Metabolic engineering

1. Metabolic engineering

2. Metabolic engineering • Targeted and purposeful alteration of metabolic pathways found in an organism in order 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 enzymes • Current focus is to target the regulatory networks in a cell to efficiently engineer the metabolism

4. Biosynthetic pathway of L-Thr in E. coli Glucose Phosphenolpyruvate ppc Pyruvate aspC aceBAK Oxaloacetate TCA cycle mdh metL L-Aspartate thrA lysC L-Aspartyl phosphate asd dapA L-Aspartate semidaldehyde L-Lysine thrA metA Homoserine L-Methionine thrB Homoserine phosphate thrC L-Threonine ilvA Feedback repression L-Isoleucine

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

6. Fatty Acid Biosynthesis • 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

8. 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

10. Nature Vol. 463 (2010)

11. 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 Ascophyllumnodosum

12. 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 biology and seeks to transform biology in the same way that synthesis transformed 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 components (parts of enzymes, genetic circuits, metabolic pathways, etc.) that can be modeled, understood, and tuned to meet specific performance criteria, and the assembly of these smaller parts and devices into larger integrated systems that solve specific problems.

13. 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 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. Theparasite 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

14. 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

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

16. New pathway in yeast for artemisinicacid production • Strategy to engineer the yeast cell to produce the artemisinic acid at cheaper cost • Engineering the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production: HMG-CoAreductase (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, commonly known as sweet wormwood • Cloning a novel cytochrom P450 that performs a three-step oxidation of amorphadiene to Artemisinic acid • from A. annua Production level : ~ 1.6 g/L by yeast

17. 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. Paddonet al., Nature (2013)

18. Overview of artemisinic acid production pathway • Overexpressed genes controlled by the GAL induction system are shown in green. Copper- or methionine-repressed • squalenesynthase (ERG9) is shown in red. DMAPP, dimethylallyldiphosphate; FPP, farnesyldiphosphate; IPP, • isopentenyldiphosphate. tHMG1 encodes truncated HMG-CoA reductase. b, The full three-step oxidation of • amorphadieneto artemisinic acid from A. annuaexpressed in S. cerevisiae. CYP71AV1, CPR1 and CYB5 oxidize • amorphadieneto artemisinic alcohol; ADH1oxidizes artemisinic alcohol to artemisinic aldehyde; ALDH1 oxidizes • artemisinicaldehyde to artemisinic acid.

19. Chemical conversion of artemisinic acid to artemisinin

20. Cell factory for valuable compounds from renewable biomass Bio-Nylon Production of Bio adipic acid from renewable source(C6 feed stock) Petroleum Adipic acid Bioprocess Pretreatment of biomass Chemical process Adipic acid New Strain Biomass Sugars

21. Use and Applications Bio Nylons production : Worldmarket $ 10 Billion

22. Muconic acid derivatives

23. Biosynthesis of cis,cis-muconic acid tryptophan phenylalanine tyrB, aspC Design of new metabolic pathway in Corynebacterium 4-hydroxy phenylpyruvate phenylpyruvate tryptophan Glucose pheA::aroFm tyrA::aroGm trpC~A trpG pyruvate prephenate Shikimic acid pathway pps Δcsm ΔtrpE PEP aro, aroII aroK aroB aroD aroE aroA aroC DAHP SA S3P DHQ DHS EPSP Chorismic acid E4P Dihydroxyacetone phosphate ubiC pobA:p-hydroxybenzoate hydroxylase cis,cis-muconic acid pobA aroY catA Chemical synthesis catechol protocatecheuate p-Hydroxybenzoic acid Adipic acid

24. 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

25. Critical point : Balanced synthesis of PEP and E4P Glucose Pentose phosphate pathway PTS Glucose 6-P Glucono-1,5-lactone 6-P 6-P-Gluconate Ribulose 5-P zwf pgl gnd ru5p pgi Fructose 6-P tkt Xylulose 5-P Erythrose 4-phosphate (E4P) pfk Fructose 1,6-P tka tal Sedoheptulose 7-P Digydroxy acetone P Glyceraldehyde 3-P tis pgk Glycolysis 3-P Glycerate aroF,G eno Phosphoenolpyruvate (PEP) DHAP Dihydroxyacetone phosphate