1
Michael Smith Laboratories Monoterpene Production In Yeast Laura Bain, Gurpal Bisra, Joe Ho, Daisy Ji, Vicki Ma, Marianne Park, Jacob Toth, Samuel Wu Alina Chan, Rafael Saer, Shing Zhan Joerg Bohlmann, Leonard Foster, Joanne Fox, Phil Hieter, Chris Keeling C Pine Beetle Epidemic monoterpenes HMG-CoA MVA K6R-hmg2 I-PP DMA-PP FPP GPP IDI1 synthase erg20-2 Monoterpene Production in Budding Yeast Galactose pAG415-Synthase GAL Promoter BBa_K517000 Alpha-Pinene Synthase Gene BBa_K517002 pAG413-erg20-2 GPD Promoter BBa_K517001 erg20-2 Gene BBa_K517005 Galactose Signaling Pathway Alpha- Pinene Synthase erg20-2 Mevalonate Pathway IPP + DMAPP 75% GPP + 25% FPP 85% Alpha-Pinene 15% Beta-Pinene Monoterpenes Purication of desired monoterpene 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 communities 2. Reduction of trade and economic revenue 3. Depletion of pine forest carbon sink 4. 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 monoterpenes known 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 Practices Monoterpene Factory Metabolic Model iSynthase Trap Box Strategy Objectives: 1) To simulate the spread of MPB in British Columbia from year 2011-2020 2) Derive a strategy for containment of the MPB outbreak by implementation of trap boxes at sub- population centres Left image: Trees infected by MPB in Year 2010 based on data from the British 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 eect. 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 simplied version of the mevalonate pathway with targeted metabolic genes highlighted in red. The erg20-2 1 and K6R-hmg2 2 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 literature 1 . 1 Oswald, M; Fischer, M; Dirninger, N; Karst, F. Monoterpenoid biosynthesis in Saccharomyces cerevisiae. FEMS Yeast Res. 2007. 7: 413-421. 2 Gardner 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 Model Achievements 40 50 60 70 80 90 100 110 120 130 140 0 Abundance Beta-Pinene from our Beta-Pinene Synthase Sample 93 136 40 50 60 70 80 90 100 110 120 130 140 0 m/z Abundance Beta-Pinene from the Compound Library 93 136 m/z Monoterpene Data: We puried alpha-pinene, beta-pinene and limonene synthases from E. coli and incubated with GPP substrate. Products were analysed by GC-MS, conrming that the expected monoterpenes were produced for each of the synthases. Left: Gas chromatograph of beta-pinene synthase products. Below: Comparison of mass spectrometry prole of beta-pinene product (top panel) and beta-pinene from a library (bottom panel). Characterized Biobricks New biobrick parts: BBa_K517000-BBa_K517003 Existing parts: BBa_K118025, BBa_J63006, BBa_K115056. Demonstrated functionality of monoterpene synthases in vitro. Future Directions Characterize codon-optimized beta-pinene synthase in S. cerevisiae. Co-culture monoterpene-producing yeast with blue stain fungus to investigate potential detrimental interaction. Pine Tree Host Mountain Pine Beetle Vector Blue Stain Fungus 2 4 6 8 10 12 14 16 18 0 1 2 3 4 5 concentra on [x10 M] me [x10 s] beta-pinene K6R-hmg2+erg20-2 beta-pinene HMG2+ERG20 alpha-pinene K6R-hmg2+erg20-2 alpha-pinene HMG2+ERG20 5 -6 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 science Activity: 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 Columbia undergraduate students at orientation were surveyed for their denitions 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 Registry Characterized previously uncharacterized BBa_J63006 GAL1 promoter and found it to be non- functional. As an alternative, we submitted our own BBa_K517000 GAL promoter which is more exible and 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 High School team, (ii) Synthetic Biology in the Open and (iii) iGEM Dictionary. UBC Department of Chemical and Biological Engineering 2010 2011
