daniel klein-marcuschamer, parayil kumaran ajikumar and gregory stephanopoulos
TRANSCRIPT
Daniel Klein-Marcuschamer, Parayil Kumaran Ajikumar
and Gregory Stephanopoulos
Table of Contents
• Introduction• Taxol
• Lycopene, Isoprenoids, Carotenoids• Background, Pathway
• Three Problems• Substrate availability• Intermediate build-up• Increase storage capacity
• Conclusion
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Taxol
https://upload.wikimedia.org/wikipedia/commons/thumb/5/59/Taxol.svg/1280px-Taxol.svg.png
Taxol
Taxol
It’s Complicated
Taxol
It’s Complicated=
$$$$
Metabolic Engineering:‘de novo engineered microbes’
http://www.earthtimes.org/nsimages/files/genetic_engineering_gm_encyclopaedia.jpg
Metabolic Engineering:‘de novo engineered microbes’
http://www.earthtimes.org/nsimages/files/genetic_engineering_gm_encyclopaedia.jpg
Metabolic Engineering:‘de novo engineered microbes’
http://www.earthtimes.org/nsimages/files/genetic_engineering_gm_encyclopaedia.jpg
Metabolic Engineering:‘de novo engineered microbes’
http://www.earthtimes.org/nsimages/files/genetic_engineering_gm_encyclopaedia.jpg
Metabolic Engineering:‘de novo engineered microbes’
http://www.earthtimes.org/nsimages/files/genetic_engineering_gm_encyclopaedia.jpg
Metabolic Engineering:‘de novo engineered microbes’
http://www.earthtimes.org/nsimages/files/genetic_engineering_gm_encyclopaedia.jpg
Metabolic Engineering:‘de novo engineered microbes’
http://www.earthtimes.org/nsimages/files/genetic_engineering_gm_encyclopaedia.jpg
Metabolic Engineering:‘de novo engineered microbes’
http://rsif.royalsocietypublishing.org/content/10/78/20120671
Metabolic Engineering:‘de novo engineered microbes’
http://www.rcsb.org/pdb/101/motm.do?momID=152
http://rsif.royalsocietypublishing.org/content/10/78/20120671
Metabolic Engineering:‘de novo engineered microbes’
http://www.brooklyn.cuny.edu/bc/ahp/LAD/C7/graphics/C7_atp_1.GIF
http://www.rcsb.org/pdb/101/motm.do?momID=152
http://rsif.royalsocietypublishing.org/content/10/78/20120671
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Isoprenoids
Isoprenoids
x
Isoprenoids
x
Isoprenoids
x
Isoprenoids
x
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http://www.inkworldmagazine.com/contents/displayImage/29681
Isoprenoids
x
http://gizmodo.com/5887825/what-are-steroids
Carotenoids
8
Carotenoids
Tetraterpenoid
8
Lycopene
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MEP & MVA PathwaysMethylerythritol Phosphate
Mevalonic Acid
MEP & MVA PathwaysMethylerythritol Phosphate
Mevalonic Acid
IPP & DMAPPIsopentenyl Pyrophosphate
Dimethylallyl Diphosphate
MEP & MVA PathwaysMethylerythritol Phosphate
Mevalonic Acid
IPP & DMAPPIsopentenyl Pyrophosphate
Dimethylallyl Diphosphate
Isoprenoids
The big picture
The big picture
The Three Problems:
1. Ensuring Substrate Availability2. Balancing Intermediate Pools
3. Improving Storage Capacity for End Products
Ensuring Substrate Availability
• Focus on increasing the availability of substrates (IPP, DMAPP, FPP).
Ensuring Substrate Availability
• Focus on increasing the availability of substrates (IPP, DMAPP, FPP).
A) Stoichiometric Flux Balance Analysis
• A method of modelling a metabolic network to identify enzymes which heavily influence carbon flux.
(PT5-idi / PT5-ispDF / PT5-dxs)
A) Stoichiometric Flux Balance Analysis
• A method of modelling a metabolic network to identify enzymes which heavily influence carbon flux.
(PT5-idi- / PT5-ispDF- / PT5-dxs-)
B) Metabolic Control Structure
• Diverts carbon flux to lycopene during the stationary phase of growth.• PT5-idi and PT5-pps under glnAp2.
B) Metabolic Control Structure
• Diverts carbon flux to lycopene during the stationary phase of growth.• PT5-idi and PT5-pps under glnAp2.
C) Rational Strain Design
• Mevalonate pathway from S. cerevisiae cloned into E. coli.• Amorphadiene synthase.
C) Rational Strain Design
• Mevalonate pathway from S. cerevisiae cloned into E. coli.• Amorphadiene synthase.
C) Rational Strain Design
• Mevalonate pathway from S. cerevisiae cloned into E. coli.• Amorphadiene synthase.
C) Rational Strain Design
• Mevalonate pathway from S. cerevisiae cloned into E. coli.• Amorphadiene synthase.
D) Modification of Native Proteins•
• Monoterpenes and diterpenes produced from substrates geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP).
D) Modification of Native Proteins•
• Monoterpenes and diterpenes produced from substrates geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP).• FPP synthase (ispA) point mutant.
This isn’t right!
E) Randomized Overexpression
• Eight clones of random enzymes in the MEP/DOXP pathway.• T5-phage promoter.
E) Randomized Overexpression
• Eight clones of random enzymes in the MEP/DOXP pathway.• T5-phage promoter crtEBIY operon.• Screened for increased B-carotene accumulation.
http://www.discoveryandinnovation.com/BIOL202/notes/images/generalized_transduction.jpg
F) Knockout Library Approach
• Screened an E. coli genomic library in the presence of carotenogenic genes.• appY and crl.
F) Knockout Library Approach
• Screened an E. coli genomic library in the presence of carotenogenic genes.• appY and crl.
Who would’ve thought?
G) Investigation of Carotenogenic Genes• Tao et. al (2005) produced a transposon mutagenesis
library in E. coli.• crtEXYIB operon on a ColE1 plasmid.
G) Investigation of Carotenogenic Genes• Tao et. al (2005) produced a transposon mutagenesis
library in E. coli.• crtEXYIB operon on a ColE1 plasmid.
Balancing Intermediate Pools
http://aje.oxfordjournals.org/content/162/10/953/F1.large.jpg
A) mRNA Stability
• Smolke et. al (2001) altered mRNA processing signals on crtY /crtI. • 300-fold variation in ϐ-carotene and lycopene
production.
• Library of mRNA processing signals.• Auxotrophic reporter strain expressing GFP.
Decreased HMG-CoA synthase / HMG-CoA reductase mRNA levels = more mevalonate
production!
• Library of mRNA processing signals.• Auxotrophic reporter strain expressing GFP.
Decreased HMG-CoA synthase / HMG-CoA reductase mRNA levels = more mevalonate
production!
B) Operon mRNA Levels and PermutationS• FPP zeaxanthin, Bacillus subtilis.
B) Operon mRNA Levels and PermutationS• FPP zeaxanthin, Bacillus subtilis.• One variant showed a 35% increase in zeaxanthin
production.
C) Global mRNA Manipulation
• Random mutagenesis of housekeeping E. coli sigma factor (σD).• Some strains demonstrated increased carotenoid yields
by up to 50%.
C) Global mRNA Manipulation
• Random mutagenesis of housekeeping E. coli sigma factor (σD).• Some strains demonstrated increased carotenoid yields by
up to 50%.
Take home message: gene deletion and overexpression are not always ideal solutions for managing
intermediate pools.
Improving Storage Capacity FOR End Products
• Isoprenoid end products can potentially be toxic at high concentrations.
Improving Storage Capacity FOR End Products
• Isoprenoid end products can potentially be toxic at high concentrations.
Detergents and the absence of light implicated in affecting lycopene yields.
Improving Storage Capacity FOR End Products
• Isoprenoid end products can potentially be toxic at high concentrations.
Detergents and the absence of light implicated in affecting lycopene yields.
IN CONCLUSION
Increased energy costs + environmental concerns with traditional methods of chemical synthesis = microbes!
IN CONCLUSION
Increased energy costs + environmental concerns with traditional methods of chemical synthesis = microbes!
IN CONCLUSION
Increased energy costs + environmental concerns with traditional methods of chemical synthesis = microbes!