TBT4170 v2014. Kapittel 4 White Biotechnology: Cells as Synthetic Factories. Part 1

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1 TBT4170 v2014 Kapittel 4 White Biotechnology: Cells as Synthetic Factories Part 1 1

2 The single definition, OECD Biotechnology The application of science and technology to living organisms, as well as parts, products and models thereof, to alter living or non-living materials for the production of knowledge, goods and services. Red biotechnology Green biotechnology White biotechnology Blue biotechnology healthcare biotechnology, biomedicines, gene therapy, genetic testing, stem cell research agrifood biotechnology, GM seeds, GM plants industrial biotechnology, bioprocesses, biomaterials, contained use of GM microorganisms and use of renewable raw materials to substitute fossil raw materials Marine 2

3 Industrial Bioprocessing - Industrial Biotechnology - Industrial Microbiology 3

4 100 ml m3

5 Industrial fermentation products Traditional products (e.g. bread, beer) Agricultural products (e.g. fungicide) Amino acids (glutamate, lysine) Enzymes (carbohydrases, cellulases, lipases, pectinases, proteases) Fuels and chemical feedstock (butanol, ethanol, methane) Nucleotides (IMP, GMP) Organic acid (acetic, citric, lactic) Pharmaceutical and related products (alkaloids, antibiotics, vitamins, polymers) SCP (single cell protein) From g to ton scale l to l scale??? / kilo to??? / g 5

6 4.1 An Overview - reading material Anabolism vs Catabolism

7 Figure 4.14 Brock glycolytic enzymes/ cell Glucose + 2NAD 2Pyruvate + 2ATP + 2NADH 2Pyruvate + 2NADH 2Ethanol + 2CO 2

8 Figure 4.21 Pyruvate (three carbons) Acetyl-CoA C 2 C 4 C 5 C 6 Energetics Balance Sheet for Aerobic Respiration Oxalacetate 2 Citrate 3 Aconitate 3 Malate 2 Isocitrate 3 Fumarate 2 Succinate 2 -Ketoglutarate 2 Succinyl-CoA NADH Elektrontransportkjede Protongradient ATPase ATP dannelse

9 Glukose + 2ADP + 2NAD + + 2P i 2Pyr + 2ATP + 2NADH + 2H + Glukose + 2ADP + 4NAD + + 2P i + 2CoA 2Ac-CoA + 2CO2 + 2ATP + 4NADH + 4H + Glukose + 4ADP + 8NAD + + 2NADP + + 2FAD + 4P i 6CO2 + 4ATP + 8NADH + 2NADPH +2FADH H + or C 6 H 12 O 6 + 6H 2 O 24(H) + 6CO 2 Fig Comparison of respiration and fermentation of a glucose molecule

10 4.2 Tactical adaption -uses the enzymes available, activating or inhibiting them chemically or physically. These process permit short-term reactions, sometimes within a fraction of a second. Control of enzyme activity Inactivated/ activated by covalent modification Activity is modulated by reversible association with another molecule (ligand is small molecule, modulator if it is a large one) Allosteric enzymes: proteins that have, in addition to active site, another site with affinity for binding small molecules. Allosteric effectors are thought to work by changing the conformational state of the enzyme, Result: the enzyme exist in two conformational states: one with a hig affinity for the substrate and one low affinity - Negative allosteric effectors stabilize low-affinity conformation - Positive allosteric effectors stabilize high-affinity conformation Important: allosteric interactions provide a means for the activity of an enzyme to be modified by substances not even remotely resembling the substrates or products of the enzyme itself Feed-back inhibition: the first enzyme in a biosynthetic pathway is allosteric and its negative allosteric effector is the end product in the pathway. Generally, the end product acts as a noncompetitive step in the pathway Feed-back inhibition maintains constant internal concentrations of building blocks in the face of changes in demands and availability 13

4.3 Strategic adaptation: Enzyme production on demand -is a response to general changes in living conditions and is genetically controlled Constitutive enzymes - produced at an approximately steady

11 4.3 Strategic adaptation: Enzyme production on demand -is a response to general changes in living conditions and is genetically controlled Constitutive enzymes - produced at an approximately steady rate, essential metabolic enzymes Inducible enzymes - only produced when needed Three enzymes for lactose degradation induced by addition of lactose and in absence of a more preferred carbon source (catabolic repression) Repression: a mechanism that stops the further production of a compound when large amounts of the end product are already available (see 4.5) 16

12 4.4 An Allosteric Molecular Computer Glutamine Synthetase -reading material 4.5 Catabolite Repression or Fishing for Polymerase Whenever glucose is abundant, bacteria usually ignore other sources of carbohydrate camp - a second messenger CAP catabolite activator protein is activated by camp, then activates and stimulates enzymes that degrade nonglucose nutrients In contrast to the negative lactose induction process, catabolite repression is a positive control mechanism The camp/cap complex enhances the reading of DNA by polymerase 50-fold 17

13 Reading Material Box 4.1 Biotech History: Aspergillus niger The End of the Italian Monopoly Box 4.2 Biotech History: Boyd Woodruff and the Second Important Antibiotic Box 4.3 The Expert s View: High-grade Cysteine No Longer Has to Be Extracted From Hair 4.6 Mold Replacing Lemons - Aspergillus niger (fungi) based process to replace citrus fruit extraction - Acidic medium and reduction of free iron < 0.5 mg/l enhances citric acid production (inhibit the citric acid degrading enzyme aconitase and low ph facilitate the excretion of citric acid). The process is not that vulnerable for contamination either. See Fig to 500 m 3 fermenters, capacity to turn 85% of raw material into product (what about time?) 18

14 Traditional development of bioprocesses: (In particular microbial ) 1. Identification and isolation of organism which produces the particular biochemical 2. Strain development overproducing strains* with commercial potential, classical mutagenesis and creative selection strategies to isolate promising overproducing strains 3. Lab-and pilot scale, optimization of fermentation conditions and development of downstream processing 4. Industrial scale and commercial production * Wild type strains isolated from Nature do not produce significant amounts of wanted biochemcial, eg. Penicillin production has been increased from mg/l to > 90 g/l see Box

15 A. Wild type B. Optimization of fermentation conditions A. Glucose B. Glucose Product Z Product Z Biomass + CO 2 Biomass + CO 2 C. Metabolic engineering Classical mutagenesis D. Genetic/ metabolic engineering C. Glucose D. Glucose Product Z Product X Biomass + CO 2 Biomass + CO 2 21

16 4.7 Overproduction of Lysine How Mutants Outwit the Feedback Inhibition of Aspartate Kinase The overproducing strains of Corynebacterium glutamicum selected for lysine production have defects in feedback control mechanisms in the lysine biosynthetic pathway. They lack homoserine dehydrogenase activity (hom) and are thus homoserine auxotrophs. These auxotrophs convert all aspartate semialdehyd (ASA) to lysine, and because of the lack of threonine synthesis there is no longer feedback control. However, carefully measured amounts of threonine must be added to the culture medium to enable the auxotrophic mutant to grow. 22

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18 4.8 L-Glutamate «Levorotory» Soup Seasoning in Abundance Glutamate (E620-glutamic acid, E621-monosodium glutamate) flavour enhancer, has key role in the umami taste (in addition to disodium inosinate (IMP) and disodiumguanylate (GMP)) Largest producer in the world is the Japanese company Ajinomoto (means flavour enhancer in japanese) using the same bacterium as for lysine production: Corynebacterium glutamicum, other «glutamic acid producing» bacteria have been isolated later For genetic reasons, the oxoglutarate dehydrogenase (OGD) of C. corynebacterium in TCA is not very active -> 2-oxoglutarate ( - KG) accumulates OGD Transaminase/ Glutamate dehydrogenase Glutamic acid + NH 3 See Box 1.4 p

19 The overall strategy for achieving overproduction of the amino acid involves: Increasing the activity of anabolic enzymes Manipulation of regulation to remove feedback control mechanisms Blocking pathways that lead to unwanted byproducts and that results in degradation of target product Limiting the ability to process the immediate precursor of L-glutamic acid, namely oxoglutaric acid, to the next intermediate of the TCA cycle (succinyl-coa). Secretion of Glutamate is important for achieving over-production In addition, as these bacteria do not normally secrete glutamate, a range of treatments are employed to render the cells more permeable and aid release of the amino acid into the medium. These treatments include: biotin limitation, restriction of phospholipids biosynthesis by adding C 16 -C 18 saturated fatty acids during the growth phase and inclusion of surfactants and penicillin in the production media. All glutamate overproducers are natural biotin auxotrophs. 27

20 Addition of penicillin to cells grown in high biotin resulted in excretion of glutamate: i.e. growth in presence of non limiting levels of biotin result in a cell membrane permeability barrier that restricts the excretion of glutamate, and inhibition of cell wall biosynthesis by penicillin alters the permeability properties of the cell membrane and allows glutamate to flow out easily These manipulations (also addition of fatty acid surfactants) result in a phospholipiddeficient cytoplasmic membrane, which favors active excretion of glutamate of the cell. not pensum Glutamate excretion cells have very low level of cell lipids, especially phospholipids. Later found that the various manipulations leading to glutamate overproduction increased permeability of the mycolic acid layer of the cell wall 28

Chap 14 Amino Acids. I. Amino acids: Introduction

Chap 14 Amino Acids. I. Amino acids: Introduction Chap 14 Amino Acids Introduction Biotechnology has numerous applications: Medicine: the advent of genetic engineering has advanced the drug design to molecular levels. Molecular biology methods and genomics-based