Ka-Yiu San

Metabolic Engineering and Systems Biotechnology Laboratory

recombinant DNA technology/genetic engineering

Metabolic engineering

Recombinant proteins by microorganisms

Transformation

Res

tric

tio

n s

ites

Cloning vector

Ligation

Recombined plasmid

Restrictioncleavage

Restrictioncleavage

Gen

e o

f in

tere

st

Tra

nsc

rip

tio

n

Tran

slation

mR

NA

Pro

tein

Host cell

Recombinant proteins by microorganisms

Year Products Disease Company 1982 Humulin Type 1 diabetes Genentech, Inc.

(synthetic insulin) 1985 Protropin Growth hormone Genentech, Inc.

Deficiency

Some early products

What is Metabolic Engineering?

Metabolic engineering is referred to as the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology

Three major projects

1. Pathway and co-factor engineering

2. Quantitative systems biotechnology(genomics, transcriptomics, proteomics and metabolomics)

3. Plant metabolic engineering

Gene TargetsIdentified

Insert/Knockout Genes/pathways Potential

strains

Shake Flask/Bioreactor

Experiments

Analysis

Modeling

Molecular Biology TechniquesPCR, RE digest, ligation, transformation,

knockout, etc.

3 N 3 N NaOHNaOH

Air

0.2m filters

PumpsPumps3 N HNO3 N HNO33

Gas outletGas outlet

Fresh Medium

Temperaturethermocouple

pHprobe

1L Glass Vessel

Bioreactor NBS BioFlo110

ControllerController

Mass flow controller

Waste tank

3 N 3 N NaOHNaOH

Air

0.2m filters

PumpsPumps3 N HNO3 N HNO33

Gas outletGas outlet

Fresh Medium

Temperaturethermocouple

pHprobe

1L Glass Vessel

Bioreactor NBS BioFlo110

ControllerController

Mass flow controller

Waste tank

PYCPYC

aceA

Isocitrate

OAA

Malate

Succinate

NAD+

NADH

-

glyoxylate

Acetyl CoAaceB

Fumarate

NADH

NAD+

aceA

Citrate

CO2

Fructose 1,6-diP

NADH

Pyruvate

PFL

ADP ATP

Dihydroxyacetone-PGlyceraldehyde-3 P

Glycerate 1,3-diP

Glucose

Glucose-6P

PEP

Pyruvate

ADP

ATP

Pi + NAD+

PYK

PEP

Acetyl - CoA

NAD+NADH

Lactate

LDH

ACKAcetate

PTAAcetyl-P

ADP ATPADH

2NADH

2NAD+

Ethanol

H 2CO2Formate

ADP

ATP

aceA

Isocitrate

OAA

Malate

Succinate

NAD+

NADH

-

glyoxylate

Acetyl CoAaceB

Fumarate

NADH

NAD+

aceA

Citrate

aceA

Isocitrate

OAA

Malate

Succinate

NAD+

NADH

-

glyoxylate

Acetyl CoAaceB

glyoxylate

Acetyl CoAaceB

Fumarate

NADH

NAD+

aceA

Citrate

CO2

Fructose 1,6-diP

NADH

Pyruvate

PFL

ADP ATP

Dihydroxyacetone-PGlyceraldehyde-3 P

Glycerate 1,3-diP

Glucose

Glucose-6P

PEP

Pyruvate

ADP

ATP

Pi + NAD+

PYK

PEP

Acetyl - CoA

NAD+NADH

Lactate

LDH

ACKAcetate

PTAAcetyl-P

ADP ATPADH

2NADH

2NAD+

Ethanol

H 2CO2Formate

ADP

ATP

Fructose 1,6-diP

NADH

Pyruvate

PFL

ADP ATP

Dihydroxyacetone-PGlyceraldehyde-3 P

Glycerate 1,3-diP

Glucose

Glucose-6P

PEP

Pyruvate

ADP

ATP

Pi + NAD+

PYK

PEP

Acetyl - CoA

NAD+NADH

Lactate

LDH

ACKAcetate

PTAAcetyl-P

ADP ATPADH

2NADH

2NAD+

Ethanol

H 2H 2CO2CO2Formate

ADP

ATPCO2CO2

Metabolic Engineering/systems biotechnology Cycle

Metabolic evolution

metabolomics proteomics transcriptomics

Pathway design and manipulation

Succinic acid

Top Value Added Chemicals from Biomass (2004 DOE report )

Top Value Added Chemicals from Biomass (2004 DOE report )

Simplified central anaerobic pathway in Simplified central anaerobic pathway in E. coliE. coli

NADH limitationMax theoretical Yield1 mole/mole

Succinate

2NADH 2NAD+ Lactate

2NAD+ 2NADH

Formate

Pyruvate

Acetyl- CoA

Acetate

Fructose 1,6-diP

Glucose

Glucose-6-PBiomass

PEP Pyruvate

ADP ATP

Acetyl-PADP ATP

PEP

ADP ATP

Glycerate-1,3-diP

ADP ATP

OAA

CoA NADH NAD+

Ethanol

NADH

CO2

H2

NAD+

Pepc

Ldh

Pta

Ack

CO2

E. coli Wild Type

succinate

Problems: low yield; mixed acid fermentation

Resulting pathway designSimplified central anaerobic pathway in Simplified central anaerobic pathway in E. coliE. coli

Lactate

Acetyl-P

Acetate

ADP

ATP Ack

Succinate

2NADH 2NAD+

2NAD+

2NADH

Formate

Pyruvate

Acetyl- CoA

Fructose 1,6-diP

Glucose

Glucose-6-PBiomass

PEP

Pyruvate

ADP

ATP

PEP

ADP

ATP

Glycerate-1,3-diP

ADP

ATP

OAA

CoA

Ethanol

NADH

CO2

NAD+

Pepc

Ldh

PtaH2

CO2

NADH NAD+

NADH limitationMax theoretical Yield1 mole/mole

Fermentative pathway

Glyoxylate pathway(no NADH

requirements)

aceA

Isocitrate

OAA

Malate

Succinate

NAD+ NADH

glyoxylate

Acetyl-CoA aceB

Fumarate

NADH

NAD+

aceA

Citrate

Pyc

CO2

Fructose 1,6-diP

NADH

Pyruvate

Pfl

ADP ATP

Glycerate 1,3-diP

Glucose

Glucose-6P

PEP

Pyruvate

ADP

ATP

NAD+

Pyk

PEP

Acetyl-CoA

H2

CO2

Formate

ADP

ATP CO2

Experimental yield= 1.6 mol/mol

Resulting pathway design

Fermentative pathway

Glyoxylate pathway(no NADH requirements)

PYC

CO2

aceA

Isocitrate

OAA

Malate

Succinate

NAD+ NADH

-

glyoxylate

Acetyl CoA aceB

Fumarate

NADH

NAD+

aceA

Citrate

Fructose 1,6-diP

NADH

Pyruvate

PFL

ADP ATP

Dihydroxyacetone-PGlyceraldehyde-3 P

Glycerate 1,3-diP

Glucose

Glucose-6P

PEP

Pyruvate

ADP

ATP

Pi + NAD+

Pyk

PEP

Acetyl - CoA

H 2CO 2 Formate

ADP

ATP

CO2

SBS550MG (pHL413)

Succinic acid produced from 20g/L (~110 moles) of glucoseSuccinic acid produced from 20g/L (~110 moles) of glucose

E. coli Wild Type

succinate

succinate

Three routes engineered for succinate production

Strain SBS552Strain SBS552

Genes inactivated:Genes inactivated:

sdhAB sdhAB

ackA-ptaackA-pta

poxB poxB

iclRiclR

ptsG ptsG

ldhAldhA

adhEadhE

(aerobic)(aerobic)(anaerobic)(anaerobic) (anaerobic & aerobic)(anaerobic & aerobic)

PyruvatePyruvate

Acetyl-CoAAcetyl-CoA

SuccinateSuccinate

G6PG6P

PEPPEP

PyruvatePyruvate

PEPPEP

G3PG3P

COCO22

GlucoseGlucose

OAAOAA CitrateCitrate

IsocitrateIsocitrateMalateMalate

GlyoxylateGlyoxylate

Acetyl-CoAAcetyl-CoA

aceAaceAaceBaceB

COCO22

ptsGptsG

icdicd 2-ketoglutarate2-ketoglutarate

Succinyl-CoASuccinyl-CoA

COCO22

COCO22FumarateFumarate

SuccinateSuccinateSuccinateSuccinate

COCO22

Cofactor manipulations

Cofactor manipulations

• NAD+/NADH; NADP+/NADPH

• Coenzyme A (CoA)

Examples• esters• chiral compounds• terpenoids – lycopene• polyketides

• Cancer (adriamycin)• Infection disease (tetracyclines, erythromycin)• Cardiovascular (mevacor, lovastatin)• Immunosuppression (rapamycin, tacrolimus)

Importance of cofactor manipulation

Enzymes

Cofactors

+

Products

Substrate

G6P

F6P

GAP

PEP Pyr AcCoA Acetate

E4P

S7P

PEPPyr

3-PG

CO2

CO2

Glucose

GAP

F6P

F6P GAP

Mal

OAA ICT

Suc

AKG

CO2

CO2

P5P

CO22 NADP+ 2 NADPH

ATP

ADP

NAD+

NADH

ADPATP

ADP ATPADP ATP

NADH NAD+

NADP+

NADPH

NAD+

NADH

FAD+FADH2

NAD+

NADH

ADPATP

GAPA

NADP+

NADPH

GAPC

pHL621

Lycopene production is limited by NADPH availability

0

100

200

300

400

500

LB 2YTMedium

Spe

cific

lyco

pene

pro

duct

ion

( g

/g b

iom

ass)

MG1655 (pDHC29/pK19-lyco) MBS100 (pHL621/pK19-lyco)

136.0% (2.4) 136.7% (2.4)

WT mutant

Example: lycopene production

Plant metabolic engineering

Metabolic Engineering of the Terpenoid and Indole Pathways in Catharanthus roseus hairy roots

Catharanthus roseus (Madagascar Periwinkle) Produce a wide range of secondary metabolites Ajmalicine and Serpentine – hypertension Vinblastine and Vincristine – anticancer drugs used

to treat lymphomas and leukemia

vinblastine vincristine

Chorismate

Anthranilate

Tryptophan

Tryptamine

Pyruvate + G3P

1-Deoxy-D-Xylulose-5-Phosphate

2-C-Methyl-D-erythitol-4-phosphate

IPPDMAPP

GPP

Geraniol

10-Hydroxygeraniol

Loganin

SecologaninStrictosidine

Ajmalicine

Serpentine

Tabersonine

Lochnericine

Hörhammericine

Vindoline

Catharanthine

Vinblastine

Vincristine

Terpenoid Indole Alkaloid Pathway

Indole Pathway

Terpenoid Pathway

MevalonateAS

DXS

STR

G10HTDC

Clone Generation

Plasmid Construction

in E. coli

Agrobacterium

Ri

Sterile Grown

Plants

(5 weeks)

Infection

(6 weeks) Selection Media

(6 weeks)

Adapt to Liquid Media

(24 weeks)Transgene

Discovery of new compounds

• Isolated and verified the structures of 11 new phenolic compounds

• Structure very similar to existing agents that exhibit anti-fungal, anti-cancer properties

• Currently testing new compounds for biological activities

Produce useful compounds that exhibit desirable biological functions

Collaborators:

Dr. George N. BennettDepartment of Biochemistry and Cell BiologyRice University

Dr. Jacqueline V. ShanksDepartment of Chemical EngineeringIowa State University

Dr. Sue GibsonDepartment of Plant BiologyUniversity of Minnesota

Dr. Steve CoxDepartment of Computational and Applied Mathematics Rice University

Dr. Ill-Min ChungKonKuk University, Korea

Questions ?

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