Engineering of Biological Processes

Lecture 2: Biosynthesis

Mark Riley, Associate ProfessorDepartment of Ag and Biosystems

EngineeringThe 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 glutamateGlutamate- + NH4

+ Glutamine G= + 14.2 kJ/mol – not thermodynamically favored

2 step processGlutamate- + ATP 5 Phosphoglutamate + ADP5 Phosphoglutamate + NH4

+ Glutamine + Pi

Overall:Glutamate- + ATP + NH4

+ ADP Glutamine + Pi

G = -16.3 kJ/mol

Manufacturing biological products

1. Cell

2. Environment (T, pH, flow, O2)

3. Nutrients (sugars, amino acids)

4. Control schemenutrient feeding, product removal, cell growth

5. Bioseparation train

6. Integration planhow 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

Glycolysis

TCA cycle

Glucose Glucose 6-Phosphate

Fructose 6-Phosphate

Fructose 1,6-Bisphosphate

Glyceraldehyde 3-Phosphate

Pyruvate

Acetate Acetyl CoA

Citrate

-Ketoglutarate

Succinate

Fumarate

Oxaloacetate

Phosphogluconate

Glyceraldehyde 3-Phosphate

AcetaldehydeLactate

Ethanol

MalateIsocitrate

CO2+NADHFADH2

CO2+NADH

NADH

NADH

GTP

GDP+Pi

Phosphoenolpyruvate

Anabolic processes - Biosynthesis

-Ketoglutarate

Glutamate

Glutamine Proline Arginine

Oxaloacetate

Aspartate

Asparagine Methionine Threonine Lysine

IsoleucinePyruvate

Alanine Valine Leucine

Phosphoenolpyruvate

Phenylalanine Tyrosine Tryptophan

Tyrosine

3-Phosphoglycerate

Serine

Glycine Cysteine

Ribose 5-phosphate

Histidine

Amino acid biosynthesis is regulated by feedback inhibition

Threonine -Ketobutyrate Isoleucine

Inhibited by isoleucine

Types of feedback control

1) Sequential feedback control

A → B → C

D → E → Y

F → G → Z

Inhibited by Y

Inhibited by Z

Protein productionCentral 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 degradedUbiquitin-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 destructionMitosis related proteins

Abnormally folded proteins

Proteins that are not properly folded can cause disease in humansPrion disease

Creutzfeldt-Jacob disease (CJD)

Bovine spongiform encephalopathy (BSE- mad cow)

Alzheimer’s 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 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

NuUfN Pk - Pk

dt

dP

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.

PU + PN = 1

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

HO

CH3

CH3

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 Core lipids Mechanisms of lipid deliveryChylomicron triacylglycerol hydrolysis by lipoprotein lipase

Very low densitylipoprotein (VLDL) triacylglycerols hydrolysis by lipoprotein lipase

Intermediate-density receptor-mediated endocytosis bylipoprotein (IDL) cholesterol esters liver and conversion to LDL

Low-density receptor-mediated endocytosis bylipoprotein (LDL) cholesterol esters liver and other tissues

High-density transfer of cholesterol esters tolipoprotein (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 estersaids 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)

Mineralocorticoids (C21)

Androgens (C19)

Estrogens (C18)

Pregnenolone Progesterone Cortisol(hydrocortisone)

Estrone

Androstenedione

O

O

CH3

O

O

CH3

Testosterone

Estradiol

O

OH

CH3

O

OH

CH3

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?