daniel klein-marcuschamer, parayil kumaran ajikumar and gregory stephanopoulos

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

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Taxol

Taxol

It’s Complicated

Taxol

It’s Complicated=

$$$$

Metabolic Engineering:‘de novo engineered microbes’

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Metabolic Engineering:‘de novo engineered microbes’

http://www.earthtimes.org/nsimages/files/genetic_engineering_gm_encyclopaedia.jpg

Metabolic Engineering:‘de novo engineered microbes’

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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’

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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

?

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

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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!