Abstract Oscillatory Driving of an Engineered Mevalonate Network to Increase Biofuel Yields Significance Summary and Future Work Preliminary Results Catherine Shi*, Tal Danino*, Howard Chou , Jeff Hasty*, Jay Keasling Bioengineering Department, UCSD(*) and Biocircuits Institute, Department of Bioengineering, UCB (#) Previously a mevalonate pathway in E. coli was engineered for high production of terpenoids [1], which are valuable compounds of numerous commercial uses such as anti-malarial drugs and possibly biofuel candidates. However, non-native synthetic pathways often inhibit cell growth due to an unbalanced production of toxic intermediates or cause a high metabolic burden by taking up resources that would feed normal cell growth and function. The goal of this project is to use computational modeling & experimental biocircuits to determine whether oscillatory production of enzymes in this network can mitigate the metabolic load, relieve cell toxicity and thus lead to higher yields of the desired products. Using a previous synthetic oscillator [7] we constructed several plasmids that drive either the MevB, MevT, or nudF genes responsible for producing isopenteneol at a particular frequency. We simulated this network with a discrete time- delay model for the enzymes and growth of cells involved. Calibrating our model to experimental results, we were able to show that downstream production of isopentenol can be increased by a factor of 5 at high frequencies as compared to a control with average level of production. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 2 1 0 3 6 9 600 Cell growth (OD ) Time pBBR1MCS pMKPMK pMevB pMBI pMBIS 2 mM Cell growth 4 mM 6 mM 8 mM 10 mM 0 20 40 60 80 100 0 0.5 1 1.5 2 2.5 Time(hours) Mevalonate Construct 10 mM Mevalonate Cell growth ratio 2 4 6 8 10 2 4 6 8 10 2 3 4 Isopentanol ratio 2 4 6 8 10 2 4 6 8 10 2 3 4 Inhibition of mevalonate on cell growth as seen in [1] for various constructs (top) and simulations showing a similar effect on cell growth rate (bottom). Higher cell growth is achieved with oscillatory production of mevB and nudF genes for different mevalonate concentrations. Cells pMevT pMevB IPP T I A-CoA AA-CoA HMG-CoA Mev-P Mev-PP B t 1 T t 1 t T M t B Cell Growth n nudF Isopenteneol OH X F Hypothesis The mevalonate pathway puts too high a metabolic burden on the cells or IPP is toxic to cell growth. Using oscillatory promotors to drive the mevalonate pathway can alleviate these burdens. Methods Using a previous synthetic oscillator [7] we have constructed several plasmids and simulated this network with a discrete time- delay model for the enzymes and growth of cells to show a 5-fold increase in isopentenol at high frequencies of oscillation. Plasmid systems containing all three genes have yet to be completed. Systems will be experimentally tested and data analyzed to build better com- putational models of oscillatory control of bi- synthetic pathways. Cell growth Time 0 25 50 75 100 0 1 2 3 1mM 3mM 10mM 7mM oscillatory control Ratio of isopenteneol produced in oscillatory case at a given frequency and amplitude compared with that produced at 0 frequency and a mean ampli- tude. The majority of amplitudes and frequency show a gain in production, which is increased upon increasing mevalonate concentration (10mM shown). Optimization of the mevalonate pathway could result in viable biosynthetic production of isopentane and isopentene. MevT MevB nudF 1 Oscillatory promoter Inducible promoter MevT MevB nudF 2 Inducible promoter Oscillatory promoter Oscillatory promoter Inducible promoter nudF MevB 3 Additionally, the results of this research can be used to build computational models applied to the oscillatory control of many different biosynthetic pathways. Three different examples of plasmid systems with oscillatory driving of either the MevB, MevT or nudF genes responsible for producing isopenteneol. We aim to use the mevalonate pathway previous engineered in E. coli for high production of terpenoids [1] with the addition of the nudF gene to yield bio- fuel candidates. This provides a model system for analyzing the effect of oscillatory driving of biosynthetic pathways. Drive oscillatory production of mevalonate pathway intermediates at rates of oscillation to find optimal period and affect of amplitude. References: [1] Martin, V., et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology 21, 796-802 (2003). [2] Ro, D. et al. Production of the antimalarial drug precursor in artemisinic acid in engineered yeast. Nature 440, 940-943 (2006). [3] Withers, S., et al. Identification of isopentenol biosynthetic genes from Bacilus subtilis by screening method based on isoprenoid precursor toxicity. Applied and Environmental Microbiology 73, 6277 (2007). [4] Kudla, G., et al. Coding-Sequence Determinants of Gene Expression in Escherichia coli. Science 324, 255 (2009). [5] Pitera, D. et al. Balancing a heterologous mevalonate pathway for improved isoprenoid production in Eschericia coli. Metabolic Engineering 9, 193-207 (2007). [6] Anthony, J. et al. Optimi- zation of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4, 11-diene. Metabolic Engineering 11, 13-19 (2009). [7] Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516-519 (2008). [8] Lutz, R. & Bujard, H. Independent and tight regularion of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic acids research 25, 1203 (1997). [1] # # MevT
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Abstract
Oscillatory Driving of an Engineered Mevalonate Network to Increase Biofuel Yields
Significance
Summary and Future Work
Preliminary Results
Catherine Shi*, Tal Danino*, Howard Chou , Jeff Hasty*, Jay Keasling Bioengineering Department, UCSD(*) and Biocircuits Institute, Department of Bioengineering, UCB (#)
Previously a mevalonate pathway in E. coli was engineered for high production of terpenoids [1], which are valuable compounds of numerous commercial uses such as anti-malarial drugs and possibly biofuel candidates. However, non-native synthetic pathways often inhibit cell growth due to an unbalanced production of toxic intermediates or cause a high metabolic burden by taking up resources that would feed normal cell growth and function. The goal of this project is to use computational modeling & experimental biocircuits to determine whether oscillatory production of enzymes in this network can mitigate the metabolic load, relieve cell toxicity and thus lead to higher yields of the desired products. Using a previous synthetic oscillator [7] we constructed several plasmids that drive either the MevB, MevT, or nudF genes responsible for producing isopenteneol at a particular frequency. We simulated this network with a discrete time-delay model for the enzymes and growth of cells involved. Calibrating our model to experimental results, we were able to show that downstream production of isopentenol can be increased by a factor of 5 at high frequencies as compared to a control with average level of production.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
210 3 6 9
600
Cell g
rowth
(OD
)
Time
pBBR1MCS
pMKPMK
pMevB
pMBI
pMBIS
2 mM
Cell growth
4 mM 6 mM 8 mM 10 mM
0 20 40 60 80 1000
0.5
1
1.5
2
2.5Time(hours)
Mevalonate
Construct10 mM Mevalonate
Cell growth ratio
2 4 6 8 10
2
4
6
8
10
2
3
4
Isopentanol ratio
2 4 6 8 10
2
4
6
8
10
2
3
4
Inhibition of mevalonate on cell growth as seen in [1] for various constructs (top) and simulations showing a similar effect on cell growth rate (bottom).
Higher cell growth is achieved with oscillatory production of mevB and nudF genes for different mevalonate concentrations.
Cells
pMevT pMevB
Mevalonate
IPP
TI
A-CoA AA-CoA HMG-CoA Mev-P Mev-PPB
t 1
T
t 1
t TM
t B
Cell Growthn
nudF
IsopenteneolOH
X
F
HypothesisThe mevalonate pathway puts too high a metabolic burden on the cells or IPP is toxic to cell growth.
Using oscillatory promotors to drive the mevalonate pathway can alleviate these burdens.
Methods
Using a previous synthetic oscillator [7] we have constructed several plasmids and simulated this network with a discrete time-delay model for the enzymes and growth of cells to show a 5-fold increase in isopentenol at high frequencies of oscillation. Plasmid systems containing all three genes have yet to be completed. Systems will be experimentally tested and data analyzed to build better com-putational models of oscillatory control of bi-synthetic pathways.
Cell growth
Time 0 25 50 75 100
0
1
2
3
1mM
3mM
10mM
7mM
oscillatorycontrol
Ratio of isopenteneol produced in oscillatory case at a given frequency and amplitude compared with that produced at 0 frequency and a mean ampli-tude. The majority of amplitudes and frequency show a gain in production, which is increased upon increasing mevalonate concentration (10mM shown).
Optimization of the mevalonate pathway could result in viable biosynthetic production of isopentane and isopentene.
MevT MevB nudF 1
Oscillatory promoter Inducible promoter
MevT MevB nudF 2
Inducible promoter Oscillatory promoter
Oscillatory promoter Inducible promoter
nudF MevB
3
Additionally, the results of this research can be used to build computational models applied to the oscillatory control of many different biosynthetic pathways.
Three different examples of plasmid systems with oscillatory driving of either the MevB, MevT or nudF genes responsible for producing isopenteneol.
We aim to use the mevalonate pathway previous engineered in E. coli for high production of terpenoids [1] with the addition of the nudF gene to yield bio-fuel candidates. This provides a model system for analyzing the effect of oscillatory driving of biosynthetic pathways.
Drive oscillatory production of mevalonate pathway intermediates at rates of oscillation to find optimal period and affect of amplitude.
References: [1] Martin, V., et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology 21, 796-802 (2003). [2] Ro, D. et al. Production of the antimalarial drug precursor in artemisinic acid in engineered yeast. Nature 440, 940-943 (2006). [3] Withers, S., et al. Identification of isopentenol biosynthetic genes from Bacilus subtilis by screening method based on isoprenoid precursor toxicity. Applied and Environmental Microbiology 73, 6277 (2007). [4] Kudla, G., et al. Coding-Sequence Determinants of Gene Expression in Escherichia coli. Science 324, 255 (2009). [5] Pitera, D. et al. Balancing a heterologous mevalonate pathway for improved isoprenoid production in Eschericia coli. Metabolic Engineering 9, 193-207 (2007). [6] Anthony, J. et al. Optimi-zation of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4, 11-diene. Metabolic Engineering 11, 13-19 (2009). [7] Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516-519 (2008). [8] Lutz, R. & Bujard, H. Independent and tight regularion of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic acids research 25, 1203 (1997).
[1]
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MevT
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