Monoterpene Production In Yeast
Transcript of Monoterpene Production In Yeast
Michael Smith Laboratories
Monoterpene Production In YeastLaura Bain, Gurpal Bisra, Joe Ho, Daisy Ji, Vicki Ma, Marianne Park, Jacob Toth, Samuel Wu
Alina Chan, Rafael Saer, Shing ZhanJoerg Bohlmann, Leonard Foster, Joanne Fox, Phil Hieter, Chris Keeling
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Pine Beetle Epidemic
monoterpenes
HMG-CoA MVAK6R-hmg2
I-PPDMA-PP
FPPGPP
IDI1
synthase
erg20-2
MonoterpeneProduction inBudding Yeast
Galactose
pAG415-Synthase
GAL PromoterBBa_K517000
Alpha-Pinene Synthase GeneBBa_K517002
pAG413-erg20-2
GPD PromoterBBa_K517001
erg20-2 GeneBBa_K517005
Galactose SignalingPathway
Alpha-Pinene
Synthaseerg20-2
MevalonatePathway
IPP+
DMAPP
75% GPP+
25% FPP
85% Alpha-Pinene15% Beta-Pinene
Monoterpenes
Puri!cation of desiredmonoterpene products
Synthase genes Limonene Synthase, BBa_K118025 Beta-Pinene Synthase, BBa_K517003 Alpha-Pinene Synthase, BBa_K517002 (shown)
Over 17.5 hectares of pine trees in British Columbia, Canada have been killed by Mountain Pine Beetles (MPB) with serious socioeconomic and environmental consequences such as:
1. The loss of forestry jobs in small communities2. Reduction of trade and economic revenue3. Depletion of pine forest carbon sink4. Increased carbon emissions from dead trees
This epidemic has spread to American states such as California, Arizona, Montana and Colorado, even reaching the pine forests in Mexico.
Trees use chemicals called monoterpenes to !ght o" beetle attacks. The Blue Stain Fungus lives symbiotically with the MPB and can degrade certain monoterpenes to deactivate the tree’s natural defenses.
The aim of our project is to over-produce monoterpenesknown to confer resistance in trees to beetles. We chose Saccharomyces cerevisiae, a well-studied model organism that possesses an endogenous mevalonate pathway essential to terpene production. Our strategy incorporates monoterpenes synthases and variants of metabolic enzymes in the mevalonate pathway to boost production.
This serves as a proof-of-concept for future development of synthetic organisms that can be feasibly released into the environment to tackle the MPB epidemic.
Human PracticesMonoterpene Factory Metabolic Model
iSynthase Trap Box StrategyObjectives: 1) To simulate the spread of MPB in British Columbia from year 2011-20202) Derive a strategy for containment of the MPB outbreak by implementation of trap boxes at sub-population centresLeft image: Trees infected by MPB in Year 2010 based on data from theBritish Columbia Ministry of Forests, Lands, Natural Resource Operations.Right image: Model-based clustering prediction of MPB spread in Year 2020. Circled in red are sub-population centres of the MPB, strategic locations where the iSynthase Trapbox can be placed for maximum e!ect.
Our model was developed using ArcGIS, Python and R.By predicting the impact of our trap box strategy, this model can inform future conservation policies.
2010 2020
Objective: To model monoterpene output by an engineered mevalonate pathway in S. cerevisiae.
Above is the simpli!ed version of the mevalonate pathway with targeted metabolic genes highlighted in red. The erg20-21 and K6R-hmg22 are variants of endogenous enzymes.
Developed using SimBiology MatLab, our model predicts that introduction of targeted metabolic genes results in a roughly twofold increase in monoterpene production in agreement with literature1.
1Oswald, M; Fischer, M; Dirninger, N; Karst, F. Monoterpenoid biosynthesis in Saccharomyces cerevisiae. FEMS Yeast Res. 2007. 7: 413-421. 2Gardner RG, Hampton RY. A 'distributed degron' allows regulated entry into the ER degradation pathway. Embo J. 1999. 18: 5994-6004.
Monoterpene Synthases & Metabolic Enzymes- Removed illegal restriction enzyme sites- Cloned into biobrick plasmid pSB1C3- Expressed in C41 E. coli (expresses rare tRNA) and S. cerevisiae- Characterized by gas chromatography-mass spectrometry (GC-MS) Yeast GAL and GPD Promoters- Cloned into biobrick plasmid pSB1C3- Characterized by #uorescence activated cell sorting (FACS)Improved Existing Registry Parts- Characterized Limonene Generator (BBa_K118025) by expression in C41 E. coli and GC-MS analysis
Epidemic ModelAchievements
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Beta-Pinene from our Beta-Pinene Synthase Sample93
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Beta-Pinene from the Compound Library93
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Monoterpene Data:We puri!ed alpha-pinene,beta-pinene and limonenesynthases from E. coli andincubated with GPP substrate.Products were analysed by GC-MS, con!rming that the expected monoterpenes were produced for each of the synthases.Left: Gas chromatograph ofbeta-pinene synthase products.Below: Comparison of mass spectrometry pro"le of beta-pinene product (top panel) and beta-pinene from a library (bottom panel).
Characterized BiobricksNew biobrick parts: BBa_K517000-BBa_K517003Existing parts: BBa_K118025, BBa_J63006, BBa_K115056.Demonstrated functionality of monoterpene synthasesin vitro.
Future DirectionsCharacterize codon-optimized beta-pinene synthase in S. cerevisiae.Co-culture monoterpene-producing yeast with blue stain fungus to investigate potential detrimental interaction.
Pine Tree
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Mountain
Pine Beetle
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Blue Stain
Fungus
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beta-pinene K6R-hmg2+erg20-2beta-pinene HMG2+ERG20alpha-pinene K6R-hmg2+erg20-2alpha-pinene HMG2+ERG20
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Contributed to CommunityBricks:- Guide to starting a high school iGEM team- Dialogue on Synthetic Biology in the Open (a discussion about the release of synthetic organisms in the real world)- iGEM Dictionary: an iGEM-wide collaboration
Mentoring Future Science Leaders
Audience: Future Science Leaders, program for high school students with a passion for scienceActivity: Introduction to synthetic biology and our iGEM project, complemented
by a plasmid activity in which students construct plasmids aimed at solving various environmental crises. We invited high school students to join our 2012 iGEM team or start their own team.
What is Synthetic Biology to you?University of British Columbiaundergraduate students at orientationwere surveyed for their de!nitions of synthetic biology. This is our annual practice aimed at investigating the changes in perception.
Visit our wiki for...videos, comics,word clouds and outreach documentation!
Submitted and characterized 4 new standard biobrick parts: BBa_K517000 to BBa_K517003.
Demonstrated that the 4 parts above were functional using GC-MS and FACS characterization, and entered this information on the Registry.
Functionally characterized previously uncharacterized BBa_K118025 Limonene Generator in the RegistryCharacterized previously uncharacterizedBBa_J63006 GAL1 promoter and found it to be non-functional. As an alternative, we submitted our ownBBa_K517000 GAL promoter which is more #exibleand has been functionally characterized.Sequenced Registry’s BBa_K115056 Isopentenyl Diphosphate Isomerase. Based on sequence discrepancies, we recommend that this part be removed from distribution.
Outlined and detailed new human practices approaches: (i) Guide for How to Start a iGEM HighSchool team, (ii) Synthetic Biology in the Open and(iii) iGEM Dictionary.
UBC Department of Chemical and Biological Engineering
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