Metabolic engineering

1. Metabolic engineering

2. Metabolic engineering • Targeted and purposeful alteration of metabolic pathways 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 to achieve a goal. • Current focus is to target the regulatory networks in a cell to efficiently engineer the metabolism

4. Glucose Phosphenolpyruvate ppc Pyruvate aspC aceBAK Oxaloacetate TCA cycle mdh Biosynthetic pathway of L-Thr in E. coli 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 Feedback repression Feedback inhibition ilvA L-Isoleucine

5. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass • Biofuels : Relied on manufacturing ethanol from corn starch or sugarcane  Harder to transport than petroleum, low energy density 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 (biodisel), fatty alcohols, and waxes directly from sugars or hemi-cellulose - Cost-effective way of converting grass or crop waste into fuels

6. Increased production of free fatty acids and Acyl-CoAs overexpressing TES and ACL, and by eliminating b-oxidation (ΔfadE) • Fatty alcohols are produced directly from fatty acyl-CoAs by overexpressing FAR (fatty acyl-Coareductase • Esters are produced by overexpressing AT (acyltransferase) Nature, 463 (2010)

7. Alternative biomass Macro algae : Multi-cellular marine algae, sea weed (red, brown, and green algae) Switch grass Ascophyllumnodosum

8. Direct biofuel production from Brown macro-algae by an engineered microorganism • Corn and sugarcane: industrial feedstock • Food versus fuel concerns preclude their long-term use • Lignocellulosic materials: Preferred feedstock, but fermentation of the simple sugars in lignocellulose are costly and complex  Needs for energy-intensive pretreatment and hydrolysis processes

9. Marine macro-algae(seaweeds) : next generation feedstock • Brown macroalgae as an ideal feedstock for production of biofuels and renewable commodity chemicals • Requiring no arable land, fertilizer, or fresh water resources • No economic concerns associated with land management • Avoids adverse impact on food supplies • Large-scale cultivation is practices in several countries, yielding 15 million metric tons per year • Contains no lignin, and sugars can be released by simple operations such as milling or crushing • Most abundant sugars in brown macroalgae : alginate, mannitol, and glucan ( glucose polymers) • Alginate : a linear block copolymer of two uronic acids, Β-D-mannuronateand α-L-guluronate

10. Potential of ethanol production from macroalgae :  limited by the inability of industrial microbes to metabolize the alginate components • The discovery of the genes from Vibrio splendidus encoding enzymes for alginate transport and metabolism • Construction of a microorganism with the capacity for degrading, up-taking, and metabolizing alginates • Expression of the genes in E. coli for the production of ethanol from macroalgae Science, 335 (2012)

14. Production of the anti-malarial drug precursor artemisinicacid in engineered yeast 300 million to 500 million people infected with malaria each year mainly in Africa Parasite that causes malaria has become at least partly resistant to every other treatment tried so far. Artemisininis still effective, but it is costly and scarce. Artemisinin : Extracted from the leaves of Artemisia annua, or sweet wormwood, and has been used for more than 2,000 years by the Chinese as a herbal medicine called qinghaosu.

15. Artemisinin works by disabling a calcium pump in the malaria parasite • Mutation of a single amino acid was sufficient to confer resistance (Uhlemanet al. Nature Struct. Mol. Biol. 12(2005)

16. 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 • Introduction of the amorphadiene • synthase (ADS) gene from Artemisia • annua, commonly known as sweet • wormwood • Cloning a novel cytochrom P450 that perform a three-step oxidation of amorphadiene to Artemisinic acid from A. annua Production level : ~ 100 mg/L by yeast culture Ro et al., Nature (2009)

17. 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.