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ITE 25 th - 2008 November 8, 2008 G. Stephanopoulos Bioinformatics and Metabolic Engineering Laboratory ITE 25 th Birthday Celebration Heraklion, Crete, 8 November, 2008 Reflections from the explorations of an engineer in the space of life sciences Gregory Stephanopoulos MIT

Heraklion, Crete, 8 November, 2008 · G. Stephanopoulos November 8, 2008 Bioinformatics and Metabolic Engineering Laboratory Eliciting new multigenic phenotypes Eliciting tolerance

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ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

ITE 25th Birthday Celebration

Heraklion, Crete, 8 November, 2008

Reflections from the explorations of an engineer in the space of

life sciences

Gregory StephanopoulosMIT

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Mixed cultures

ProductTank

Feed TankWith glucose

Aqueous medium with cells

F (L/h) F (L/h)

Scale

Parameters:

• Dilution Rate, D=F/ V• Substrate feed, Sf

M1+

B1

S

B2 P(Glucose)

+

+ M2

-

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

• Take advantage of an enriched pool of genes by sustaining more than one species in the same system

Mixed Cultures(progenitor of Metabolic Engineering)

Science, 213, pp. 972-979 (1981)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Following the recombinant revolution

• Instead of sustaining a mixed culture, transfer relevant pathways into a single cell

(genesis of)

Metabolic EngineeringScience, 252, pp. 1675-1681 (1991)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

M1

+

B1

S

B2 P(Glucose)

+

+ M2

-

From mixed cultures …

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Asp

Lysine

CO2

Glt

Gln

AspartaticsemialdehydeHomoserine

Threonine

Methionine

Trehalose

PYR

H4D

meso-DAPSuc

OAA

GLC6P

FRU6P

GAP

Acetate

ALA, VAL, LAC

Ribu5P

Sed7PE4PFru1,6bP

G3P

GLC

PYR

PEP

AcCoA

Isocit

aKG

Mal

Fum

To metabolic pathways …

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

To complex models of multi-level regulation and metabolism

1 23

4

Stephanopoulos, et al., “Exploiting biological complexity for strain improvement through systems biology,” Nature Biotechnology, 22: 1261-1267 (2004)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Lessons from the explorations of an

engineer in the space of life sciences

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

The Engineering ethosIntegration

Making sense of large systems and a lot of dataData-driven hypotheses

QuantificationModelsStructure of interactions Patterns of characteristic phenotypes

RelevanceDoing things for a reason

Lesson 1:Define the line between engineering

and life sciences

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Asp

Lysine

CO2

Glt

Gln

AspartaticsemialdehydeHomoserine

Threonine

Methionine

Trehalose

PYR

H4D

meso-DAPSuc

OAA

GLC6P

FRU6P

GAP

Acetate

ALA, VAL, LAC

Ribu5P

Sed7PE4PFru1,6bP

G3P

GLC

PYR

PEP

AcCoA

Isocit

aKG

Mal

Fum

Back to metabolic engineering …

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Metabolic Engineering as a new Organic Chemistry

Metabolic Engineering: Making improved biocatalysts (or engineering microbes) capable of :

Enhanced production of a native product to a microorganism

Formation of a product that is new to the microorganism

Synthesizing novel products

Science, 252:1675-1681 (1991)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Asp

Lysine

CO2

Glt

Gln

AspartaticsemialdehydeHomoserine

Threonine

Methionine

Trehalose

PYR

H4D

meso-DAPSuc

OAA

GLC6P

FRU6P

GAP

Acetate

ALA, VAL, LAC

Ribu5P

Sed7PE4PFru1,6bP

G3P

GLC

PYR

PEP

AcCoA

Isocit

aKG

Mal

Fum

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Probing cellular function

DNA microarrays

Environment

DNA

mRNAFluxes

Proteins

Proteomics Metabolic Flux Analysis

Signal Transduction

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Lesson 2

Biology, at the molecular level, is a

Chemical ScienceChemical engineering is the natural

engineering discipline to explore the applied aspects of biology at the cellular and molecular levels

Stephanopoulos G., “Chemical and Biological Engineering,”Chemical Engineering Science, 58: 3291-3293 and 58: 4931-4933

(2003).

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

ths

ASP

ASA

DAP

Lys

Hom

Meth

Thr

Ile

hdh

hkdapA

Science, 252: 1675 (1991)Biotech. & Bioeng. 41 633 (1993),Biotech. Progress, 10: 320 (1994)Biotech. & Bioeng. 58: 149 (1998)Science: 292:p.2024 (2001)

Example 1: Engineering the aspartate aminoacid pathways

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Lesson 3

There are many biologies besides

molecular biology

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Schematic pathway of aminoacid biosynthesis in C. glutamicum

Asp

Lysine

CO2

Glt

Gln

AspartaticsemialdehydeHomoserine

Threonine

Methionine

Trehalose

PYR

H4D

meso-DAPSuc

OAA

GLC6P

FRU6P

GAP

Acetate

ALA, VAL, LAC

Ribu5P

Sed7PE4PFru1,6bP

G3P

GLC

PYR

PEP

AcCoA

Isocit

aKG

Mal

Fum

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Probing metabolic pathways using isotopic tracers

Can help in:

• Reconstructing metabolic networks

• Calculating pathway fluxes

• Identifying rate-controlling steps

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Schematic pathway of aminoacid biosynthesis in C. glutamicum

Asp

Lysine

CO2

Glt

Gln

AspartaticsemialdehydeHomoserine

Threonine

Methionine

Trehalose

PYR

H4D

meso-DAPSuc

OAA

GLC6P

FRU6P

GAP

Acetate

ALA, VAL, LAC

Ribu5P

Sed7PE4PFru1,6bP

G3P

GLC

PYR

PEP

AcCoA

Isocit

aKG

Mal

Fum

Simultaneous amplification of pyc and ask increased lysine qpby 100-300%Park et al.,Biotech. & Bioeng., 55: 864 (1997)

Koffas et al., Metabolic Eng., 5:32-41, (2003)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Lesson 4

Metabolic engineering is far more powerful in constructing

optimal biocatalysts than chemical catalysis

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Example 2: Indene biocatalysis for the synthesis of Crixivan precursor

• Crixivan™: Merck’s HIV protease inhibitor

N

N

NHN

OH

O NHC(CH3)3O

OH·H2SO4

Aminoindanol

• Chemical synthesis of aminoindanol requires the use of expensive catalyst and gives low yields.

• Increased production is desired to meet patient needs (1 kg/patient/year).

• Bioconversion can potentially result in 100% yield.• Project focus on production of 2R-indandiol.

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Indene Bioconversion by RhodococcusStafford et al., PNAS, 99:1801-1806, (2002);

European J. of Biochemistry, 278: 1450-1460 (2001)

Indene

OH

1-Indenol

1-indanone

O

OH

OH

trans-(1R,2R)-indandiol

OH

OH

cis-(1S,2R)-indandiol

O

indene oxide

OH

O

1-keto-2-hydroxy-indan

OH

OH

cis-(1R,2S)-indandiol

Proposed NetworkDecoupled from primary metabolism

IndeneOH

1-Indenol

1-indanone

O

OH

OH

trans-(1R,2R)-indandiol

OH

OH

cis-(1S,2R)-indandiol

O

indene oxide

OH

OH

cis-(1R,2S)-indandiol

OxygenaseActivities

OH

O

1-keto-2-hydroxy-indan

DehydrogenaseActivities

Extracellular medium

Strain I24Cell

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Rational pathway design

indene

OH

OH

1,2-indenediol

O

indene oxide

1-indenol OH

1-indanoneO

OH

O

1-keto-2-hydroxy-indan

OH

OH

cis-(1S,2R)-indandiol

OH

OH

trans-(1R,2R)-indandiol

OH

OH

cis-(1R,2S)-indandiol

1. Isolation of KY1 (Y=50%)2. Network definition3. Flux analysis4. LimA overexpression (Y=75%)5. pH optimization (Y=95%)

Stafford et al., PNAS, 99:1801-1806, (2002);European J. of Biochemistry, 278: 1450-1460 (2001)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Lesson 5

Sometimes it pays to combine some math with genetics and

molecular biology

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Record of Metabolic Engineering

Aminoacids: Increases in lysine specific productivity by 120-300%Ethanologenic E. coliBiopolymers (Metabolix-ADM)1,3 propane diol (DuPont-Tate and Lyle)Indan-diol production (precursor of Crixivan-HIV protease inhibitor): Yield increased from 25% to >95%Artemisinin (amorphadiene) production by yeast and E. coliLycopene production in E. coli: Increase from 4,500 to ~25,000 ppm of CDW, fermentations > 250 mg/LMany other applications:

Succinate3-HPAThreonineTyrosine

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Isoprenoid pathway

PYR

G3P

DXP

DMPP

IPPPdxs

idiisp

genes

Chromosome-based Genes Plasmid-based Genes

LycopenecrtEBI

crtE

crtI

crtBCm pACLYC

Other CarotenoidsWS140: dxs, isp, idi

over-expressions: ~4,000ppm

TaxolArtemisininTerpenoids

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

0

1

2

3

4

5

6

7

8

0 10 20 30 40 50 60 70

Time (h)

Glu

cose

(g/L

) , O

D60

0, p

H

0

5000

10000

15000

20000

25000

30000

Lyco

pene

(ppm

)

OD600pHAcetatelycopene

Fed batch fermentation of yjiD over-expressing strain in the background of triple

knock-outs (ΔgdhA, ΔaceE, and ΔFdhF)

0

1

2

3

4

5

6

0 20 40 60 80

Time (hour)

Glu

cose

(g/L

)

Feeding profile

Nature Biotechnology, 23: 612-616 (2005)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Constructing an oil-producing, obese microbe

Lesson 7

If an experiment does not work the first time, order new

reagents and repeat

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

How does Metabolic Engineering differ from

Genetic Engineering?

… Metabolic engineering differs from Genetic Engineering and related molecular biological sciences in that it concerns itself with the properties of the entire metabolic network as opposed to individual genes and enzymes.

"Metabolic Engineering: Issues and Methodologies," Trends in Biotechnology, Vol. 11, pp. 392-396 (1993)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

What is Metabolic Engineering?

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

The biotech revolution, and the chemical-fuels industry (White Biotech)

Fuels and chemicals were the initial biotech targetCetus (Chiron), Genex, Biogen

More challenging technical problem than insulinSwitch of emphasis to medical applications

Changing boundary conditions Emphasis on renewable resourcesRobust US federal funding ⇒ Applied mol. biologyGenomicsSystems Biology: a new mindframe in biological researchMetabolic Engineering

Exploit applications of biology beyond medicine

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

What is different now: The New Biology

Unprecedented ability to transfer and modify (mutate) genes and amplify and control gene expression by molecular biological methods (genetic engineering)Genomics: Genome sequencing and study of

genomes. From a few to lots and lots of genesGenomics-based technologies for probing

intracellular content on cell-wide basisA new mind-frame for studying biological systems

that addresses issues in an integrated manner: Systems Biology

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Eliciting new multigenic phenotypes

Eliciting tolerance to toxicity in yeast and bacteria

Engineering microbes that can tolerate high concentrations of biofuels (ethanol)

Key in emerging industry of cellulosic biofuels

G. Stephanopoulos, “Challenges in engineering microbes for biofuels production,”Science, 315: 801-804, (2007)

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Gene-to-phenotype mapping

G1

G2

Gn

Gf

P1

P2

Pn

Pf

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Transcriptome-to-phenotype mapping

T1

T2

Tn

Tf

P1

P2

Pn

Pf

Gx

TF, σ

Global Transcription machinery controls the transcriptome

Gene 1

Gene 2

Transcription machinery

(polymerase)

Promoter 1

Factor

Promote

r 2

XGene 1 mRNA

Protein 1

Factor

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Exp

ress

ion L

evel

Gene

Exp

ress

ion L

evel

Gene

global Transcription Machinery Eng’g (gTME): a tool for reprogramming the transcriptome

Native machinery Engineered machinery

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Lesson 9

Diversity pays!

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

rpoD*

rpoD*rpoD*

rpoD*

rpoD*

rpoD

rpoD*

Error-prone PCR

Cloning followed by phenotype selection or screening

Iterative, directed

application of gTME

Sigma factor engineering strategy

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Ethanol Tolerance

50 g/L Ethanol

60 g/L Ethanol

70 g/L Ethanol

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Ethanol tolerance—growth curves

20 g/L 40 g/L 50 g/L

60 g/L 70 g/L

Red =Round 3

Blue =Native rpoD

00.10.20.30.40.50.60.70.8

0 2 4 6 8 100

0.05

0.1

0.15

0.2

0.25

0 2 4 6 8 100

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 2 4 6 8 10

00.0050.01

0.0150.02

0.0250.03

0.0350.04

0 2 4 6 8 100

0.005

0.01

0.015

0.02

0.025

0 2 4 6 8 10

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Low Inoculum fermentations

Medium: YSC –URAGlucose: 20 g/LCulture: 50 ml in 250 ml flasksAgitation: 225 RPMInitial OD: 0.01

The SPT15 mutant exhibits a nearly identical growth rate to the control, but continues exponential growth longer

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

High cell density fermentations

Medium: YSC –URAGlucose: 100 g/LCulture: 40 ml in 250 ml flasksAgitation: 225 RPMInitial OD: 15

Science, 314: 1565-1568 (2006)Metabolic Engineering, 9: 258-267 (2007)

Proceedings National Academy of Sciences, 105: 2319-2324 (2008).

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

HCDC summary

0.35(86%)

0.310.221.205.394.10

Control

+15%

+14%+41%+69%+20%---

% Improvement

2.03Productivity (g/L h-1)

0.31Specific rate (g/dcw h)

0.40(98%)

True EtOH yield accounting for biomass production(Percent of 0.41 g/g theoretical)

0.36Conversion yield calculated between 6 and 21 hours

6.46Final DCW (g/L)

4.06Initial DCW (g/L)

Mutant

⎟⎟⎠

⎞⎜⎜⎝

⎛1-1-

-1-1

h L consumed Glucose ghL produced EtOH g

⎟⎟⎟⎟⎟

⎜⎜⎜⎜⎜

⎟⎟⎠

⎞⎜⎜⎝

⎛ 1-1-

1-

L producedgDCW DCW g 0.5

glucose g 1 - L utilized Glucose g

L produced EtOH g

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

Lesson 10

Exploring the applied side of life sciences during the past

25 years:An incredible journey and a life of

perpetual change

ITE 25th - 2008November 8, 2008G. Stephanopoulos

Bioinformatics and Metabolic Engineering Laboratory

The end

Questions?