The Effect Of Precursors Based On Metabolic Pathway On Mevastatin Biosynthesis

By the competitive inhibition with HMG-CoA(3-hydroxy-3-methylglutaryl-coenzyme-A) reducase,statins effectively lower the synthesis of endogenous cholesterol and add compensatively the Synthesis of low density lipoprotein(LDL)in organic dells,improve the uptake of LDL in blood and reduce the contents of total cholesterol.It is tabulated,as the optimal drugs for curing hypercholesterolemia,at the top of the list of global best-sale drugs.Mevastatin is the earlies found statins materials.At the processes of looking for the structural analogs,lovastatin,the HMG-CoA reductase inhabitor medicne as the first one that was appoved by FDA and put in the market,was found.It is also the direct substrate ofpravastatin,so mevastatin has also a nonnegligible important relation to the development of statins and occupies the market as biotransformation substrates,raw materials all the time and additive despite of the fact that it isn't finally developed as medicine.The promulgation of mevastatin biosynthesis gene clusters and putative biosynthesis pathway reveals that mevastatin is a typical polyketide compound,which provides new ideas and foundations to improve biosynthesis yields.Proved by a large number of experiments,fitting precursors can effectively increase the yields of the polyketide compounds.This thesis focuses on the morphology and molecular identification of a fungus IMBM21 whose fermentation product is suspected as mevastatin characterized by TLC,UV scanning,HPLC and LC-MS,and researches on the basic conditions of culturing IMBM21 after the product is confirmed to be mevastatin.According to the metabolic pathway of mevastatin biosynthesis, short-chain fatty acid,soybean oil,TCA intermediate and amino acids are selected as precursors with different metabolic pathway in microbe vital movement to study the impact of precursors on mevastatin biosynthesis,the main secondary metabolite produced by IMBM21.The thesis aims at finding the fitting precursors for mevastatin industrial production and provides foundational research information for further research on metabolism regulation of IMBM21 strain.As the culure time goes by,the color of IMBM21 colony changes from orange into white with evident short thin white hairs covering on its surface drape.The mycelium is short,thick and septa, and spores can be found.Strain Identification is performed by means of the ITS conserved sequence of fungus genome and the PCR amplified sequence is compared in NCBI after sequenced.The results display that the fungus has close relationship with Penicillium echinulatum.The product identification has nearly the same Rf value(TLC),wave pattern(UV),retension time(HPLC) and spectroscopy feature(MS)as reference mevastatin,which proves that mevastatin is main secondary metabolite of IMBM21.Short-chain fatty acids are biogens of polyketide,which are direct precursors when mevastatin biosynthesis.The experiments studies on acetate,propionate and butyrate,and results demonstrate that propionate can increasethe specific productivity of IMBM21 strain biosynthesis into mevastatin to 79.85%when the addition quantity of propionate is 0.010%at 132h.After soybean oil,the representative of long-chain fatty acid,is added into medium,is firstly decomposed by thalli into glycerol and different length fatty acids and then starts the mevastatin biosynthesis.As precursors of biosynthesis,fatty acids are sure influences in biosynthesis.Adding of glycerol has no significant effect on the yield of mevastatin but obvious on thalli biomass.So the experiment of adding soybean oil appear that the specific productivity can be improved to 33.35% comparing with the control on the condition of adding 0.30%soybean oil at 120h.The main effective materials in soybean oil metabolite are short-chain fatty acids.TCA cycle is the fundamental of material and energy metabolites in microbe vital movement.It could influences carbon and energy metabolite and studies the source of carbon in mevastatin biosynthesis by adding TCA intermediate.The experiment studying on the main fermentation parameters of citric acid,trisodium citrate,ferric citrate,pyruvic acid and succinic acid,shows that trisodium citrate can remarkably improve the specific productivity to 83.83%when adding 0.05%of it at 72h.Nine familiar amino acids adding in two respective concentrations are as follows:Asp,Glu,Leu, lie,Lys,Met,Phe,Thr and Val.Their comparison with the related parameters of mevastatin biosynthesis demonstrates that pH value is increased apparently at the process of biosynthesis and changes the whole thalli culture environment into weak basic condition.Asp,Lys,Leu,Met and Phe can improve thalli biomass;Lys and Leu begin to inhibit the growth of thalli when the concentration is 0.10%.Asp,Glu,Ile,Met,Thr and Val can all increase the amounts of mevastatin biosynthesis and present an ascending trend except Met.Above-mentioned results demonstrate that precursors selected on the base of metabolic pathway have the pertinence effects on the pathway of mevastatin biosynthesis and the 4 precursors can all be found precursor materials--sodium propionate,soybean oil and trisodium citrate and most of common arninophenol—to improve effectively mevastatin biosynthesis and provide efficient and cheap precursors for mevastatin's industrial production.