Engineering of Biological
Processes
Lecture 2: Biosynthesis
Mark Riley, Associate Professor
Department of Ag and Biosystems
Engineering
The University of Arizona, Tucson, AZ
2007
Objectives: Lecture 2
Biosynthetic processes (anabolic)
Precursors for structural and functional
compounds
Case studies - proteins & cholesterol
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
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)
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.
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+
DG = -16.3 kJ/mol
ADP Glutamine + Pi
Manufacturing biological products
1.
2.
3.
4.
Cell
Environment (T, pH, flow, O2)
Nutrients (sugars, amino acids)
Control scheme
nutrient feeding, product removal, cell growth
5. Bioseparation train
6. Integration plan
how does this all work?
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
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
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)
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
Anabolic processes - Biosynthesis
Glycolysis
Glucose
Glucose 6-Phosphate
Phosphogluconate
Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate
Glyceraldehyde
3-Phosphate
Phosphoenolpyruvate
Acetaldehyde
Lactate
Pyruvate
TCA cycle
NADH
Ethanol
Acetate
Acetyl CoA
Citrate
Oxaloacetate
NADH
Isocitrate
Malate
CO2+NADH
a-Ketoglutarate
Fumarate
GTP
Succinate
FADH2
GDP+Pi
CO2+NADH
a-Ketoglutarate
Oxaloacetate
Glutamate
Aspartate
Glutamine Proline Arginine
Asparagine Methionine Threonine Lysine
Pyruvate
Isoleucine
Phosphoenolpyruvate
Alanine Valine
Leucine
3-Phosphoglycerate
Tyrosine
Serine
Glycine
Phenylalanine Tyrosine Tryptophan
Cysteine
Ribose 5-phosphate
Histidine
Amino acid biosynthesis is
regulated by feedback inhibition
Inhibited by
isoleucine
Threonine
a-Ketobutyrate
Isoleucine
Types of feedback control
1) Sequential feedback control
Inhibited by Y
D→E →Y
A→B →C
F→G→ Z
Inhibited by Z
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).
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
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.
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.
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
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
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
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.
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.
2 state model
dPN
 k fPU - kuPN
dt
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.
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?
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)
Biosynthesis of lipids and
hormones
• Biological membranes are composed of
– phosphoglycerides
– sphingolipids
– cholesterol
CH3
CH3
HO
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.
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
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%).
Plasma lipoproteins carry fat and
cholesterol into cells
Lipoprotein
Chylomicron
Core lipids
triacylglycerol
Mechanisms of lipid delivery
hydrolysis by lipoprotein lipase
Very low density
lipoprotein (VLDL)
triacylglycerols
hydrolysis by lipoprotein lipase
Intermediate-density
lipoprotein (IDL)
cholesterol esters
receptor-mediated endocytosis by
liver and conversion to LDL
Low-density
lipoprotein (LDL)
cholesterol esters
receptor-mediated endocytosis by
liver and other tissues
cholesterol esters
transfer of cholesterol esters to
IDL and LDL
High-density
lipoprotein (HDL)
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
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
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
Steroid hormones are derived from
cholesterol
Cholesterol (C27)
Pregnenolone (C21)
Progestagens (C21)
Glucocorticoids (C21)
Androgens (C19)
Mineralocorticoids (C21)
Estrogens (C18)
Pregnenolone
Progesterone
Cortisol
(hydrocortisone)
Androstenedione
Testosterone
O
OH
CH3
CH3
O
O
Estrone
Estradiol
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
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?