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 G 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
G= + 14.2 kJ/mol not thermodynamically favored
2 step process
Glutamate- + ATP
5 Phosphoglutamate + ADP
5 Phosphoglutamate + NH4+
Glutamine + Pi
Overall:
Glutamate- + ATP + NH4+
G = -16.3 kJ/mol

ADP Glutamine + Pi

Manufacturing biological products

1. Cell
2. Environment (T, pH, flow, O2)
3. Nutrients (sugars, amino acids)
4. 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 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

Anabolic processes - Biosynthesis

Glycolysis
Glucose

Glucose 6-Phosphate

Phosphogluconate

Fructose 6-Phosphate
Fructose 1,6-Bisphosphate
Glyceraldehyde 3-Phosphate

Glyceraldehyde
3-Phosphate

Phosphoenolpyruvate
Lactate

Acetaldehyde

Pyruvate

TCA cycle

NADH

Ethanol

Acetate

Acetyl CoA
Citrate

Oxaloacetate
NADH

Isocitrate

Malate

CO2+NADH

-Ketoglutarate

Fumarate

GTP

Succinate
FADH2

GDP+Pi

CO2+NADH

-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

-Ketobutyrate

Isoleucine

Types of feedback control

1) Sequential feedback control
Inhibited by Y

DE Y
AB C
FG 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 (helices, -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
-helix, -sheet, -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)

Alzheimers disease (20 M people)

Forms amyloid 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 3 100
= 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
Mechanisms of lipid delivery
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

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 + CO 2 + Pi + 3 ADP
C6
(C5, contains 2 Pi)
3 isopentyl pyrophosphate farnesyl pyrophosphate
C5 C15
2 farnesyl pyrophosphate squalene + 4 P i
C15
C30
squalene cholesterol + 3 CO 2
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

OH

CH3

CH3

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?