Oscillatory Driving of an Engineered Mevalonate Network to Increase Biofuel Yields
Oscillatory Driving of an Engineered Mevalonate Network to Increase Biofuel Yields Catherine Shi*, Tal Danino*, Howard Chou , Jeff Hasty*, Jay Keasling # # Bioengineering Department, UCSD(*) and Biocircuits Institute, Department of Bioengineering, UCB (#) Abstract Significance Preliminary Results Previously a mevalonate pathway in E. coli was engineered for high production of terpenoids , Optimization of Inhibition of mevalonate on 1.4 Cell growth 10 mM Mevalonate which are valuable compounds of numerous the mevalonate 1.2 Construct cell growth as seen in  Cell growth (OD600) 1 pBBR1MCS 0.8 pMKPMK commercial uses such as anti-malarial drugs and pathway could pMevB for various constructs (top) 0.6 pMBI 0.4 pMBIS result in viable 0.2 possibly biofuel candidates. However, non-native and simulations showing a 0 0 3 6 Time(hours) 9 12 biosynthetic similar effect on cell growth 2.5 synthetic pathways often inhibit cell growth due to 2 Mevalonate rate (bottom). 2 mM 1.5 an unbalanced production of toxic intermediates or production of 4 mM 6 mM 1 8 mM 0.5 10 mM cause a high metabolic burden by taking up isopentane and Cell growth 0 0 20 40 Time 60 80 100 resources that would feed normal cell growth and isopentene. 3 oscillatory control Higher cell growth is function. The goal of this project is to use Additionally, the results of this research can be used to build 2 1mM achieved with computational modeling & experimental biocircuits computational models applied to the oscillatory control of many oscillatory production of to determine whether oscillatory production of different biosynthetic pathways. 3mM mevB and nudF genes 1 for different mevalonate enzymes in this network can mitigate the metabolic 7mM 10mM concentrations. load, relieve cell toxicity and thus lead to higher 0 0 25 50 75 100 Methods Time yields of the desired products. Ratio of isopenteneol produced Cell growth ratio Using a previous synthetic oscillator  we in oscillatory case at a given 10 8 4 constructed several plasmids that drive either the  We aim to use the mevalonate frequency and amplitude 6 3 MevB, MevT, or nudF genes responsible for pathway previous engineered compared with that produced at 4 2 2 in E. coli for high production of 0 frequency and a mean ampli- producing isopenteneol at a particular frequency. terpenoids  with the addition 2 4 6 Isopentanol ratio 8 10 We simulated this network with a discrete time- tude. The majority of amplitudes 10 of the nudF gene to yield bio- and frequency show a gain in 8 4 delay model for the enzymes and growth of cells fuel candidates. This provides production, which is increased 6 4 3 involved. Calibrating our model to experimental 2 a model system for analyzing upon increasing mevalonate 2 results, we were able to show that downstream the effect of oscillatory driving concentration (10mM shown). 2 4 6 8 10 production of isopentenol can be increased by a of biosynthetic pathways. factor of 5 at high frequencies as compared to a control with average level of production. Summary and Future Work Drive oscillatory production of Using a previous synthetic oscillator  we mevalonate pathway intermediates have constructed several plasmids and Hypothesis at rates of oscillation to find optimal simulated this network with a discrete time- period and affect of amplitude. delay model for the enzymes and growth of M tT X The mevalonate tB T A-CoA AA-CoA B I HMG-CoA pathway puts too Mevalonate Mev-P Mev-PP cells to show a 5-fold increase in isopentenol 1 Three different examples at high frequencies of oscillation. Plasmid high a metabolic OH IPP Isopenteneol MevT MevB nudF Cells t t 1 F 1 Oscillatory promoter Inducible promoter of plasmid systems with systems containing all three genes have yet to burden on the cells pMevT pMevB nudF oscillatory driving of either n Cell Growth T or IPP is toxic to 2 MevT MevB nudF the MevB, MevT or nudF be completed. Systems will be experimentally cell growth. Inducible promoter Oscillatory promoter genes responsible for tested and data analyzed to build better com- Using oscillatory promotors to drive the mevalonate 3 MevT MevB nudF producing isopenteneol. putational models of oscillatory control of bi- pathway can alleviate these burdens. synthetic pathways. Inducible promoter Oscillatory promoterReferences:  Martin, V., et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology 21, 796-802 (2003).  Ro, D. et al. Production of the antimalarial drug precursor in artemisinic acid in engineered yeast. 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