production of the antimalarial drug precursor artemisinic acid in engineered yeast february 12, 2007...
TRANSCRIPT
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Production of the Antimalarial Drug Precursor Artemisinic Acid in
Engineered Yeast
February 12, 2007Patrick Gildea
By J.D. Keasling et all.
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In A Nutshell
• Metabolic Engineering – The alteration of metabolic pathways found in an organism in order to understand/utilize cellular pathways for chemical transformation, energy transduction, supramolecular assembly– Antibiotics– Biosynthetic precursors – polymers
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Motivation• Diseases like
diabetes treated via recombinant proteins
• However, protein therapeutic approaches have not been applicable for infectious diseases
• Synthetic chemistry is far too expensive and inefficient
The structure of insulin
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Design Concept of the Engineered Biological System
• Overall Goal: engineer a microorganism to produce artemisinin from an inexpensive, renewable resource
• Find & clone (or synthesize) the genes that produce the precursor artemisinic acid in Artemisia annua leaves
• Identify the chemistry of the enzyme reactions• Express genes of different organisms in a host (difficult)• Balance metabolic pathways to optimize production• Well characterized genetic control system
– Chassis (stable)– Parts– Metabolic Engineering Tools
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Key Elements of the Metabolic Pathway in Yeast
• Artemisinic acid in yeast is produced in 3 steps in the metabolic pathway
• Modifications to host strain (expression vector) via chromosomal integration (ensure genetic stability)
• Yeast is used as the chassis because the codon usage between yeast and A. Annua are very similar
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Process for the microbial production of artemisinic acid in the biosynthetic pathway in S. cerevisiae strain EPY224
Starting from acetyl-CoA the microbes produce: mevalonate, farnesyl pyrophosphate (FPP), amorphadiene, and finally, artemisinic acid
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The follow up synthesis procedures for after artemisinic acid is purified and converted into artemisinin via chemical conversions for artemisinin-based combination therapies
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Optimization• Through modifying the pathway in yeast through adjusting
the expression of specific genes in the pathway, production was increased
• Native metabolic intermediates can be toxic at high concentrations
• “Pulling” on a pathway is just as important as “pushing”• DNA arrays and proteomics• Library-based engineering of intergenic regions of operons
Production of amorphadiene by S. cerevisiae strains
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Optimization Contd.• Functional genomics analyzes the dynamic aspects such
as gene transcription, translation, and protein-protein interactions in cells
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How Big of a Deal is this?
• Metabolic Engineering – 1970-80’s• For synthetic biology, production of artemisinic acid
in yeast and E. Coli is the “poster child” for cheaper drugs
• Difficult to synthesize and expensive molecules can be manufactured cheaply via synthetic biology
• Enzymes can catalyze in a single step what might take many steps using synthetic chemistry (expensive and difficult)
• Coupling multiple enzymes in a metabolic pathway, purification of chemical intermediates are not necessary before proceeding to the next reaction.
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End result?Pockets are much lighter as well as a curative for malaria
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Artemsinic Acid in Yeast Particularly Novel? You Bet!
• A biological system that can convert cheap resources (i.e. glucose) into a high quality precursor of artemisinic acid
• Use of a host that is easily obtainable and cheap to maintain as a microbial chassis
• The critical idea is the use of enzymes to catalyze complex molecules in a number of small steps
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Integration of Existing Parts? • Genes for producing artemisinic acid (A.A.) from sweet wormwood
• Stable chassis that is modified to produce high yields of A.A. (yeast)
• Modification/adjustment of metabolic pathway for high yields
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Science: Relevant?
• Principles of metabolic engineering applicable toward synthetic biology
• Possible to use intracellular metabolites for the production of chemicals from simple starting materials (i.e. glucose)
• Possible to insert the gene for making a complex molecule into a different organism where the gene will successfully be expressed
• Understanding of how different genes from different organisms can affect metabolic system of host organism
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Technology: Applicable?
• Applicable in the industrial setting• Well-characterized biological parts
– Cytochrome P450’s, etc.
• Methodology for optimization of the mevalonate pathway can be applied for other processes
• Enzymes are powerful!• Library-based engineering/functional genomics
– CAD and debugging tools aid biological design
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Example of Industrial Process for Mass Manufacture of Artemisinic Acid
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Outlook for VGEM Team
• The tools and techniques used in synthetic biology for metabolic engineering are similar to other tools/techniques for other components (cells, circuits)– Chassis– Vectors– Promoters– Simultaneous engagement of multiple genes– CAD and debugging
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What is Impossible/Possible
• Impossible:– Trip to Amazon to find cool genes in some obscure plant that
produce molecules that suppress cancer or something along those lines
• Possible: – In literature: find a gene that manufactures a complex molecule
and determine whether the codon usage of the genes and a host are compatible
– Insert the genes via a vector and adjust the expression levels of the genes via promoters
– Tweak the system in different ways to maximize the production of target chemical by using tools such as functional genomics, etc.
– However, even without the Amazon trip – this will be expensive
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Credits
• Jay D. Keasling– Resource for research into the production of
artemisinic acid