Engineering of Biological Processes Lecture 2: Biosynthesis

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Engineering of Biological ProcessesLecture 2
Biosynthesis

• Mark Riley, Associate Professor
• Department of Ag and Biosystems Engineering
• The University of Arizona, Tucson, AZ
• 2007

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Objectives Lecture 2
Biosynthetic processes (anabolic) Precursors for
structural and functional compounds Case studies
- proteins cholesterol
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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/protein
production.shtml
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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)

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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.

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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

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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?

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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

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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

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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)

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Formation of AAs

• 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

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Anabolic processes - Biosynthesis
Glucose
Glucose 6-Phosphate
Phosphogluconate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Glyceraldehyde 3-Phosphate
Phosphoenolpyruvate
Acetaldehyde
Pyruvate
Lactate
TCA cycle
Acetyl CoA
Acetate
Ethanol
Citrate
Oxaloacetate
Isocitrate
Malate
a-Ketoglutarate
Fumarate
Succinate
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Amino acid biosynthesis is regulated by feedback
inhibition
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Types of feedback control

• 1) Sequential feedback control

Inhibited by Y
Inhibited by Z
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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).

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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

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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.

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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.

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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

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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)
• Alzheimers disease (20 M people)
• Forms amyloid b plaques
• Mis-folded (or un-folded) proteins which are
  remarkably resistant to proteolysis

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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

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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.

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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.

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2 state model
PU PN 1
PN is 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.
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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?

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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)

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Biosynthesis of lipids and hormones

• Biological membranes are composed of
• phosphoglycerides
• sphingolipids
• cholesterol

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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.
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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

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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 (lt 0.94 g/ml) because they
  are rich in triacylglycerols and low in protein
  (lt2).

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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

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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

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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

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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

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

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HW 1 questions

1. 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.
2. 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?