Food Bioengineering

LIFS324

Lecture 5
How cells work:
Cellular metabolism and regulations

October 30 & November 1, 2017

Prof. Kyoung Heon Kim
 1. Introduction
A cell
Much more than a bag filled with lipids, amino acids,
sugars, enzyme, and nucleic acids
Cell must control how these components are made and
interact with each other

Metabolic regulation
Key is the flow and control of information
Human society depends on electronic signals for
information storage, processing, and transmission
Cells use chemical signals for the same purpose
Molecular biology is the study of information flow and
control
 2. Central Dogma
Living systems
To have the same core approach to the storage,
expression, and utilization of information
Information is stored in the DNA molecule
Central dogma of biology
Information is stored on the DNA molecule
That information can be replicated directly to
form a second identical molecule
That information can be transcribed to yield
RNAs
 2. Central Dogma
Central dogma of biology (continued)
Using a variety of RNAs, this information is
translated into proteins
Proteins then perform a structural or enzymatic
role
Enzymes meditate all the metabolic functions in
the cell
 Translation and Transcription
Which species of RNA that are present and in
what amount varies with time and with changes in
culture conditions => Transcriptomics
Proteins that are present will change with time
but on a different time scale than for RNA species
=> Proteomics
Key omes in a cell
 Metabolome analysis
Number of microbial metabolites
E. coli: 4400 genes; 442 metabolites (Edward and
Palsson, 2000)
S. cerevisiae: 6200 genes; over 700 metabolites
(Forster et al., 2000)
A. thaliana: 25000 genes (Nature 408, 796-815,
2000); 5000 metabolites
 Application of metabolomics
Detection of biomarkers
Disease diagnosis, prognosis and drug discovery
e.g. COMET (Consortium for Metabonomics
Technology) established by 6 big pharmaceutical
companies in Europe
Characterization of cellular metabolic
phenotypes
Understanding of cell physiology
Metabolic networking
Clustered heat map of 133 metabolites
(agarose metabolism)
 Metabolism with polymers (agarose)
Nucleosides
(psuedourindin
e, guanosin) by
salvage
pathway to
nucleotides
Carbon
channeled to
amino acids:
enzyme protein
production
Agarose metabolism
Galactose metabolism
 Metabolomics work by Min Hye Shin in KH Kim Lab.

Shin, M. et al. Analytical Chemistry, 2010

Shin, M. et al. Biotechnology & Bioengineering, 2010 Shin, M. et al. New Biotechnology, 2010
 Codon
Language for information storage and exchange
Four-letter alphabet made up of the nucleotides: A
(adenine), T (thymine), G (guanine), C (cytosine)
All codons are three-letter words with having a
maximum of 64 words
These words represent a particular amino acid or
stop protein synthesis
 Gene
The words are put into a sequence: sentence: gene
The complete set of information in an organisms
DNA (i.e., book): genome
Totality in Greek
 3. DNA Replication
DNA double helix
G hydrogen-binds only to C
A hydrogen-binds only to T
Each daughter chromosome contains one parental
strand and one newly generated one
Replication is semiconservative
 DNA polymerases in E. coli
Covalent linking of monomer DNA: DNA
polymerases
E. coli has three DNA polymerases: Pol I, Pol II,
and Pol III
Pol I: Hydrolyzes an RNA primer; duplicates
single-stranded regions of DNA; the repair of
DNA molecules
Pol II: Exact role is still unclear
Pol III: Meditates the addition of nucleotides to an
RNA primer
 Replication process
Replication begins at a predetermined site: Origin
of replication
In E. coli, Ori is attached to the plasma membrane
at the start of replication
Initiator proteins bind to DNA at Ori, break H
bonds and force the two DNA strands apart
Once replication starts, two strands separate to
form a Y-shaped structure: Replication fork
 Replication process (continued)
To initiate DNA synthesis, an RNA primer is
required
RNA polymerase requires no primer to initiate
chain-building process
Once a RNA primer complementary to one of the
DNA strands is made, DNA synthesis begins with
Pol III
RNA primer is degraded by Pol I
 DNA polymerase
DNA polymerase works only on 5to-3 direction
The next nucleotide is always added to the exposed
3-OH group of the chain
Leading strand: formed continuously since it is
synthesized in the same direction (5 to 3 direction)
as the replication fork is moving
Lagging strand: must be synthesized
discontinuously
 Leading strand

Lagging strand
 DNA polymerase (continued)
Okazaki fragment: short pieces of DNA attached to
RNA are formed on the lagging strand
DNA ligase :joins two short pieces of DNA on the
continuous strand; important in genetic engineering
 3. Transcription

Primary products of transcription

m-RNA
t-RNA
r-RNA
Their rates of synthesis determine the cells
capacity to make proteins
RNA synthesis from DNA is mediated by RNA
polymerase
 RNA polymerase
Two subcomponents: core enzyme + sigma factor
= holoenzyme
Core enzyme: contains catalytic site
Sigma factor: a protein to locate the appropriate
beginning for the message
RNA polymerase always read in the 3-to 5-
direction => the direction of reading on each
strand is opposite
 opposite

5
3
 Initiation
Transcription: initiation, elongation, termination
Sigma factor: involved only in initiation
Sigma factor recognizes a specific sequence of
nucleotides on a DNA strand (e.g., promoter
region)
Strong promoter: high affinity for the holoenzyme
Rate of formation of transcript
Frequency of initiation of transcription
Strength of promoter
 Elongation
After the initiation site is recognized, elongation
of transcript begins
As soon as elongation is established, sigma factor
is released so it can be reused
 Termination
Transcript is made until RNA polymerase
encounters a stop signal, transcription terminator
At that point, RNA polymerase dissociates from
DNA template and RNA transcript is released
In some cases, rho protein is additionally required
for termination
Fig. Messenger RNA synthesis
 Transcripts
Stable RNA species: r-RNA, t-RNA
Unstable RNA species: m-RNA
m-RNA of E. coli has 1-min half-life
In higher plants and animals, m-RNA is more
stable
 Transcription in procaryotes
Procaryote
No nuclear membrane
No separation of chromosome from cytoplasma and
ribosomes
Polygenic
Related proteins are often encoded in a row without
interspacing terminators
Thus, transcription from a single promoter results in a
polygenic message
Polygenic: many genes
Each single gene encodes a separate protein
Regulation of transcription from a single promoter
 Transcription in procaryotes
Polygenic
Regulation of transcription from a single promoter
=> Efficient regulation of functionally related
proteins
Such a strategy => Important for relatively small and
simple cells
Eucaryotic cells do not produce polygenic messages
TATA box + TTGAC: specific
for RNA polymerase
Effector
 Corepressor

Negative regulation
 Transcription in eucaryotes
Eucaryotic cells do not produce polygenic
messages
In eucaryotes, the nuclear membrane separates
chromosomes and ribosomes
m-RNA is subject to processing before translation
DNA encodes for an intervening sequence in the
middle of transcript: Intron
Ends of the remaining fragments are joined by
RNA splicing
 Transcription in eucaryotes (continued)
Then the spliced message is translated into an
actual protein
The role of introns is not well understood, but
likely to play a role in either evolution or cellular
regulation
m-RNA processing: m-RNA splicing, RNA
capping, polyadenylation
RNA capping: 5 end is modified by the addition
of a guanine nucleotide with a methyl group
attached
 Transcription in eucaryotes (continued)
Polyadenylation: A string of adenine nucleotides
are added to the 3 end
RNA capping & polyadenylation
To increase m-RNA stability
To facilitate transport across the nuclear membrane
TATA box + CCAT (CAT box)
+ GC box
= Promoter sequences
Fig. RNA splicing in eucaryotes
 4. Translation: Message to Product

Genetic code: Universal code

Blueprint for living cells: genetic code
The code is made of three-letter words (codons)
64 words are possible
Many of them are redundant
Sufficient to serve as a complete blueprint
 Dictionary for the genetic language
 Genetic code
The code is degenerate
More than one codon can specify a particular
amino acid
Serine: UCU, UCC, UCA, UCG
Nonsense codons do not code for amino acids
(UAA, UAG, and UGA)
act as stop points in translation
encoded at the end of each gene
 Universality of genetic code
The language used to make a human protein is
understood in E. coli and yeast
The microbial cells produce the same amino acid
sequence as a human cell
The universality facilitates genetic engineering
Codon optimization
 Translation: How the machinery works
Primary steps: Initiation, elongation, and termination
Initiation
m-RNA binds to ribosome
All protein synthesis begins with a AUG (or GUG) codon on
m-RNA
This AUG encodes N-formylmethionine
In the middle of a protein, AUG encodes for methionine
How does the cell knows AUG is for N-formylmethionine?
 Translation: How the machinery works
Initiation (contd)
Shine-Delgano box: approx. 10 base upstream of the
AUG where ribosome binding site (RBS) is located
Initiation complex: required in the initiation of
polymerization in prokaryotes
Initiation complex is composed of
A 30S ribosomal unit with an N-formylmethionine
bound to its initiation region
A 50S ribosomal unit
Three proteins called initiation factors
 Translation: How the machinery works
Elongation
t-RNA used as decoders
One end of the t-RNA contains anticodon
Anticodon is complementary to the codon on the m-
RNA
The other end of the t-RNA binds a specific amino
acid
Anticodon

Codon
 Translation: How the machinery works
Termination
When a nonsense or stop codon is reached, the protein
is released from the ribosome
The release needs the aid of a protein release factor
(RF)
Then 70S ribosome dissociates into 30S and 50S
subunits
What is S here?
 Translation: How the machinery works
Termination
When a nonsense or stop codon is reached, the protein
is released from the ribosome
The release process needs the aid of a protein release
factor (RF)
Then 70S ribosome dissociates into 30S and 50S
subunits
S: sedimentation velocity coefficient
1 S (svedberg) = 10-13 (sec)
Range: 10-13 10-11 sec (1 100 S)
 Posttranslation processing: Making the
product useful
Further processing of formed polypeptide
Newly formed chain fold into the proper structure
In some cases, several chain associate to form a
particular enzyme or structural protein
Chaperones
A class of protein that assist in the proper folding of
peptides
Level of chaperone increases in response to
environmental stresses such as high temp.
 Heat shock protein
The first chaperones to be identified are heat shock
proteins
Synthesis of heat shock proteins induced by high temp.
The monomeric proteins bind to newly synthesized
polypeptides and to existing cellular proteins
Therefore, can prevent as well as reverse improper
folding
e.g., E. coli DnaK
 E. Coli DnaK
(Only substrate protein
binding domain shown)

A polypeptide
segment of a
substrate protein
 Inclusion body
Often misfolded proteins aggregate and form insoluble
particles: Inclusion body
High levels of expression of foreign protein through
recombinant DNA technology in E. coli
overwhelm the processing machinery
result in inclusion bodies
Formation of proteins in inclusion bodies
complicates many bioprocess
in vitro methods to unfold and refold the protein
product must be employed
 Secretion of proteins
In many cases, the translocation of the protein across
membrane is done cotranslationally (i.e., during
translation)
Signal sequence
20-25 amino acids
clipped off during secretion
pre-form = signal sequence + mature form
 Secretion of proteins
Prokaryotes
Secretion of proteins occurs through the cytoplasmic
membrane
In E. coli and G(-) bacteria, the outer membrane
blocks the release of the secreted protein into the
extracellular compartment
In G(+) bacteria, secreted proteins readily pass the
cell wall into the extracellular compartment
Whether a protein product is retained in a cell or
released: major impact on bioprocess design
 Total
intracellular
proteins

D: IPTG at 30C at 0 h

Soluble E: IPTG at 37 at 0h
intracellular
proteins G: IPTG at 37 after 3 h

H: IPTG at 43C after 3 h

Insoluble
intracellular
proteins
How to avoid inclusion body?
 Other modifications
Particularly in eukaryotes
Addition of nonamino acid components (e.g., sugars
and lipids)
Phosphorylation
Glycosylation: addition of sugars
These modifications are important consideration in the
choice of host organisms for the production of proteins
 N-linked glycosylation
Particularly important posttranslational processing
The glycosylated pattern serve to target the protein to a
particular compartment
Or to control its degradation and removal from the
organism
Therapeutic proteins
If the N-linked glycosylation product pattern is not
human like
The protein product may be ineffective
Glycoform of a protein product is a key issue in
bioprocesses to make therapeutic proteins
 5. Metabolic Regulation

Metabolic regulation
Metabolic regulation is the heart of any living cell
Takes place at the genetic level and at the cellular
level
Cellular level
Control of enzyme activity
Cell surface receptors
 Genetic-level control
Which proteins are synthesized?
Transcriptional control of protein synthesis is the most
common control strategy used in bacteria
Regulation
i) Feedback repression
ii) Induction
Feedback repression
End product of enzymatic activity accumulates and
blocks transcription
Induction
A metabolite accumulates and acts as an inducer of
transcription
 Genetic-level control (continued)
Operon
A set of contiguous genes, encoding proteins with
related functions, under the control of single promoter-
operator
Central to understanding microbial regulation
 Genetic-level control (continued)
Repression
Active repressor (with corepressor) binds to operator
region
Active repressor binds to the operator region => Hinders
RNA polymerase binding
Corepressor (typically the end product of the pathway)
required
Active repressor combined with corepressor can block
transcription
Regulation by repressor proteins: Negative
regulation (e.g. lac operon)
Regulation by activator proteins: Positive control
(e.g. ara regulon for arabinose metabolism in E. coli)
 Genetic-level control (continued)
Induction
In normal condition (e.g. no inducer) => Active
repressor => No transcription => Negative control

Inducer (typically a substrate for a reaction) combines

with repressor

The complex (inducer + repressor) is inactive as a

repressor
 Lac operon: the first control

I stands for Inducibility

Transfers acetyl
CoA to galactotose
 Allolactose is an isomer of lactose and
is the inducer of the lac operon. Lactose
is galactose-(1->4)-glucose, whereas
allolactose is galactose-(1->6)-glucose

or IPTG
 Catabolite repression
the second control of lac operon
Catabolite repression (glucose effect)
E. coli on glucose and lactose
Less easily metabolize lactose
Glucose preferentially consumed
Glc => cAMP (cyclic 3,5-adenosine monophosphate)
=> induction in lac operon
ATP to cAMP: inhibited by glucose
Amount of cAMP is reduced by catabolite products of
glucose => Catabolite repression
 Catabolite repression in lac operon
Repressor Promotor Operator Structural
gene gene

lac operon
cAMP-CAP (i.e. glue)

RNA polymerase

cAMP + CAP cAMP-CAP => This complex controls the

activity of promoter sequence

Glucose => cAMP => glue => RNA polymerase binding

affinity => Transcription of structural gene
 Strongly
expressed

Not expressed

Not expressed

Low expression
 Diauxic growth
E. coli on glucose and lactose
-Galactactosidase
E. coli

Glucose
Lactose
 Diauxic growth
Yeast on glucose
Ethanol is consumed after glucose is exhausted
Yeast growth

Glucose
Ethanol
Lac Promoter in a plasmid vector
 Metabolic pathway control
Metabolic pathway control
How the activities of a group of enzymes controlled
Cell: the most efficient use of its resources
Human: cause the cell to overproduce the product of
commercial interest
Overproduction in bioprocesses
Disrupt the cells control strategy
Understanding of how cells control their pathway
 Metabolic pathway control (continued)
The simplest case: linear pathway
A linear pathway making a product, P1

The first reaction inhibited by accumulation of the

product

Feedback inhibition or End-product inhibition

Sufficient supply of P1, it deactivates the pathway

Can be extended to more complicated pathways with

many branch points
 Metabolic pathway control (continued)
Case 1: Isozymes
Isozymes: isofunctional enzymes

Two separate enzymes to carry out the same

conversion

Each enzyme inhibited by a different end product

If P1 is in excess,

i) It inhibits E2 while E1 is fully active

ii) Sufficient activity to synthesize P2 still remains
through E2
 Metabolic pathway control (continued)
Case 2: Concerted feedback inhibition
A single enzyme with two allosteric binding sites for
P1 and P2 controls entry into the pathway

Only one site occupied by an effector: enzyme activity

not affected

Both sites occupied by effectors: enzyme becomes

inactive

Textbook: A high level of either P1 and P2 is not

sufficient by itself to inhibit enzyme E2
 Metabolic pathway control (continued)
Case 3: Sequential feedback inhibition
An intermediate at the branch point accumulates

The intermediate acts as the inhibitor of metabolic flux

into the pathway

A high level of P1 inhibits enzyme E4

A high level of P2 inhibits enzyme E5

If either E4 or E5 is blocked, M3 accumulates, but not as

rapidly as when both E4 and E5 are blocked

If intermediate flux levels allowed it either P1 or P2 is

high, but the pathway inactivated if both P1 and P2 are
high
 Metabolic pathway control (continued)
Case 4: Cumulative feedback inhibition
A single allosteric enzyme has effector sites for several
end products of a pathway

Each effector causes only partial inhibition

Full inhibition is a cumulative effect

Cumulative inhibition or Cooperative feedback

inhibition
 Metabolic pathway control (continued)
Feedback inhibition
Occurs at the enzyme level

Rapid

Feedback repression
Occurs at the molecular level

Slower than inhibition