungus to synthesize lovastatin can be influenced by many factors,such as strain properties,fermentation conditions and physiological and biochemical characteristics of fungi during fermentation.The investigation on the relationship between mycelial morphologies and lovastatin synthesis efficiency in the process of fermentation,as well as the relationship between the expression efficiency of histone-modifying enzymes and lovastatin synthesis efficiency,will be helpful for improving the efficiency of lovastatin synthesis by Aspergillus terreus,and has an important prospect of application for the production of lovastatin in industrial fermentation.In this study,in order to study the effect of the mycelial morphology on lovastatin biosynthesis in Aspergillus terreus,the inoculum size was lowered by the dilution of seed culture using sterile water,with the aim to change the mycelial morphology by the change in inoculum size.Spores with a total number of 108 were transferred into 30 ml seed medium to grow for 48 hours,and then the seed culture was diluted by sterile water to different levels at the inoculum size of 1% 、 0.1% 、 0.01%,while the 10% was the normal group.The experimental results showed that at the inoculum size of 10%,the hyphae were dispersed;in the lower inoculum size groups,pellets were formed,and the diameter of the pellet was larger with the less inoculum;the diameter of mycelium pellets were on average 368.8±87.7,541.3±100.7,809.0±260.1(μm)respectively.To determine the most optimal mycelial pellet diameter for lovastatin production,the seed culture was diluted to 21-210 times by gradient,and 10 groups of smaller gradient inoculum amount were set to obtain pellets with different diameters.The experimental results showed that when the seed solution was diluted to 28 with sterile water,the inoculum size was 0.078%,and the yield of lovastatin was the highest,970.5μg/ml,which was 5.37 times higher than that of 10%(184.1 g/ml),and the diameter of pellets was 582.4μm on average.Then while the inoculum amounts were 0.156% and 0.039%,and the concentrations of lovastatin were 893.7μg/ml and 897.0μg/ml,and the average diameter were 406.4μm and 607.4μm,respectively.Therefore,the optimal pellet size for lovastatin production in Aspergillus terreus was considered to be 400-600μm.For the investigation into its internal structure,the pellets of different diameters were cut into 12-μm slices by the frozen sectioning technique.The structure showed that the density of mycelium in pellets with a diameter of 600μm was relatively uniform and high;while hollow rings appeared in the center of pellets with diameters greater than 1500μm,and mycelium was looser in pellets with a diameter of 300μm.The phenomenon of promoting lovastatin synthesis by the formation of pellets in Aspergillus terreus could be reasonably explained by quorum sensing.Pellets caused a great increase in the density of part of mycelium,which could trigger quorum sensing to increase the production of a signaling molecule(Butyrolactone I),and then the molecule could enhance lovastatin and other secondary metabolites synthesis.The concentration of a signal molecule Butyrolactone I in the fermentation culture with inoculums of 0.1% and 10% was determined.The results showed that the concentration of Butyrolactone I in fermentation fluid with the inoculum of0.1% was about 18 times that of inoculums of 10%.Thus the interpretation proposed in this work can be verified.In addition,to investigate the effect of histone modification on lovastatin synthesis,a gene encoding histone methyltransferase in Aspergillus terreus ATCC 20542 was successfully cloned.The 1000 bp and 500 bp DNA fragments on the gene encoding glyceraldehyde3-phosphate dehydrogenase were used as promoter and terminator to construct overexpression and RNA interference expression cassettes.The overexpression histone methyltransferase Rmt A plasmids were transformed to protoplast by PEG-mediated transformation method,and8 transformants were selected and 3 of them were identified to be positive transformants.These positive transformants were cultivated for submerged fermentation,and then the yield of lovastatin was measured.The result showed that the yield of lovastatin of engineered strain OE-Rmt A-3 was more than 50% higher than the parent strain,which indicated that histone methyltransferase Rmt A can up-regulate the production of lovastatin.In this work,it was found that filamentous mycelium can be transformed into pellets by dilution of seed culture with sterile water.The dense structure of pellets could trigger quorumsensing and significantly increase the yield of lovastatin.The efficiency of lovastatin synthesis could be effectively improved by improving the expression efficiency of histone methyltransferase,through the efficient constitutive expression cassette.These results provide a theoretical basis for improving the production efficiency of lovastatin.