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Production of the Antimalarial Drug Precursor Artemisinic Acid in Engineered Yeast

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Production of the Antimalarial Drug Precursor Artemisinic Acid in Engineered Yeast By J.D. Keasling et all. February 12, 2007 Patrick Gildea In A Nutshell Metabolic ... – PowerPoint PPT presentation

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Title: Production of the Antimalarial Drug Precursor Artemisinic Acid in Engineered Yeast


1
Production of the Antimalarial Drug Precursor
Artemisinic Acid in Engineered Yeast
By J.D. Keasling et all.
  • February 12, 2007
  • Patrick Gildea

2
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

3
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
4
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

5
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

6
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
7
The follow up synthesis procedures for after
artemisinic acid is purified and converted into
artemisinin via chemical conversions for
artemisinin-based combination therapies
8
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
9
Optimization Contd.
  • Functional genomics analyzes the dynamic aspects
    such as gene transcription, translation, and
    protein-protein interactions in cells

10
How Big of a Deal is this?
  • Metabolic Engineering 1970-80s
  • 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.

11
End result? Pockets are much lighter as well as a
curative for malaria
12
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

13
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

14
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

15
Technology Applicable?
  • Applicable in the industrial setting
  • Well-characterized biological parts
  • Cytochrome P450s, 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

16
Example of Industrial Process for Mass
Manufacture of Artemisinic Acid
17
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

18
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

19
Credits
  • Jay D. Keasling
  • Resource for research into the production of
    artemisinic acid
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