Metabolic engineering

Metabolic engineering

• Targeted and purposeful alteration of metabolic pathways found in an organism in order to better understand and use cellular pathways for the production of valuable products

• Practice of optimizing genetic and regulatory processes within cells to increase the cells' production of a substance.

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

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

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

- Since cells use these metabolic networks for their survival, changes can have drastic effects

on the cells' viability. Therefore, trade-offs in metabolic engineering arise between the cells

ability to produce the desired substance and its natural survival needs

• The ultimate goal of metabolic engineering is to design the organism to produce valuable substances on an industrial scale in a cost effective manner

Strategy for metabolic engineering

• Redesign of a existing metabolic pathway: addition, amplification, and/or deletion of enzymes • Broadening substrate spectrum • Deregulation of inhibition / repression by products • Blocking/knock-out of a branch pathway: construction of auxotroph

• Setting up a metabolic pathway for analysis - Identify a desired goal to achieve through the improvement or modification of an organism's metabolism - Databases and references are used to research reactions and metabolic pathways for a target product - If the organism does not contain the complete pathway for the desired product, then genes that produce the missing enzymes are incorporated.

• Analyzing a metabolic pathway - The completed metabolic pathway is modeled mathematically to find the theoretical yield of the product or the reaction fluxes in the cell - A flux is the rate at which a given reaction in the network occurs. - Simple metabolic pathway analysis can be done by hand, but most require the use of software to perform the computations

• Determining the optimal genetic manipulations - Once the fluxes of reactions are solved in the network, then determine which reactions should be altered to maximize the yield of the desired product. - To determine what specific genetic manipulations to perform, computational algorithms, such as OptGene or OptFlux, are used. - Determine which genes should be overexpressed, knocked out, or introduced in a cell to increase the production of the desired product.

General procedure

• Experimental measurements - In order to create a solvable model, it is often necessary to have certain fluxes already known or experimentally measured - To verify the effect of genetic manipulations on the metabolic network (to ensure they align with the model), it is necessary to experimentally measure the fluxes in the network. - To measure reaction fluxes, carbon flux measurements are made using carbon-13 isotopic labeling

Metabolic flux analysis

• Experimental method to examine production and consumption rates of metabolites in a biological system.

• Employing stoichiometric models of metabolism and mass spectrometry methods with isotopic mass resolution, the transfer of moieties containing isotopic tracers from one metabolite into another can be elucidated

• Information about the metabolic network thus derived

• Prediction of limiting steps Maximize the production of a target product.

Metabolic balance analysis

• Important tool for elucidating the flux of certain elements through the metabolic pathways and reactions within a cell.

• An isotopic label is fed to the cell, then the cell is allowed to grow utilizing the labeled feed. For stationary metabolic flux analysis, the cell must reach a steady state (the isotopes entering and leaving the cell remain constant with time) or a quasi-steady state (steady state is reached for a given period of time).

• The isotope pattern of the output metabolite is determined.

• The output isotope pattern provides valuable information, which can be used to find the magnitude of flux, rate of conversion from reactants to products, through each reaction.

Metabolic flux analysis (MFA) using stable isotope labeling

• Technique used to track the passage of an isotope, or an atom with a variation, through a reaction, metabolic pathway, or cell.

• The reactant is 'labeled' by replacing specific atoms by their isotope. • The reactant is then allowed to undergo the reaction. • The position of the isotopes in the products is measured to determine the sequence

the isotopic atom followed in the reaction or the cell's metabolic pathway

Isotopic labeling

• NMR and MS detects isotopic differences, which allows information about the position of the labeled atoms in the products' structure to be determined.

• With the information on the positioning of the isotopic atoms in the products, the reaction pathway the initial metabolites utilize to convert into the products can be determined

• Stable isotope labeling involves the use of non-radioactive isotopes that can act as a tracers used to model several chemical and biochemical systems.

• The chosen isotope can act as a label on that compound that can be identified through nuclear magnetic resonance(NMR) and mass spectrometry(MS).

• Some of the most common stable isotopes are 2H, 13C, and 15N, which can further be produced into NMR solvents, amino acids, nucleic acids, lipids, common metabolites and cell growth media

• The compounds produced using stable isotopes are either specified by the percentage of labeled isotopes (i.e. 30% uniformly labeled 13C glucose contains a mixture that is 30% labeled with 13 carbon isotope and 70% naturally labeled carbon) or by the specifically labeled carbon positions on the compound (i.e. 1-13C glucose which is labeled at the first carbon position of glucose).

• A network of reactions adopted from the glycolysis pathway and the pentose phosphate pathway : the labeled carbon isotope rearranges to different carbon positions throughout the network of reactions.

• The network starts with fructose 6-phosphate (F6P), which has 6 carbon atoms with a label 13C at carbon position 1 and 2.

• 1,2-13C F6P becomes two glyceraldehyde 3-phosphate (G3P), one 2,3-13C G3P and one unlabeled G3P. • The 2,3-13C G3P is reacted with sedoheptulose 7-phosphate (S7P) to form an unlabeled erythrose 4-

phosphate(E4P) and a 5,6-13C F6P. • The unlabeled G3P will react with the S7P to synthesize unlabeled products.

Labeling patterns: Demonstrate the use of stable isotope labeling to discover the carbon atom rearrangement through reactions using position-specific labeled compounds

G3P G3P

Biosynthetic pathway of L-Thr in E. coli

L-Aspartyl phosphate

Homoserine phosphate

Glucose

Phosphenolpyruvate

Pyruvate

TCA cycle Oxaloacetate

ppc

mdh

aceBAK aspC

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

Feedback inhibition Dotted line

Development of an L-Threonine-overproducing strain

• Conventional mutagenic method

• Metabolic engineering:

- Release of product repression/inhibition

- Knock-out of branch pathways: Auxotroph

- Amplification of aspC (aspartate aminotransferase) that

converts oxaloacetate to L-aspartate

• Production level of L-threonine - W3110 (Wild-type E. Coli ) : < 0.001 g/L - Industrial strain: > 150 g/L

Broadening the substrate ranges : 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) - Switchgrass

Ascophyllum nodosum

Construction of organisms capable of utilizing the alternative carbon sources by incorporating the related enzymes

large, common brown algae Switchgrass can be converted into biofuel

Engineering of organisms for biosynthesis of drug precursors

• 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 shortage and price fluctuation.

• Artemisinin works by disabling a calcium pump in the malaria parasite, Plasmodium falciparum. Mutation of a single amino acid confers the 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 : As of 2006, quinine is no longer recommended by the WHO as a first-line

treatment for malaria, and it should be used only when artemisinins are not available

• Nobel prize in 2015: Dr. Tu Youyou for discovering artemisinin from sweet wormwood in China

Anti-malarial drug precursor artemisinic acid in engineered yeast

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

Malaria

• Mosquito-borne infectious disease of human and other animals. - caused by protist (a type of microorganism) of the genus Plasmodium.

• It begins with a bite from an infected female Anopheles mosquito, which introduces protists through saliva into the circulatory system. • A motile infective form (called the sporozoite) is introduced 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 (The form of the malaria parasite that invades red blood cells)

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

• Symptoms: fever and headache, which in severe cases can progress to coma or death.

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

Life cycle of Malaria parasite Plasmodium. Morphology of a sporozoite (The form that enters humans and other vertebrates from the saliva of female mosquitoes)

Strategy to engineer the yeast cell to produce the artemisinic acid at cheaper cost: Isoprenoid biosynthesis pathway - Naturally occurring organic chemicals derived from five-carbon isoprene units

• Engineering the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production

• Introduction of the amorphadiene

synthase (ADS) gene from Artemisia annua, known as sweet wormwood

• Cloning a novel cytochrome P450 that

perform a three-step oxidation of amorphadiene to Artemisinic acid

from Artemisia annua

Production level : ~ 1.6 g/L by yeast

New pathway in yeast for artemisinic acid production

Ro et al. Nature (2009) Hydroxymethylglutaryl-CoA

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 titre 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. - Because all intellectual property rights have been provided free of charge, the technology

has the potential to increase provision of first-line anti-malarial 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. (a) 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

Manufacturing of semisynthetic artemisinin on a commercial scale

• Synthetic sequence by Sanofi • Process development to achieve an efficient, large-scale access to an anti-malaria drug for a price that is at least competitive with a simple extraction from the plant

Biosynthesis of natural alkaloids by yeast

• Alkaloids: Heterocyclic nitrogenous compounds of plant origin that are physiologically active

- The first individual alkaloid, morphine, was isolated in 1804 from the opium poppy

• Natural compounds are widely used as pharmaceuticals, but are still manufactured from

plant extracts:

- Too structurally complex to be cost-effectively produced by total organic synthesis.

- Target metabolites often accumulate at low levels in plants

- Increase in yields : hindered by limitations in plant metabolic engineering, namely

complex pathway regulation, a lack of genetic tools and long development cycles

• Microbial production systems: Overcome many of the barriers and are easy to transform

the manufacturing and drug discovery processes for many natural products

Opiate

• Alkaloid compounds found naturally in the resin of opium poppy plant (Papaver somniferum) • Psychoactive compounds found in the opium plant: morphine, codeine, thebaine

• Opioids: - Substances that act on opioid receptors to produce morphine-like effects. - Most often used medically to relieve pain • All opioids, including the opiates: drugs of high abuse potential and are listed under the Controlled Substances Act of the US.

Morphine

Codeine

Heroin

used to treat pain, as a cough medicine, and for diarrhea

Engineering of yeast

• Opiate-synthesis pathway is long: roughly 18 steps and biochemically complex.

• No whole sequenced genome for the opium poppy is available

• Identifying the enzymes that catalyze the synthesis reactions has been difficult.

• Creation of a yeast strain containing the first half of a biochemical pathway that turns

simple sugars into morphine : mimicking the process by which poppies make opiates

Yeast can convert glucose into the intermediate compound (S)-reticuline : The first half of the poppy’s morphine-production pathway

Pathway potholes

• Key step in the morphine assembly line

- Conversion of L-tyrosine to L-DOPA (L-3,4-dihydroxyphenylalanine) by tyrosine hydroxylase - L-DOPA: precursor to the neurotransmitters dopamine and used to increase the dopamine concentration in the treatment of Parkinson's disease

• Enzymes from other plants immediately convert L-DOPA to dopaquinon, unwanted by-product, that many organisms use to make the pigment melanin

- Copper-containing tyrosinases (EC 1.14.18.1), used by many organisms for melanin

production, exhibit both tyrosine hydroxylase and DOPA oxidase activities to produce L-dopaquinone from L-tyrosine.

• Approach: Search for a yeast-active tyrosine hydroxylase from plants - Cytochrome P450 from the sugar beet and its molecular evolution

L-Tyrosine L-DOPA

First-half of the opiate assembly line

CYP: Cytochrome P450 mutant DODC:DOPA decarboxylase NCS: Norcoclaurine synthase 6OMT: 6-O-methyltransferase CNMT : Coclaurine N-methyltransferase 4′OMT: 4′-O-methyltransferase NMCH: N-methylcoclaurine hydroxylase

Engineered yeast paves way for home-brew heroin

Maximum titer of (S)-reticuline by a 96-h fermentation : ~ 80.6 μg /L

Benefit and issues

• The opium poppy : only commercial source of morphine and opioid painkillers such as oxycodone and hydrocodone.

- The crop must be grown in highly regulated conditions, only in a few countries. - Outside those boundaries, it is grown to supply the illegal heroin trade. - Producing opiates in industrial facilities from yeast eliminate the need for the tightly controlled legal plant-production chain • Proactive examination of the risks and benefits of engineering organisms to make

compounds that are both useful and dangerous

• Drug and biosecurity regulators, law-enforcement agencies, scientists and public-health officials : establish safeguards that minimize risk without quashing research

• Besides providing the alternative morphine-production process, the advance could lead to more-effective, less addictive and cheaper painkillers that could be brewed under

tight controls in fermentation vats.

• Raises concerns about illicit use.

- It could enable widespread, localized production of illegal opiates such as heroin,

increasing people’s access to such drugs.

- Need to prevent the technology’s misuse without hampering further research.

Host cells for the production of valuable molecules

• The use of appropriate expression system for specific products : Each expression system displays its own unique set of advantages and disadvantages

• Many of therapeutic agents currently on the market : produced by recombinant DNA technology using various expression systems such as bacteria, yeast, fungi, and mammalian cells

• Many therapeutic proteins are produced by recombinant DNA technology

Escherichia coli (E. coli)

• Most common microbial species used to produce heterologous proteins

- Heterologous protein : protein that does not occur in host cells

• Some important molecules: Amino acids, organic acids, vitamins

ex) recombinant human insulin (Humulin) in 1982

tPA (tissue plasminogen activator in 1996

L-Threonine, L-Tryptophan, L-lactic acid

• Major advantages of E. coli

- Molecular biology is well characterized

- High level expression of heterologous proteins :

- High expression promoters

- ~ 30 % of total cellular protein

- Rapid growth, simple and inexpensive media, appropriate fermentation technology, large scale cultivation

• Intracellular accumulation of proteins in the cytoplasm

complicate downstream processing compared to extracellular production

additional primary processing steps : cellular homogenization,

subsequent removal of cell debris by filtration or centrifugation

extensive purification steps to separate the protein of interest

• Inclusion body (insoluble aggregates of partially folded protein)

formation via intermolecular hydrophobic interactions

- high level expression of heterologous proteins overload the

normal cellular protein-folding mechanisms

- Nonetheless, inclusion body displays one processing advantage

easy and simple isolation by single step centrifugation

denaturation using 6 M urea

refolding via dialysis or diafiltration

Drawbacks of E. coli

- Prevention of inclusion body formation

- growth at lower temperature (30 oC)

- expression with fusion partner : GST, Thioredoxin, GFP,

- high level co-expression of molecular chaperones

• Inability to undertake post-translational modification, especially glycosylation

: limitation to the production of glyco-proteins

Typical examples of glyco-proteins

• Presence of lipopolysaccharide on its surface : pyrogenic nature

more complicate purification procedure

Yeast

• Saccharomyces cerevisiae, Pichia pastoris :

• Major use for the production of glycoproteins and alcohols

• Major advantages

- Well-characterized molecular biology easy genetic manipulation

- Regarded as GRAS-listed organisms (generally regarded as safe)

Long history of industrial applications ( e.g., brewing and baking)

- Fast growth in relatively inexpensive media, outer cell wall protects them from physical damage

- Suitable industrial scale fermentation equipment/technology is already available

- Post-translational modifications of proteins, especially glycosylation

• Drawbacks

- Glycosylation pattern usually differs from the pattern observed in the native

glycoprotein : highly mannosylation pattern

- Low expression level of heterologous proteins : < 5 %

• Many therapeutic proteins are produced in Yeast

Fungal production system

• Aspergillus niger

• Mainly used for production of industrial enzymes : a-amylase, glucoamylase, cellulase, lipase, protease etc..

• Advantages

- High level expression of heterologous proteins

- Secretion of proteins into extracellular media

easy and simple separation procedure

- Post-translational modifications : glycosylation

- different glycosylation pattern compared to that in human

Animal cells

• Major advantage : Post-translational modifications, especially glycosylation

• Chinese Hamster Ovary (CHO) and Baby Hamster Kidney (BHK) cells

• Many glycoproteins are produced in animal cells

• Drawbacks

- Complex nutritional requirements : Many kinds of growth factors

Expensive

Complicate the purification procedure

- Slow growth rate

- Far more susceptible to physical damage or contamination

- Increased production costs

CHO cells