Metabolic engineering

Metabolic engineering

• Targeted and purposeful alteration of metabolic pathways found in an organism in or-der to better understand and use cellular pathways :

- To increase the production rate of the existing products - To produce new valuable products - To expand the substrates that can be assimilated by organisms

• Practice of optimizing genetic and regulatory processes within the cells to maximize the production of a target material by the cells.

- Expression and release of repression

• Metabolic engineers commonly work to reduce cellular energy use (i.e, the energetic cost of cell reproduction or proliferation) and to reduce the waste production.

• Direct deletion and/or over-expression of the genes that encode the metabolic en-zymes

• Current focus is to target the regulatory networks in a cell to efficiently engineer the metabolism

Biosynthetic pathway of L-Thr in E. coli

L-Aspartyl phosphate

Homoserine phosphate

Glucose

Phosphenolpyruvate

Pyruvate

TCA cycleOxaloacetate

ppc

mdh

aceBAKaspC

L-Lysine

L-Methionine

L-Aspartate

L-Aspartate semidaldehyde

Homoserine

L-Threonine

L-Isoleucine

thrA lysC

metL

asd

thrA

thrB

thrC

ilvA

dapA

metA

Feedback repression

Microbial production of fatty-acid-derived fuels and chemicals from plant biomass

• Biofuels: Production of ethanol from corn starch or sugarcane Harder to transport than petrol Raise of global food prices• Need for high-energy fuel : Fatty-acid derived fuels Energy-rich molecule than ethanol Isolated from plant and animal oils

• More economic route starting from renewable sources - Engineering E. coli to produce fatty esters(bio-disel), fatty al-

chols, and waxes directly from sugars or hemi-cellulose - Cost-effective way of converting grass or crop waste into fuels

• Synthesis takes place in the cytosol• Intermediates covalently linked to acyl carrier protein - Activation of each acetyl CoA. - Acetyl CoA + CO2 Malonyl CoA• Four-step repeating cycle, extension by 2-carbons /cycle – Condensation – Reduction – Dehydration – Reduction

Fatty Acid Biosynthesis

Fatty Acid Synthase (FAS)

• Polypeptide chain with multiple domains, each with distinct enzyme activities required for fatty acid biosynthesis.• ACP( Acyl carrier protein ): - Activator in the fatty acid biosynthesis

- Part of FAS complex• FAS complex: The acyl groups get anchored to the CoA group of ACP by a thioester linkage• Condensing enzyme/β-ketoacyl synthase (K-SH): Part of FAS, CE has a cysteine SH that participates in thioester linkage with the carboxylate group of the fatty acid.• The growing FA chain alternates between K-SH and ACP-SH

Nature Vol. 463 (2010)

Alternative biomass

• Corn starch, sugar cane: currently used• Cheaper renewable sources - Cellulose - Macro algae : Multi-cellular marine algae, sea weed (red, brown, and green algae) - Switch grass

Ascophyllum nodosum

Synthetic Biology

• Design and construction of new biological entities such as enzymes, genetic circuits, and cells or the redesign of existing biological systems.

• Synthetic biology builds on the advances in molecular, cell, and systems bi-ology and seeks to transform biology in the same way that synthesis trans-formed chemistry and integrated circuit design transformed computing.

• The element that distinguishes synthetic biology from traditional molecular and cellular biology is the focus on the design and construction of core com-ponents (parts of enzymes, genetic circuits, metabolic pathways, etc.) that can be modeled, understood, and tuned to meet specific performance crite-ria, and the assembly of these smaller parts and devices into larger inte-grated systems that solve specific problems.

• Artemisinin : extract from the leaves of Artemisia annua, or sweet wormwood. - used for more than 2,000 years by the Chinese as a herbal medicine called qinghaosu.

• The parasite that causes malaria has become partly resistant to every other treatment tried so far. • Artemisinin is still effective, but it is costly and scarce. The supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations• Artemisinin works by disabling a calcium pump in the malaria parasite, Plasmodium falciparum.

Mutation of a single amino acid confers resistance (Nature Struct. Mol. Biol. 12, 628–629; 2005).

• 200 million people infected with malaria each year mainly in Africa, and at least 655,000 deaths in 2010 Treatment : Intravenous or intramuscular quinine

Production of the anti-malarial drug precursor artemisinic acid in engineered yeast

• US $ 43-million dollar grant from the Seattle-based Bill & Melinda Gates Foundation

Malaria

Mosquito-borne infectious disease of humans and other animals caused by protists (a type of unicellular microorganism) of the genus Plasmodium.

Malaria causes symptoms that typically include fever and headache, which in severe cases can progress to coma or death : No effective vaccine exists2

In 2012, 219 million documented cases. Between 660,000 and 1.2 million people died

It begins with a bite from an infected female Anopheles mosquito, which introduces the protists through saliva into the circulatory system.

A motile infective form (called the sporozoite) to a vertebrate host such as a human (the secondary host), thus acting as a transmission vector. A sporozoite travels through the blood vessels to liver cells (hepatocytes), where it reproduces asexually (tissue schizogony), producing thousands of merozoites.

These infect new red blood cells and initiate a series of asexual multiplication cycles (blood schizogony) that produce 8 to 24 new infective merozoites ( 낭충 )

Only female mosquitoes feed on blood; The females of the Anopheles genus of mosquito prefer to feed at night

A Plasmodium in the form that enters humans and other vertebrates from the saliva of female mosquitoes (a sporozoite)

Strategy to engineer the yeast cell to pro-duce the artemisinic acid at cheaper cost

• Engineering the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production: HMG-CoA reductase (3-hydroxy-3-

methyl-glutaryl-CoA reductase); rate-controlling enzyme in the mevalonate pathway that produces cholesterol and other isoprenoids

• Introduction of the amorphadiene synthase (ADS) gene from Artemisia annua, com-monly known as sweet wormwood

• Cloning a novel cytochrom P450 that per-forms a three-step oxidation of amorphadi-ene to Artemisinic acid

from A. annua

Production level : ~ 1.6 g/L by yeast

New pathway in yeast for artemisinic acid production

Improvement of production yield of artemisinic acid

- Production level is too low to be economically feasible

- Discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 g/L of artemisinic acid by yeast.

- Practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment.

- The strains and processes form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization

into active pharmaceutical ingredients.

- Because all intellectual property rights have been provided free of charge, the technology has the potential to increase provision of first-line antimalarial treatments to the

developing world at a reduced average annual price.

Paddon et al., Nature (2013)

Overexpressed genes controlled by the GAL induction system are shown in green. Copper- or methionine-repressed squalene synthase (ERG9) is shown in red. DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; IPP, isopentenyl diphosphate. tHMG1 encodes truncated HMG-CoA reductase. b, The full three-step oxidation of amorphadiene to artemisinic acid from A. annua expressed in S. cerevisiae. CYP71AV1, CPR1 and CYB5 oxidize amorphadiene to artemisinic alcohol; ADH1 oxidizes artemisinic alcohol to artemisinic aldehyde; ALDH1 oxidizes artemisinic aldehyde to artemisinic acid.

Overview of artemisinic acid production pathway

Chemical conversion of artemisinic acid to artemisinin

Cell factory for valuable compounds from renewable biomass

Production of Bio adipic acid from renewable source (C6 feed stock)

Petro-leum

Biopro-cess

Pretreatment of biomass

Biomass

Sugars New Strain Chemi-

cal process

Adipic acid

Adipic acid

Bio-Nylon

Raw material for Carpet Raw material for Nylon

Raw material for various polymers

Electronic materials

Use and Applications

Bio Nylons production : World market $ 10 Billion

Muconic acid derivatives

PEP

E4P

DAHP Chorismic acidDHQ

aro, aroII aroB aroD aroE aroK aroA aroCDHS SA S3P EPSP

p-Hydroxybenzoic acid

tryptophan

prephenate

phenylpyruvate4-hydroxyphenylpyruvate

trpC~AtrpG

Δcsm

pheA::aroFm tyrA::aroGm

phenylalaninetryptophan

tyrB, aspC

protocatecheuate

aroYcatA

catechol

cis,cis-muconic acid

Adipic acid

Chemical synthesis

ubiC

ΔtrpE

pobA

pyruvate

pps

Biosynthesis of cis,cis-muconic acid

pobA: p-hydroxybenzoate hydroxylase

Design of new metabolic pathway in Corynebacterium

Glucose

Shikimic acid pathway

Dihydroxyacetone phosphate

Design and construction of new strain

• Synthetic promoter• Incorporation of new enzymes• Deletion/knock-out of waste pathway• Incorporation of transporter• Control of carbon flux • Cofactor balance

24

Critical point : Balanced synthesis of PEP and E4P

Glucose

Glucose 6-P

Fructose 6-P

Fructose 1,6-P

Digydroxy acetone P

Glyceraldehyde 3-P

3-P Glycerate

Phosphoenolpyruvate(PEP)

Glucono-1,5-lactone 6-P 6-P-Gluconate Ribulose 5-P

Erythrose 4-phosphate(E4P)

Sedoheptulose 7-P

DHAP

Xylulose 5-P

PTS

zwf pgl gnd ru5p

tkt

tal

tka

pgi

pfk

pgk

eno

tis

aroF,G

Pentose phosphate pathway

Glycolysis

Dihydroxyacetone phosphate