Study Of Fermentation Strategy For Enhancing MPA Production And Metabolic Engineering

Mycophenolic acid(MPA),produced by Penicillium brevicompactum,is an antibiotic with diverse biological properties,including antifungal,antineoplastic,and immunosuppressive activity.In the 21st century,MPA is a reversible inhibitor of hypoxanthine single-nucleotide dehydrogenase and shows selective inhibition of lymphocyte activation.Mycophenolate mofetil(MMF),a 2-ethyl ester derivative of MPA,is a new generation of immunosuppressant.It shows prospects for clinical organ transplantation and autoimmune disease treatment.Therefore,it is of great importance to establish a high-efficient production process for MPA fermentation enterprises.This study has conducted a series of researches in view of enhancing yield of MPA produced by Penicillium brevicompactum ATCC16024 in liquid fermentation environment.We verified the law of scale-up of stirred-tank fermenter to improve the production performance of industrial strains by genetic modification,which could be as reference for engineering practices.(1)Optimization of initial medium for MPA fermentationIn this study,the initial medium for MPA production was optimized by the Plackett-Burman design and response surface methodology.The optimized fermentation medium comprised(g/L):glucose,93.4;glycine,13.4;methionine,0.5;KH2PO4,2.0;MgSO4·7H2O,and trace element solution.The MPA concentration reached 1.72 g/L with the optimized medium in a 7 L fermenter.The MPA production was enhanced by 63.8%compared with the initial medium.(2)A combined feeding strategy for enhancing MPA production by fed-batch fermentation in P.brevicompactumWe investigated a combined feeding strategy for enhancing MPA production by fed-batch fermentation in P.brevicompactum.In a 7 L fermenter,the relevant parameters of CN-feeding strategy(F-CN),pH shift strategy(F-pH),and Met-feeding strategy(F-Met)were inspected and analyzed during the synthesis phase of MPA fermentation after 120 h.Compared to the fed-batch fermentation,the three strategies improved the MPA production to 2.23,1.92,and 2.03 g/L,increased by 29.65,11.63,and 18.02%,respectively.Based on the above results,a combined strategy(F-CPM)was designed.Under F-CPM strategy,the maximum dry cell weight(DCWmax)reached 45.77 g/L at 240 h,and the maximum MPA production(Pmax)reached a higher level of 2.68 g/L,increased by 55.81%.The combined feeding strategy can effectively reduce the impact of substrate inhibition and improve MPA productivity.Thus,our work offered a new idea for industrialization of MPA fermentation with conventional industrial equipment.(3)Cloning and expression of vgb gene in P.Brevicompactum by ATMTWe developed a novel recombinant AV3.Agrobacterium tumefaciens-mediates transformation(ATMT)was applied for expressing the Vitreoscilla Hemoglobin gene(vgb)in P.Brevicompactum ATCC160224.In this study,the effects of cloning vgb gene in ATCC16024 on the oxygen utilization and the MPA production were studied.The results showed that vgb gene was successfully inserted into the transformant genome,verifying by hygromycin-resistance selection,PCR and fermentation experiments.The growth rate and cell density of transformants were higher than control.The MPA production reached 2.18 g/L,increased by 27.5%than the host strain.(4)Insertion mutation in HMG-CoA lyase increases the production yield of MPA through ATMTWe studied on the genes of key enzymes in the metabolic pathway of MPA biosynthesis.One of the key enzymes ?-hydroxy-?-methylglutaryl-CoA(HMG-CoA)lyase in the bypass metabolic pathway was chosen to modified metabolic pathways.The inhibitory activity of HMG-CoA lyase increases MPA biosynthetic flux by reducing the generation of by-products.In this study,we cloned the P.brevicompactum HMG-CoA lyase gene using TAIL-PCR and gene walking technology.ATMT technology was used to insert a mutated HMG-CoA lyase gene into P.brevicompactum.Successful insertion of the HMG-CoA lyase gene was confirmed by hygromycin screening,PCR,Southern blot analysis,and enzyme content assay.The maximum MPA production by transformants was 2.94 g/L.This was 70.9%higher than wild-type ATCC16024.Our results demonstrate that ATMT may be an alternative practical genetic tool for directional transformation of P.brevicompactum.In conclusion,we present a series of novel approaches for improving MPA production.A new combined feeding strategy based on CN-feeding strategy was designed.MPA production in different growth stages was optimized by ratio of carbon and nitrogen source.This study expounded the biosynthetic pathway and metabolic regulation mechanism of MPA,and discussed how the fermentation conditions affected the accumulation of MPA,morphology of mycelium and pellet and other parameters such as DO,pH,liquor color.The genes of key enzymes were studied based on metabolic engineering in the metabolic pathway of MPA biosynthesis.The sequences of the key enzymes gene were cloned by technologies of thermal asymmetric interlaced polymerase chain reaction(Tail-PCR)and gene walking,which was significant for enhancing MPA fermentation.