Engineering of Biological Processes Lecture 2: Biosynthesis

1. Engineering of Biological ProcessesLecture 2: Biosynthesis Mark Riley, Associate Professor Department of Ag and Biosystems Engineering The University of Arizona, Tucson, AZ 2007

2. Objectives: Lecture 2 Biosynthetic processes (anabolic) Precursors for structural and functional compounds Case studies - proteins & cholesterol

3. Anabolic processes • Biosynthesis – builds larger molecules from smaller ones • formation of cellular components • amino acids for proteins • storage of sugars (glycogen) • nucleic acids • lipids and hormones • cholesterol and vitamins • growth and mineralization of bone and increase of muscle mass. http://www.doegenomestolife.org/technology/proteinproduction.shtml

4. Integration of metabolism • Universal energy currency • ATP generated by oxidation of fuel molecules (glucose, fatty acids, amino acids) • Biosynthesis vs. degradation • NADH primary reducing power for degradative reactions • NADPH is the major electron donor in reductive biosyntheses • Biosynthetic and degradative pathways are almost always distinct • Biomolecules are constructed from a small set of building blocks (often components of catabolic cycles)

5. Is ATP a high energy compound? • No, it has an intermediate level of energy compared with other biological molecules. • The DG for hydrolysis is intermediate compared to that for other reactions. • The energy released in cleaving ATP is used to support reactions that are normally thermodynamically unfavorable.

6. Example • Synthesis of glutamine from glutamate • Glutamate- + NH4+ Glutamine • DG= + 14.2 kJ/mol – not thermodynamically favored • 2 step process • Glutamate- + ATP 5 Phosphoglutamate + ADP • 5 Phosphoglutamate + NH4+ Glutamine + Pi • Overall: • Glutamate- + ATP + NH4+ ADP Glutamine + Pi • DG = -16.3 kJ/mol

7. Manufacturing biological products • Cell • Environment (T, pH, flow, O2) • Nutrients (sugars, amino acids) • Control scheme • nutrient feeding, product removal, cell growth • Bioseparation train • Integration plan • how does this all work?

8. How to stimulate production of desired compounds • Generate a lot of precursor molecules • Turn off degradative pathways and / or pathways which consume precursor to make other products

9. Hormones - molecular signals that switch metabolism • Classic anabolic hormones include • * Growth hormone • * IGF1 and other insulin-like growth factors • * Insulin • * Testosterone • * Estrogen • Classic catabolic hormones include • * Cortisol • * Glucagon • * Adrenaline and other catecholamines • * Cytokines

10. Amino acids are precursors for many biomolecules • Building blocks for proteins (of course) • Purines (adenine, Base A in DNA) • Pyrimidines (cytosine, Base C in DNA) • Histamine (potent vasodilator) • Nicotinamide (NAD) • The amino acid glycine + acetate is used to form porphyrins (heme groups, hemoglobin)

11. Formation of AA’s • Non-essential amino acids • formed by fairly simple reactions • Essential amino acids • produced through complex pathways • humans and most mammals do not have the necessary enzymes to produce these

12. Glycolysis NADH NADH CO2+NADH GTP CO2+NADH GDP+Pi FADH2 Anabolic processes - Biosynthesis Glucose Glucose 6-Phosphate Phosphogluconate Fructose 6-Phosphate Fructose 1,6-Bisphosphate Glyceraldehyde 3-Phosphate Glyceraldehyde 3-Phosphate Phosphoenolpyruvate Acetaldehyde Lactate Pyruvate TCA cycle Acetyl CoA Acetate Ethanol Citrate Oxaloacetate Isocitrate Malate a-Ketoglutarate Fumarate Succinate

13. Oxaloacetate a-Ketoglutarate Aspartate Glutamate Asparagine Methionine Threonine Lysine Glutamine Proline Arginine Isoleucine Pyruvate Phosphoenolpyruvate 3-Phosphoglycerate Alanine Valine Leucine Serine Phenylalanine Tyrosine Tryptophan Ribose 5-phosphate Glycine Cysteine Tyrosine Histidine

14. Inhibited by isoleucine Threonine a-Ketobutyrate Isoleucine Amino acid biosynthesis is regulated by feedback inhibition

15. D → E → Y A →B → C F → G → Z Types of feedback control • 1) Sequential feedback control Inhibited by Y Inhibited by Z

16. Protein production • Central dogma of biology • DNA → RNA → Protein • Proteins are composed of 20 base amino acids arranged in a specific sequence • After being produced, proteins must fold properly (a-helices, b-sheets) and be post-translationally modified (phosphoryl, carboxy, carbohydrates).

17. Steps in protein production • DNA is transcribed by RNA polymerase generating an mRNA sequence • In prokaryotes, the mRNA requires no further processing • Since prokaryotes lack a nucleus, transcription and translation to protein occur in a common compartment • Translation often begins before mRNA synthesis has been completed • In eukaryotes, the mRNA receives a 5’ cap, 3’ poly-A tail, and is spliced to remove introns from the primary RNA transcript

18. Steps in protein production • Protein synthesis is performed by the ribosome which reads the base sequence of the mRNA • Ribosomes in bacteria add 20 amino acids / sec. • Ribosomes are composed of 2/3 RNA and 1/3 protein making them really ribozymes • In general, the synthesis of most protein molecules can occur in 20 sec – 5 min, although multiple ribosomes may act on each mRNA, thus speeding production.

19. Steps in protein production • Proteins must fold into the proper 3-D shape in order to be functional. • Secondary structures • a-helix, b-sheet, b-turn, random coil • Folding begins while the protein is being synthesized. • Molecular chaperones help guide the folding of many proteins. • Classified as heat shock proteins (hsp60, hsp70) • Recognize exposed hydrophobic patches on proteins and serve to prevent protein aggregation (hydrophobic protein-protein interactions) • Synthesized at higher rates after cells are exposed to elevated temperatures.

20. Steps in protein production • Incompletely folded proteins are digested and degraded • Ubiquitin-conjugation marks proteins for degradation • Roughly 1/3 of all newly made proteins are marked for degradation using quality control processes. • Some proteins (and their activity) are controlled by a regulated rate of destruction • Mitosis related proteins

21. Abnormally folded proteins • Proteins that are not properly folded can cause disease in humans • Prion disease • Creutzfeldt-Jacob disease (CJD) • Bovine spongiform encephalopathy (BSE- mad cow) • Alzheimer’s disease (20 M people) • Forms amyloid b plaques • Mis-folded (or un-folded) proteins which are remarkably resistant to proteolysis

22. Kinetics of protein folding • Proteins do not fold by trying all of the available possible conformations (takes MUCH too long). • Must be some rational process through which proteins fold • Many small, monomeric proteins show wide variation in folding rates, from microseconds to seconds. • What determines the rate of folding? • chain length (# of amino acids) • topology (shape and structure formed) • Proteins with similar shapes (topology) may have different amino acid sequences and so have different folding rates

23. Kinetics of protein folding • Consider a protein with 100 AA's (residues). • If each residue can assume 3 different positions, the total number of structures is 3100 = 5x1047. • If it takes 10-13 seconds to test each structure, the protein would reach its native configuration in 1.6x1027 years.

24. Kinetics of protein folding • 3 state • unfolded, intermediate (partially folded), folded • this was the long standing assumption of how proteins searched through the possible folded states • the intermediate can consist of microdomains that are properly folded • 2 state • unfolded, folded • stable intermediates are not a prerequisite for the fast, efficient folding of proteins and may in fact be kinetic traps and slow the folding process.

25. 2 state model PU + PN = 1 PNis the fraction of protein in its native state N; PU is the fraction of protein in the unfolded state U. The folding rate is kf the unfolding rate is ku.

26. What controls the amount of protein produced? • The answer depends on what type of protein you are trying to produce • Is it constitutively produced? • Is it linked to the cell's normal metabolic or reproductive properties? • Have you engineered the microbe to generate the protein? If so, what kind of promoter is used and how is it induced?

27. Inhibitors of protein synthesis • Many of the most effective antibiotics work by inhibiting protein synthesis in prokaryotic cells • Tetracycline – blocks binding of aminoacyl tRNA • Streptomycin – prevents chain elongation • Chloramphenicol – blocks peptidyl transferase • Erythromycin – blocks translocation of ribosomes • Cycloheximide - blocks translocation of ribosomes (but only in eukaryotes)

28. CH3 CH3 HO Biosynthesis of lipids and hormones • Biological membranes are composed of • phosphoglycerides • sphingolipids • cholesterol

29. Cholesterol is synthesized from acetyl coenzyme A (acetyl CoA) Acetate → mevalonate → isopentenyl pyrophosphate → C2 C6 C5 squalene → cholesterol C30 C27 Squalene is composed of 6 isoprene (C5) units. Synthesis of mevalonate is the committed step in the process. This reaction is the site of feedback regulation.

30. Cholesterol synthesis • Cholesterol can be obtained through the diet or produced in the liver • An adult on a low cholesterol diet typical will produce 800 mg of cholesterol per day • Most mammalian cells (except liver) do not produce cholesterol, but need to uptake from their environment • The liver is the primary source of cholesterol, but some is also made in the intestine

31. Cholesterol uptake • Triacylglycerols (fat), cholesterol, and other lipids obtained from the diet are carried from the intestine to adipose tissue and liver by large chylomicrons (80-500 nm in size). • Their density is low (< 0.94 g/ml) because they are rich in triacylglycerols and low in protein (<2%).

32. Plasma lipoproteins carry fat and cholesterol into cells • Lipoprotein Core lipids Mechanisms of lipid delivery • Chylomicron triacylglycerol hydrolysis by lipoprotein lipase • Very low density • lipoprotein (VLDL) triacylglycerols hydrolysis by lipoprotein lipase • Intermediate-density receptor-mediated endocytosis by • lipoprotein (IDL) cholesterol esters liver and conversion to LDL • Low-density receptor-mediated endocytosis by • lipoprotein (LDL) cholesterol esters liver and other tissues • High-density transfer of cholesterol esters to • lipoprotein (HDL) cholesterol esters IDL and LDL

33. High-density lipoprotein (HDL) • Circulate continuously in plasma • Contain an enzyme, • phosphatidyl choline cholesterol acyltransferase • that converts free cholesterols to cholesterol esters • aids in the transport of cholesterol

34. Low density lipoprotein (LDL) • The LDL receptor on the cell surface controls the uptake of LDL • The cholesterol content of cells having an active LDL pathway is regulated by: • injected and released cholesterol suppresses production of new LDL receptors • the LDL receptor itself is subject to feedback regulation

35. Biosynthesis of cholesterol • Acetoacetyl CoA + Acetyl CoA → mevalonate + CoA • C4 C2 C6 • mevalonate + 3 ATP → isopentyl pyrophosphate + CO2 + Pi + 3 ADP • C6 (C5, contains 2 Pi) • 3 isopentyl pyrophosphate → farnesyl pyrophosphate • C5 C15 • 2 farnesyl pyrophosphate → squalene + 4 Pi • C15 C30 • squalene → cholesterol + 3 CO2 • C30 C27

36. Steroid hormones are derived from cholesterol Cholesterol (C27) Pregnenolone (C21) Progestagens (C21) Glucocorticoids (C21) Androgens (C19) Mineralocorticoids (C21) Estrogens (C18)

37. Pregnenolone Progesterone Cortisol (hydrocortisone) Androstenedione Testosterone Estrone Estradiol

38. How to stimulate production of hormones • Generate a lot of cholesterol • By: • Turning off degradative pathways or pathways which consume precursor to make other products

39. HW #1 questions • What kind of cell would you use to produce androstenedione? Your answer should describe the attributes of such a cell (don't just state, "a cell that produces andro"). An answer longer than 4 sentences is too much. • Producing cholesterol is an energy intensive process. How much energy (in terms of # of ATP molecules) is consumed in producing one cholesterol molecule from a source of glucose?