Fed batch fermentation a practical guide to scalable recombinant protein production 2015

Fed-batch fermentation


Related titles
Therapeutic risk management of medicines
(ISBN 978-1-907568-48-0)
An introduction to pharmaceutical sciences: Production, chemistry,
techniques and technology
(ISBN 978-1-907568-52-7)
Formulation tools for pharmaceutical development
(ISBN 978-1-907568-99-2)


Woodhead Publishing Series in Biomedicine: Number 42

Fed-batch
fermentation
A practical guide
to scalable recombinant
protein production in
Escherichia coli
Garner G. Moulton

amsterdam • boston • cambridge • heidelberg • london
new york • oxford • paris • san diego
san francisco • singapore • sydney • tokyo
Woodhead Publishing is an imprint of Elsevier


Woodhead Publishing is an imprint of Elsevier
80 High Street, Sawston, Cambridge CB22 3HJ, UK

25 Wyman Street, Waltham, MA 02451, USA
Langford Lane, Kidlington, OX5 1GB, UK
Copyright © 2014 Woodhead Publishing. All rights reserved
No part of this publication may be reproduced, stored in a retrieval system or
transmitted in any form or by any means electronic, mechanical, photocopying,
recording or otherwise without the prior written permission of the publisher.
Permissions may be sought directly from Elsevier’s Science & Technology Rights
Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865
853333; e-mail: Alternatively, you can submit your
request online by visiting the Elsevier website at />permissions, and selecting Obtaining permission to use Elsevier material.
Notice
No responsibility is assumed by the publisher for any injury and/or damage to
persons or property as a matter of products liability, negligence or otherwise,
or from any use or operation of any methods, products, instructions or ideas
contained in the material herein. Because of rapid advances in the medical sciences,
in particular, independent verification of diagnoses and drug dosages should be
made.
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Control Number: 2014938060
ISBN 978-1-907568-92-3 (print)
ISBN 978-1-908818-33-1 (online)
For information on all Woodhead Publishing publications
visit our website: />Typeset by RefineCatch Limited, Bungay, Suffolk
Printed and bound in the United Kingdom
Cover illustration: From the U.S. Department of Energy Genomic program website,



List of figures and tables

Figures
1.1

Nucleotide bases made up of pyrimidines and
purines as well as the addition of the sugar
ribose (RNA) or deoxyribose (DNA) and a
phosphate group

5

1.2

Chargaff’s rule

6

1.3

Base pairing in DNA is complementary

7

1.4

Conversion of simple sugars to ethanol and
carbon dioxide

9

1.5

The E. coli cell

15

1.6

Glycolytic pathway and acetyl-CoA
formation

16

TCA cycle and the formation of acetyl CoA
from acetate

18

Isopropyl β-D-1-thiogalactopyranoside
(IPTG)

21

2.1

Generic plasmid

34

2.2

Typical cloning of foreign gene into
recombinant plasmid

37

Isoproply-β-D-thio-galactoside (IPTG) shown
with the arrow pointing to the sulfur–carbon
bond that is not hydrolysable

39

1.7
1.8

2.3

vii


Fed-batch fermentation

2.4

Transcription of DNA

40

2.5

Micrograph of many transcription events

taking place on a DNA molecule

41

2.6

E. coli micrograph

42

2.7

E. coli cell wall structure and components

45

2.8

Transformation of a bacterial cell culture with
a plasmid

47

Draw a “T” on the bottom of your Petri dish,
as shown

58

2.9

2.10 Touch the inoculating loop to the upper
left-hand corner and then move it across the
agar from left to right, as shown

59

2.11 Touch the loop to the area previously
streaked and then move the loop across
the agar, as shown

60

2.12 Touch the loop on the previously streaked
area and then move the loop across the agar
onto the third area, as shown

60

2.13 Incubate the streak plate until you can see
individual colonies

61

3.1

Exponential growth curve for bacterial growth

64

3.2

Oxygen transport within the cell

76

3.3

%DO versus time

79

3.4

ln (C* − CL) versus Δtime (s)

79

3.5

Oxygen transfer rate and KLa determination

80

3.6

10-liter bioreactor for E. coli fermentation

84

3.7

Dissolved oxygen electrode: polarographic
sensor

88

3.8

The pH electrode: Calomel electrode

91

3.9

Typical fed-batch fermentation growth curve
viii

105


List of figures and tables

3.10 Analysis of residual acetate, glucose and
phosphate during the growth of the
recombinant culture

106

3.11 Typical induction gel at prior to induction and
at 3 hours post-induction

108

4.1

Prokaryotic ribosomal composition

112

4.2

Translation of protein in prokaryotes

114

4.3

A tetrapeptide (V-G-S-A) with the amino
terminus of the peptide on the left and the
carboxyl terminus on the right

118

Amino acid names, structures and one letter
symbol associated with each

120

Primary, secondary, tertiary and quaternary
structures of proteins

122

4.6

Bacterial GroES/GroEL complex

123

4.7

Aggregation pathways in vivo

133

4.4
4.5

Tables
4.1
4.2

Codons for amino acids and start and stop
sequences

113

Protein complexes within prokaryotic and
eukaryotic cells

126

ix


About the author
Gus G. Moulton is Chief Scientific Officer of BioBench LLC,
a contracting facility for purification and fermentation
development in Seattle, USA. Gus started the company in
2011 and is now pursuing this full time. BioBench’s primary
focus is initial development for product screening and vaccine
Phase I clinical trials.
Moulton has more than 20 years of process development
experience in the biotechnology community. During the last
13 years he has been responsible for setting up and running
fermentation labs to generate medium to high cell density
fermentations. He performed these services for both Corixa
Corporation, a former cancer vaccine company bought by
GlaxoSmithKline plc, and the Infectious Disease Research
Institute (IDRI), a nonprofit organization which develops
diagnostic tests and vaccines to diagnose and treat diseases
in third-world countries, such as India, Brazil and in Africa.
During Moulton’s career at Corixa he was initially
responsible for purification development of the most critical
antigens, and subsequently for setting up and developing
recombinant E. coli fermentation processes at the 30 liter
scale for Phase I clinical vaccine trials for HER2/neu. He also
developed an upstream and downstream process for the
purification of the recombinant antigen TcF to be used in the
diagnostic test for Chagas disease. The upstream process was

designed per GLP standards for in-house use, while the
downstream process was designed for and successfully
transferred to Viral Antigens, Inc.
xi


Fed-batch fermentation

During Moulton’s tenure at IDRI he again set up a
fermentation lab for development of recombinant E. coli
production of foreign antigens. Most fermentation
development work Moulton performed at IDRI was for
vaccine development against leishmaniasis – a disease caused
by protozoan parasites of the genus Leishmania and
transmitted by the bite of certain species of the sand fly
(subfamily Phlebotominae) – and tuberculosis caused by
Mycobacterium tuberculosis. While at IDRI, Moulton
developed a unique feed recipe in which he supplemented
phosphate for a recombinant E. coli fermentation using rich
media that tripled the final cell density without any significant
increase in process cost or time. Moulton also developed an
M. smegmatis recombinant system that should easily be
scalable using a wave reactor. This project can be used to
produce Mtb antigens for both diagnostics and vaccine
development.
Over the last 13 years Moulton has successfully developed
over 30 fermentation processes.

xii

1

Introduction to fermentation
DOI: 10.1533/9781908818331.1
Abstract: The use of yeast or microbial cells for the
production of a foreign protein has changed the approach
of medical research to finding healthcare solutions. The
application of recombinant systems has become
mainstream in treatment of disease. One of the most
important aspects of this new scientific discipline is the
ability to design a cell line or strain, in the case of bacterial
or yeast recombinant systems that can be grown under
controlled conditions, to produce significant quantities of
a recombinant protein. Recently, E. coli has been the
predominant bacteria in research and production
laboratories and plays a key role in the development of
modern biological engineering and industrial microbiology,
enabling foreign proteins to be produced in a prodigious
and cost-effective way. This type of cell growth and
production is called fermentation and its history and use
will be discussed along with current developments and
applications of recombinant technology.
Key words: E. coli, fermentation, recombinant DNA,
yeast, nucleic acids, bacteria, RNA, phosphate plasmid
DNA, recombinant protein, media, fed-batch, inclusion
body, acetate, glucose, IPTG, cell factory.

1

© Elsevier Limited, 2014


Fed-batch fermentation

1.1 A brief history of early
fermentation and the discovery
of DNA
It has been known for thousands of years that the fermentation
of carbon sources from grain and/or honey (for beer or
mead) and grapes or other fruit (for wine) will yield a
beverage, which when fermented correctly is quaffable as
well as entertaining to the senses (a feeling of well-being or
intoxication). In fact, scientists have shown through chemical
analysis that jars found in northern China contained a mixed
fermented beverage made from rice, honey and fruit made
9000 years ago [1]. Throughout human history, cultures
from Greece, Egypt, China and the Americas have produced
fermented concoctions for many reasons, including religious,
celebratory or personal consumption. In Egypt, the god
Osiris was believed to have invented beer. Because of this,
beer was thought of as an important part of society and
family and brewed on a daily basis [2].
In Greece, by the 16th century bc , the fermentation of
grapes into wine was common. By the 3rd century bc , the
moderate use of wine was thought of by many, including Plato
and Hippocrates, as both beneficial to health and happiness
and of therapeutic or medicinal value [3]. During this time,
the poet Eubulus stated that three bowls (glasses) of wine were
the ideal amount to consume, which roughly equals one

750 ml bottle of wine. The cult of Dionysus believed strongly
that wine or intoxication from wine would bring the consumers
closer to their deities. Along these lines, Eubulus, who wrote
the play “Dionysus”, has Dionysus saying to his patrons:
Three bowls do I mix for the temperate: one to health,
which they empty first; the second to love and pleasure;
the third to sleep. When this bowl is drunk up, wise
2


Introduction to fermentation

guests go home. The fourth bowl is ours no longer, but
belongs to violence; the fifth to uproar; the sixth to
drunken revel; the seventh to black eyes; the eighth is
the policeman’s; the ninth belongs to biliousness; and
the tenth to madness and the hurling of furniture [4].
Interestingly, these words of wisdom and warning have held
up through the thousands of years since they were first penned.
In China, one of the first alcoholic drinks made from rice,
honey and fruit was thought of as a spiritual sustenance
rather than a physical one. It was also believed that the
moderate use of fermented alcoholic substances was a
mandate from heaven and important for inspiration,
hospitality and medicinal uses.
Needless to say, the fermentation of a few carbon sources by
different yeast strains has had a profound effect on the world’s
societies, culturally and, albeit much later, scientifically. The
historical aspect of fermentation will be commented on in this
introduction but first we need to look at one of the most

important scientific discoveries in modern times, the discovery
of the cellular molecule, deoxyribonucleic acid (DNA). The
DNA molecule was first identified and isolated by the Swiss
physician and biologist Friedrich Miescher in 1869, with his
work being published in 1871 [5]. He had isolated “phosphate
rich” molecules from white blood cells, but did not understand
the molecules’ significance. This came later when Ludwig Karl
Martin Leonhard Albrecht Kossel, a German biochemist and
pioneer in the study of genetics, worked out the chemical
composition of the DNA molecule. He was awarded the
Nobel Prize for Physiology or Medicine in 1910 for this work.
Kossel had isolated and described the five organic compounds
that are present in nucleic acid: adenine (A), cytosine (C),
guanine (G), thymine (T) and uracil (U). Eventually these
compounds were to become known as nucleobases, the
3


Fed-batch fermentation

foundation for the formation and structure of DNA and
ribonucleic acid (RNA) in all living cells.
During this same time other scientists were working on
determining the structures and chemical nature of these
compounds. One of these was the Russian biochemist Phoebus
Levene, who published many papers on cellular molecules and
is credited with the discovery of the order of the three major
components of a nucleotide, the phosphate, the sugar and the
base [6]. He also identified the sugar components of both the
RNA and DNA molecules as ribose and deoxyribose,

respectively. Levene worked extensively with yeast nucleic
acids to identify the components and ultimately (in 1919)
proposed that the nucleic acids were made up of one distinct
base, a sugar and a phosphate molecule (Figure 1.1) [7].
After nearly 30 years of nucleic acid research, the scientific
community received three important contributions. In the
1920s, Frederick Griffith was studying the differences between
two Pneumococcal strains (R (non-virulent) and S (virulent)),
and while doing so came upon an interesting finding. When he
heat-killed the virulent S strain and mixed it with a live nonvirulent R strain and then injected this mixture into mice, the
mice died of pneumonia. Griffith did not realize it at the time,
but he had discovered bacterial transformation through the
transfer of DNA to a host bacterium. In 1944, Oswald Avery
and his Rockefeller University colleagues published work
along these same lines but with a more definitive result. They
demonstrated a link between DNA and virulence of these
same two strains, by transferring DNA from a heat-killed S
strain that was treated with proteases (destroys protein),
RNAses (destroys RNA) or DNAses (destroys DNA) to a
living non-virulent strain (R strain). What they found was that
only R cells, transformed with protease or RNAse treated
DNA from the S strain, were shown to be virulent. The DNAse
treated mixture did not convert the R cells to a virulent strain.
4


Introduction to fermentation

Figure 1.1

Nucleotide bases made up of pyrimidines and
purines as well as the addition of the sugar
ribose (RNA) or deoxyribose (DNA) and a
phosphate group

Soon after this work was presented, the Austrian
biochemist, Erwin Chargaff, made a startling discovery when
he analyzed DNA from different species. He noticed that the
nucleotide composition was not the same from one species to
the next. He also discovered that within the structure of a
DNA molecule, the purines (A and G) and the pyrimidines
(C and T) are in equal amounts to the other ([A] = [G] and
[C] = [T]). This finding of equality between base pairs is
known as “Chargaff’s” rule (Figure 1.2) [8].
In 1956, James Watson and Francis Crick co-discovered
the structure of the DNA and RNA molecules (Figure 1.3).
Along with Maurice Wilkins, they were awarded the Nobel
Prize in Physiology or Medicine in 1962. They leaned heavily
on the previous findings of Chargaff and his colleagues at the
5


Fed-batch fermentation

Figure 1.2

Chargaff’s rule. In DNA, the total abundance
of purines is equal to the total abundance of
pyrimidines

time, as well as having the benefit of the X-ray crystallography
work on the DNA molecule done by Rosalind Franklin and
Maurice Wilkins. This crucial work led them to define the
DNA molecular structure as a double helix [9].

1.2 The rise of biotechnology I
1.2.1 The gene
Approximately 20 years after the determination of the
structure of the DNA molecule, the term “biotechnology”
was established. Wikipedia states that biotechnology is “the
application of scientific and engineering principles to the
processing of materials by biological agents to provide goods
and services”. Biotechnology has its beginnings in what we
call zymotechnology, which are the processes/techniques
used for the production of beer. Soon after World War I,
with the advent of industrial fermentation taking a firm
6


Introduction to fermentation

Figure 1.3

Base pairing in DNA is complementary [4].
The purines (A and G) pair with the pyrimidines
(T and C, respectively) to form equal-sized base
pairs resembling rungs on a ladder (the sugarphosphate backbones). The ladder twists into a
double-helical structure.

hold on the current larger industrial issues, the path was

paved for the increase in scientific research in the area of
product formation from the single cell.
By the 1970s, the term “genetic engineering” was becoming
commonplace, ironically being used for the first time in Jack
Williamson’s science fiction novel Dragon’s Island [10], prior
to the connection of DNA as a hereditary molecule and the
7


Fed-batch fermentation

confirmation of its structure as a double helix. In 1972, the
first recombinant DNA molecule was made by combining
the DNA from the lamda virus and the SV40 virus. This
initial work was done by Paul Berg, which was followed by
Herbert Boyer and Stanley Cohen creating the first transgenic
organism by inserting antibiotic resistance genes into a
plasmid of E. coli [11].
Before 1983, the name Kary Mullis was little known at
best. Dr Mullis was a writer of fiction, a baker, but not a
candlestick maker. He was, in fact, a very good biochemist
who worked for the Cetus Corporation in California for
seven years after his initial wanderings. In this time he
worked as a DNA chemist and eventually improved on the
already existing polymerase chain reaction (PCR), although
improvement is not a strong enough word for the contribution
Mullis made to the PCR reaction [11].
A concept similar to that of PCR had been described before
Mullis’ work. Nobel Prize laureate H. Gobind Khorana and
Kjell Kleppe, a Norwegian scientist, authored a paper 17

years earlier describing a process they termed “repair
replication” [12]. Using repair replication, Kleppe duplicated
and then quadrupled a small synthetic molecule with the
help of two primers and DNA-polymerase. The difference
between Khorana and Kleppe’s work and Mullis’s is the fact
that Mullis used the heat stable taq DNA polymerase instead
of the heat labile DNA polymerase (had to be added anew to
each heat cycle). This new PCR method relies on thermal
cycling, consisting of cycles of repeated heating and cooling
of the reaction for DNA melting and enzymatic replication
of the DNA. Primers (short DNA fragments) containing
sequences complementary to the target region along with a
DNA polymerase (after which the method is named) are key
components to enable selective and repeated amplification.
As PCR progresses, the DNA generated is itself used as a
8


Introduction to fermentation

template for replication, setting in motion a chain reaction in
which the DNA template is exponentially amplified. PCR
can be extensively modified to perform a wide array of
genetic manipulations [11].
As can be appreciated, within a short time, fermentation
and the concept of recombinant protein production has
matured and evolved to the point where protein products are
produced for modern-day medicine. The work of Boyer and
Cohen, as discussed above, using plasmids and restriction
enzymes to manipulate DNA (recombined with foreign

genes) laid the groundwork for what is now known as
biotechnology [13,14].

1.2.2 Controlled fermentation
Man has harvested the energy produced by fermentation to
generate new and exciting products, used not only in
medicine but also in bioremediation and agriculture
(Figure 1.4). Surprisingly, even therapeutic antibodies are
now being produced using recombinant expression hosts,
other than Chinese Hamster Overy cells (CHO), such as yeast
and E. coli. Significant progress has been made in antibody
engineering, with a particular focus on Fc engineering and

Figure 1.4

Conversion of simple sugars to ethanol and
carbon dioxide
9


Fed-batch fermentation

glycol-engineering for improved functions, as well as cellular
engineering for enhanced production of antibodies in yeast
and bacterial hosts such as E. coli [14]. Bacteria, yeast and
some mammalian cell systems have been used to produce
essential therapeutics such as insulin, as well as recombinant
antigens for vaccines, diagnostic and therapeutic purposes
[15,16].
E. coli is a Gram-negative, rod-shaped bacterium that is

commonly found in the lower intestine of warm-blooded
organisms (endotherms). Most E. coli strains are harmless,
but some serotypes can cause serious food poisoning in
humans, and are occasionally responsible for product recalls.
The harmless strains are part of the normal flora of the gut,
and can benefit their hosts by producing vitamin K2, and by
preventing the establishment of pathogenic bacteria within
the intestine.
E. coli was one of the first organisms to have its genome
sequenced with the complete genome of E. coli K12
(MG1655) [17]. It was 4.6 million base pairs in length,
encoding 4288 protein genes, organized into 2584 operons.
It was circular in structure with a large amount of DNA
coded for genes (high genetic density) with only 118 base
pairs distance between the genes. Along with ribosomal
RNA and transfer RNA genes, the genome was also shown
to contain a large number of repeat elements, transposable
elements, and prophage and bacteriophage sequences.
In microbiology studies, E. coli has been used to study
metabolic pathways, cell division and mechanisms of cell
death. In 1946, Lederberg and Tatum discovered bacterial
conjugation using E. coli as a model bacterium [18]. Phage
genetics studies by early researchers such as Seymour Benzer
were used to understand the topography of the gene structure;
to date, Escherichia and Shigella species comprise over 60
complete genomic sequences that are available [15]. Only
10


Introduction to fermentation

about 20% of each genome is present in each genomic
species, representing a fantastic amount of diversity within
the genre. The genes present in each individual genome
number between 4000 and 5500 genes, while the number
of different genes found among all the E. coli strains that
have been sequenced is greater than 16 000! This is called a
pan-genome and is thought to have gained its diversity
through the process of horizontal gene transfer from other
species [19].
Prior to the discovery of restriction enzymes in the 1970s,
researchers used inefficient ways to modify genetic material,
such as what happens when a bacterium is infected by a
bacteriophage or a foreign plasmid. With the discovery and
isolation of the restriction enzyme Hind III in 1970 [20,21]
and the subsequent discovery and characterization of
numerous restriction endonucleases [22], the 1978 Nobel
Prize for Physiology or Medicine was awarded to Daniel
Nathans, Werner Arber and Hamilton O. Smith [23]. During
the 1970s, recombinant DNA technology and its use
exploded onto the scientific scene. One of the first important
products made with this new technology was the large-scale
production of human insulin for diabetes, using E. coli as the
recombinant host.
Since the 1920s, animal insulin was used to treat Type II
diabetes, along with forms of insulin such as zinc insulin
and the lente insulins for Type I diabetes. In the 1960s,
insulin was chemically synthesized in China, Germany and
the United States. By the mid-1970s, the separation
technology advanced enough to be able to isolate animal

(porcine, bovine) insulin to a single component by Novo
and Eli Lilly. By 1978, scientists from one of the first
biotechnology companies, Genentech (San Francisco, CA),
used a genetically engineered plasmid of the E. coli bacterium
carrying the foreign gene for human insulin. They were able
11


Fed-batch fermentation

to produce the recombinant insulin with the same genetic
sequence as human insulin, meaning the E. coli transcribed
and translated the foreign gene as it was in humans [15]. By
1980, the first recombinant DNA insulin product was
injected into a healthy control group in England. In 1982,
the FDA awarded Eli Lilly the first approved genetically
engineered insulin (Humulin R and Humulin N) to be sold
on the US market [24].
After this initial success with recombinant technology, the
sky was the limit, or so many scientists thought. This new
technology was to supply the world with any relevant
recombinant protein, which was deemed necessary to address
a medical need. Recombinant enzymes, hormones and
immunogens (for vaccines) were going to be produced easily
and cost-effectively. But these expectations were quickly
realized as much too grand and efforts to produce such
proteins were constantly being stymied. The scientific
community started to realize that most of the proteins made
in the E. coli recombinant system were not comparable
to the same protein made from natural source and thus

these recombinant proteins were not safe for human use.
The recombinant proteins had two major obstacles to
overcome:
1. proteolysis by host cell proteases [25]; and
2. the formation of inclusion bodies [26].
Ironically, human insulin was one of the first recombinant
proteins produced to show formation of inclusion bodies
[27]. Both of these issues, either combined or separate,
interfere with the ability of the process development scientist
to produce recombinant protein products in their native
state or at least produced with a consistent product character.
Part of the problem has been the lack of understanding from
a cell physiology standpoint, how the recombinant E. coli
12


Introduction to fermentation

cell is affected by the conditions of a standard fermentation
process.
As mentioned earlier, recombinant E. coli can be used to
develop antigens in vaccine development and proteins for
therapeutic uses. However, E. coli has limitations, and
cannot be used to produce large multimeric heterologous
proteins or proteins that require complex disulfide bond
formation or unpaired thiols or proteins that natively contain
post-translational modifications. There is a caveat to these
stated limitations, in that in the presentation of a vaccine
antigen, the secondary and tertiary structures are important
but not essential for an immune response.

The epitopes of protein antigens are divided into two
categories, conformational epitopes and linear epitopes,
based on their structure and interaction with the antibody. A
conformational epitope is composed of separated sections of
the antigen’s amino acid sequence. Although these epitopes
are separated within the linear sequence of the protein, they
are closely oriented spatially in the secondary or tertiary
structure of the antigenic protein. They interact with the
antibody and the surface of a certain type of cell based on
their 3-D surface features and shape or tertiary structure.
By contrast, linear epitopes interact with the antibody
based on their primary structure (amino acid sequence). A
linear epitope, usually 8 to 11 amino acids in length, is
formed by a continuous sequence of amino acids from the
antigen. Even though it is thought that most epitopes
recognized by the immune system are conformational, it has
been shown that non-conformational or aggregated proteins
can elicit an immune response. Among the critical factors in
inducing antibody responses are molecular weight and the
insoluble nature of the aggregate [28].
An epitope, also known as an antigenic determinant, is the
part of an antigen that is recognized by the immune system,
13


Fed-batch fermentation

specifically by antibodies, B cells or T cells. Although epitopes
are usually thought to be derived from non-self proteins,
sequences derived from the host can be recognized and are

also classified as epitopes. This happens in autoimmune
diseases, of which there are many, such as Lupus, Crohns
Disease and Diabetes mellitus Type I.
T cell A greater
understanding of the molecular mechanisms within the
recombinant cell host will significantly contribute to
advances in the areas of stress responses to the host cell
during expression of the recombinant protein. These stress
148


The future of Escherichia coli recombinant fermentation

responses to some recombinant proteins limit their
productivity during expression. It is obvious that there is a
need to learn how to manipulate growth conditions to
alleviate these stresses and increase recombinant protein
production. As our understanding of the physiological
processes of the host cell matures, discoveries of relevant
molecular and/or environmental tools will be employed to
this end. As mentioned above, along with genetic engineering,
other recombinant bacterial hosts need to be fully explored
and hopefully incorporated into the industrial pipeline. Their
presence in our recombinant portfolio as potential
recombinant hosts is important in fulfilling the increasing
demand for the production of therapeutics, vaccines and
diagnostics for the bio-pharmacological industries.

149

References
1. Heath, D. (1995) International Handbook on Alcohol
and Culture. Westport, CT: Greenwood Publishing
Group; p. 131.
2. Dietler,
M.
(2006)
Alcohol:
Archaeological/
anthropological perspectives. Annual Review of
Anthropology, 35: 229–49.
3. Mok, M. (1932) Stone Age had booze and prohibition.
Popular Science, 120(5): 44–6, 122–4.
4. Johnson, H. (1989) Vintage: The Story of Wine.
Rockefeller Center, New York: Simon and Schuster;
pp. 35–46.
5. Eubulus. Semele or Dionysus, fr. 93.
6. Miescher, W.G.F. (2003) The man who discovered DNA.
Chemical Heritage, 21: 10–11, 37–41.
7. Levene, P.A. (1919) The structure of yeast nucleic acid.
IV: Ammonia hydrolysis. Journal of Biological
Chemistry, 40: 415–24.
8. Chargaff, E. (1950) Chemical specificity of nucleic acids
and mechanism of their enzymatic degradation.
Experientia, 6: 201–9.
9. Watson, J.D. and Crick, F.H.C. (1953) A structure for
deoxyribose nucleic acid. Nature, 171: 737–8.
10. Stableford, B.M. (2004) Historical Dictionary of Science
Fiction Literature. Scarecrow Press, p. 133 (rev. pbk. ed.

19 April 2004).
11. Arnold, P. (2009) History of Genetics: Genetic
Engineering Timeline. Encyclopedia Wikipedia.
151