Impact of glucose uptake rate on recombinant

protein production in Escherichia coli

Emma Bcklund
M. Sc.
Royal Institute of Technology
Stockholm 2011

 Emma Bcklund
Stockholm, 2011
School of Biotechnology
Royal Institute of Technology
SE-106 91 Stockholm
Sweden
Printed at AJ E-print AB
Oxtorgsgatan 9
SE-111 57 Stockholm
Sweden
ISBN 978-91-7415-994-3
ISSN 1654-2312
TRITA-BIO Report 2011:18

Emma Bcklund (2011). Impact of glucose uptake rate on recombinant protein production
in Escherichia coli. School of Biotechnology, Royal Institute of Technology (KTH),
Albanova University Center, Stockholm, Sweden.
ABSTRACT
Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it
generally provides a rapid and economical means to achieve high product quantities. In this
thesis, the impact of the glucose uptake rate on the production of recombinant proteins was
studied, aiming at improving and optimising production of recombinant proteins in E. coli.
E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch
technique, which offers a means to control the glucose uptake rate. One objective of this study
was to find a method for control of the glucose uptake rate in small-scale cultivation, such as
microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system
(PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting
in lower growth rates and lower accumulation of acetic acid in comparison to the wild type.
By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental
to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use
of the mutant strains represent a novel, simple alternative to fed-batch cultures.
The PTS mutants were applied for production of integral membrane proteins in order to
investigate if the reduced glucose uptake rate of the mutants was beneficial for their
production. The mutants were able to produce three out of five integral membrane proteins
that were not possible to produce by the wild-type strain. The expression level of one selected
membrane protein was increased when using the mutants and the expression level appeared to
be a function of strain, glucose uptake rate and acetic acid accumulation.
For production purposes, it is not uncommon that the recombinant proteins are secreted to the
E. coli periplasm. However, one drawback with secretion is the undesired leakage of
periplasmic products to the medium. The leakage of the product to the medium was studied as
a function of the feed rate of glucose in fed-batch cultivations and they were found to
correlate. It was also shown that the amount of outer membrane proteins was affected by the
feed rate of glucose and by secretion of a recombinant product to the periplasm.
The cell surface is another compartment where recombinant proteins can be expressed.
Surface display of proteins is a potentially attractive production strategy since it offers a
simple purification scheme and possibilities for on-cell protein characterisation, and may in
some cases also be the only viable option. The AIDA-autotransporter was applied for surface
display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z
was expressed in an active form and was accessible to the medium. Expression was favoured
by growth in minimal medium and it seemed likely that expression was higher at higher feed
rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where
relatively high cell densities were achieved whilst maintaining a high expression level of Z.

Keywords: AIDA-autotransporter, Escherichia coli, fed-batch, glucose uptake rate, integral

membrane proteins, outer membrane proteins, periplasmic retention, phosphotransferase
system, recombinant proteins, specific growth rate, surface expression.

LIST OF PUBLICATIONS
The thesis is based on the following papers, referred to in the text by their Roman
numerals:

I. Bcklund E, Markland K, Larsson G (2008). Cell engineering of Escherichia coli

allows high cell density accumulation without fed-batch process control. Bioprocess
and Biosystems Engineering 31:11-20
II. Bcklund E, Reeks D, Markland K, Weir N, Bowering L, Larsson G (2008).
Fedbatch design for periplasmic product retention in Escherichia coli. Journal of
Biotechnology 135:358-365
III. Bcklund E, Ignatuschenko M, Larsson G (2011). Suppressing glucose uptake
and acetic acid production increases membrane protein overexpression in Escherichia
coli. Accepted for publication in Microbial Cell Factories.
IV. Gustavsson M, Bcklund E, Larsson G (2011). Optimisation of surface
expression using the AIDA autotransporter. Manuscript.

CONTENTS
1 INTRODUCTION ................................................................................................................................... 1
1.1 ADAPTIVE RESPONSES TO CHANGES IN GROWTH RATE ................................................................3
1.2 THE MEMBRANE STRUCTURE OF E. COLI ......................................................................................4
1.3 CONTROL OF GLUCOSE UPTAKE RATE ..........................................................................................6
1.3.1 Cultivation techniques .........................................................................................................6
1.3.2 Glucose uptake ....................................................................................................................8
1.3.3 Acetic acid formation a result of high glucose uptake rate ............................................10
1.3.4 Reduction of acetate formation .........................................................................................11
1.4 LIMITING FACTORS IN RECOMBINANT PROTEIN PRODUCTION ....................................................12
1.4.1 Cytoplasmic production ....................................................................................................14
1.4.2 Periplasmic production .....................................................................................................21
1.4.3 Production in the inner membrane ....................................................................................25
1.4.4 Surface display of proteins in E. coli ................................................................................27
1.4.5 Extracellular production ...................................................................................................33
2 PRESENT INVESTIGATION ............................................................................................................ 34
2.1 A CELLULAR ALTERNATIVE TO FED-BATCH CULTURES (I, III) ...................................................35
2.1.1 Strain evaluation (I) ..........................................................................................................35
2.1.2 Production of integral membrane proteins by the PTS-mutants (III) ...............................41
2.2 IMPACT OF FEED-RATE ON PROCESSES WITH OTHER PRODUCT LOCALISATIONS THAN THE
CYTOPLASM (II, IV) .............................................................................................................................46
2.2.1 Leakage of periplasmic products in relation to the glucose uptake rate (II) ....................46
2.2.2 Optimisation of surface expression using the AIDA autotransporter (IV) ........................52
3 CONCLUDING REMARKS ............................................................................................................... 58
4 ABBREVIATIONS ............................................................................................................................... 62
5 ACKNOWLEDGMENT ...................................................................................................................... 64
6 REFERENCES ...................................................................................................................................... 65

1 INTRODUCTION
Since the first announcement on microbial production of a protein of human origin,
insulin, was made in 1978 by researchers at the company Genentech (Genentech,
press release, 1978), recombinant protein production has become a routine business.
Today, it is a common strategy for production of many high-value proteins used in for
example various medical applications (e.g., vaccines, recombinant factor VIII for
treatment of haemophilia, insulin for treatment of diabetes and tissue-plasminogen
activator against stroke). The principle behind this technology is relatively simple: a
foreign gene, encoding a target protein of interest, can be introduced into a host cell
that will use its own cellular machinery to translate the gene into the desired protein
product. However, this important breakthrough would never have been realised
without the pioneering work made in the early seventies on how to isolate and amplify
genes (or DNA) and then insert them into specific genetic locations to create
transgenic organisms (Cohen, et al., 1973; Lobban and Kaiser, 1973; Morrow, et al.,
1974), hence forming the ground for what we today know as recombinant DNA
technology. Although Escherichia coli (E. coli) was used for expression of insulin in
this first example, the use of host cells is not restricted to bacteria such as E. coli but
many other prokaryotic (e.g., Bacillus) and eukaryotic (yeast, insect and mammalian)
cells can be used as well. Regardless of the specific host cell type used, the
productivity is influenced by environmental conditions (e.g., temperature, pH,
availability of oxygen and nutrients) as well as by genetic factors (e.g., the
gene/protein itself, the promoter strength, mRNA stability, codon usage, gene copy
numbers, availability of co-factors and various helper systems that aid in the
expression). Thus, in the end, process optimisation boils down to finding the
appropriate combination of environmental conditions and genetic factors that will
result in the highest amount of active protein.
The focus when producing recombinant proteins in the pharmaceutical industry today
is generally either on i) large-scale production of particular commercial protein
products or on ii) small scale high-throughput production (HTP) of proteins that are
used either for structural and functional studies or for high-throughput screening
(HTS) against potential drug leads in the drug development process. E. coli, with its
ability to grow rapidly to high cell densities on inexpensive substrates, its well1

characterised genetics and the availability of numerous cloning vectors and mutant
host strains, is an attractive host for production of recombinant proteins (Schmidt,
2004).
The goal for the large-scale industrial production of a protein is to achieve a high total
productivity in order to produce a large amount of the protein in a cost efficient
manner. Operator supervised bioreactors, which offer a high level of control and
regulation of the process are used and parameters like temperature, pH and dissolved
oxygen tension (DOT) are measured and regulated. The fed-batch technique is usually
applied, where a glucose feed is continuously added to the bioreactor during the
cultivation, making it possible to control the glucose uptake rate of the cells. The fedbatch technique enables the establishment of high cell densities, since growth rate and
by-product formation are controlled.
The goal for high-throughput production of proteins is to achieve sufficient
amounts of soluble proteins for functional or structural studies. This production
usually relies on unsupervised batch cultivation in small scale, most commonly micro
titre plates or shake flasks. The cells grow exponentially until limitations related to
e.g., oxygen availability or by-product formation arises. The level of control of the
environmental conditions is low in this type of small scale shaken systems and the
conditions changes rapidly as a consequence of the exponential biomass increase. For
practical reasons, the fed-batch technology is not applicable for control of the glucose
uptake rate in such systems, and there consequently is a need for other innovative
strategies for controlling the glucose uptake rate; especially as the glucose uptake rate
has been shown to have an impact on product-associated parameters such as specific
productivity, solubility and proteolysis of the recombinant protein in earlier studies
(Bostrm, et al., 2005; Ryan, et al., 1996; Sandn, et al., 2005; Sandn, et al., 2002).
In general, recombinant proteins are preferably produced in active, soluble forms. One
problem with production of recombinant proteins in E. coli is that the overexpressed
proteins are not always properly folded and then associate into mainly non-active and
insoluble aggregates, which are termed inclusion bodies. Proteins in inclusion bodies
may regain activity after a refolding process. Refolding is, however, a complicated

process, and the process has to be optimised for each protein in question (Hauke, et
al., 1998) and is therefore not an applicable strategy in HTP applications. However,
some proteins fold properly if they are secreted to the periplasm. Other difficult-toexpress proteins such as membrane proteins and toxic proteins are also preferably
transported to other parts of the cell than the cytoplasm. In order to understand the
impact of the glucose uptake rate on these kinds of processes where the product is
localised to other parts of the cell than the cytoplasm, further investigations are
needed.

1.1 Adaptive responses to changes in growth rate

The growth rate of cells varies depending on the growth conditions, i.e. the growth
rate is influenced by factors such as substrate concentration, growth medium,
temperature, pH and the supply of oxygen. In general, fast growing cells contain more
DNA, RNA, ribosomes, proteins, phospholipids and cell wall material, and tend to
increase in size (Lengeler and Postma, 1999). Cells respond to carbon or amino acid
limitation by inhibited RNA and protein synthesis. Also the DNA replication as well
as the biosynthesis of carbohydrates, phospholipids and cell wall constituents is
inhibited and the cell size decreases. This set of responses, with a tight coupling
between growth rate, ribosomal synthesis and cell size is referred to as the stringent
response and is mediated by the production of the alarmone guanosine tetraphosphate
(ppGpp). E. coli uses two different pathways to produce ppGpp. The lack of amino
acids results in the binding of uncharged tRNA to the ribosome, which activates the
RelA enzyme leading to the formation of ppGpp. The lack of carbon, on the other
hand, leads to the activation of the alternative pathway for ppGpp formation involving
SpoT (Lengeler and Postma, 1999). The ppGpp bind to the RNA polymerase core
enzyme, which affects the expression of a plethora of genes. In general, genes
involved in cell proliferation and growth are negatively regulated by ppGpp, whereas
genes involved in maintenance and stress defence are positively regulated
(Magnusson, et al., 2005). The starvation sigma subunit, S, accumulates in the cell
whenever the growth rate is lowered and not only when the growth ceases. The
synthesis of S is positively controlled by ppGpp (Booth, 1999).

1.2 The membrane structure of E. coli

The lipid structure of the membranes depends on the glucose uptake rate (Shokri, et
al., 2002). However, before discussing this dependency, an overview of the basic
structures of the membranes will be given. The basic unit of a membrane is a bilayer
that is formed by phospholipids organized in two layers with their polar head groups
along the two surfaces and the acyl chains forming the nonpolar domain between.
Membranes are dynamic with movement both across and in the plane of the bilayer.
The bilayer serves as matrix and support for many proteins that are involved in
transmembrane processes, including translocation of proteins and other molecules
across membranes (Dowhan, 1997). The fluid mosaic model (Singer and Nicolson,
1972) has been used for many years for describing the nature of the membranes. The
membranes proteins are in this model more or less viewed as icebergs floating in a sea
of lipids. However, in the last years, this view has been shifting and the importance of
transient, specialized regions called membrane rafts, which are enriched in special
lipids or proteins has become clear (Luckey, 2008).
Pore
LPS
Phospholipid

Outer
Membrane

Lipoprotein
Periplasm
Peptidoglycan
Protein
Inner
Membrane

Figure 1. Model of Escherichia coli cell envelope. The cell has a two-membrane
structure composed of the cytoplasmic and the outer membrane. The periplasm, the
space between theses membranes, contains the cell wall made of peptidoglycans.

The cell envelope of gram-negative bacteria, to which E. coli belongs, is a twomembrane structure of cytoplasmic and outer membrane (Fig 1). The space between
the membranes is the periplasm where a thin cell wall consisting of peptidoglycan is
situated. The cell wall gives shape and rigidity to the cell, and prevents the cell from
lysing in dilute environments. The outer membrane contains lipopolysaccharide
(LPS), a structure that is not found in the cytoplasmic membrane.
Escherichia

coli

membranes

contain

three

major

phospholipids:

phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). PE,

which is the major phospholipid, constitutes roughly 75 % of the total phospholipid
content, PG 18% and CL 5 % (Cronan and O.Rock, 1996). The phospholipid
composition of the cytoplasmic and outer membrane is similar, but with a slight
enrichment of PE in the outer membrane (Nikaido, 1996). PE is zwitterionic and does
not carry a net charge at physiological pH, while PG and CL are anionic.
Phospholipids contain both saturated and unsaturated fatty acids as well as cyclic fatty
acids, which are formed by methylation of unsaturated ones. E. coli adjusts the fatty
acid composition of its phospholipids in response to growth temperature in order to
preserve a more or less constant degree of membrane fluidity. The proportion of
unsaturated fatty acids increases as the temperature decreases and vice versa for the
saturated ones. This change results in more or less constant fluidity of the membranes
since the melting point of lipids decreases as the proportion of unsaturated fatty acids
increases (Neidhardt, et al., 1990). The total amount of phospholipids does not vary
with the growth rate but the composition of the phospholipids does (Cronan and
O.Rock, 1996). PG reaches a maximum at a specific growth rate of 0.3 h-1 (Shokri, et
al., 2002) and this growth rate is associated with a high permeability of the
membrane. The membrane becomes more rigid as the growth rate declines (Shokri, et
al., 2003).

1.3 Control of glucose uptake rate

1.3.1 Cultivation techniques
Batch
In a batch process all substrate components are available at high enough
concentrations to make the reaction rate unrestricted with respect to substrate
concentration (Fig 2). The biomass concentration increases exponentially, i.e. the
specific growth rate () is constant at max, until some factor e.g., by-product
concentration, oxygen supply or low substrate concentration reduces the specific
growth rate.
Oxygen limitation and the formation of the by-product acetic acid limit the usefulness
of the batch technique. The batch technique is however the easiest cultivation method
and it is used when proteins are produced either for functional and structural studies
or for HTS. Further, screening for new production methods in the industrial process
development is usually performed in batch mode.
!"#$%#

&'()*#

!+,-.'()*#

Figure 2. The difference between batch and fed-batch cultivation.

F= feed rate of limiting substrate (l h-1), Si=concentration of the
substrate (g l-1)

Fed-batch
The fed-batch technology is a common industrial method for recombinant protein
production. One substrate component, usually glucose, is added to the bioreactor at a
rate so that its concentration is growth rate limiting and thus the growth rate can be
controlled via the feed (Fig 2). The condition for growth rate limitation in a fed-batch
process is:

F/V(t)*Si < qs,max X(t)

Where F (l h-1) is the feed rate, V (l) the cultivation volume, Si (g l-1) the concentration
of the substrate in the feed solution, qs,max (g g-1 h-1) the maximal specific consumption
rate of the limiting substrate and X (g l-1) the cell mass at that time point (Enfors and
Hggstrm, 2000). There are two main reasons to apply the fed-batch technique. First,
the substrate limitation offers a tool for reaction rate control to avoid engineering
limitations with respect to cooling and oxygen transfer. Secondly, the substrate
limitation also permits a sort of metabolic control by which overflow metabolism,
resulting in formation of acetic acid, can be avoided (Enfors and Hggstrm, 2000).
The fed-batch technique makes is possible to obtain high cell densities and thus to
reach a high total productivity.
The feed profile can be designed in different ways. A constant feed results in a
continuously decreasing growth rate of the cells since the amount of substrate per cell
decreases as a function of time. An exponential feed results in a constant growth rate
of the cells, since each cell receives the same amount of substrate during the whole
cultivation. By using exponential feed-profiles, exponential growth can be
maintained, but at a lower rate than the maximal rate (max). A steady state with
respect to substrate concentration and growth rate is established in exponential fedbatch cultures.
Fed-batch cultivations may be started as batch cultures and the feed of substrate is
then started when the initial substrate is depleted. In the industry, however, it is
common to start the feed of substrate directly after the inoculation of the reactor.
Usually, an exponential feed is used until the oxygen limitation of the reactor is
reached. To further increase the biomass, a constant feed is then applied, which results
in gradually reduced specific growth rates of the cells.

1.3.2 Glucose uptake

Diffusion of carbohydrates through the outer membrane occurs mainly through the
outer membrane channel forming proteins OmpC, OmpF and LamB (Nikaido, 1996).
The phosphotransferase system (PTS) transports carbohydrates such as glucose,
fructose and mannose from the periplasm to the cytoplasm. The PTS system is
composed of both soluble proteins and proteins that are integrated in the cytoplasmic
membrane (Fig 3). Enzyme I (EI) and phosphohistidine carrier protein (HPr) are
general PTS proteins, while the enzymes IIs (EIIs) are sugar specific. EIIGlc is specific
for glucose and EIIMan for mannose. At least one of the EII-domains is bound to the
membrane. The glucose EII consists of the parts IIAGlc and IICBGlc where the latter is
membrane bound, while the mannose EII consists of the IIABMan and the membrane
bound parts IICMan and IIDMan (Fig 3). A series of reactions are involved when the
sugar molecule enters the cytoplasm. A phosphate group is transferred from
phosphoenolpyruvate (PEP) to EI and further to HPr. The phosphate group is then
transferred to the EIIA domains and further to the EIIB/EIICB domains. These
domains perform phosphorylation of the incoming sugar molecule (Postma, et al.,
1996).

Figure 3. A model for PTS mediated uptake of carbohydrates. The scheme shows
the general enzymes of the PTS: Enzyme I (EI), Phosphohistidine carrier protein
(HPr) and two Enzymes II (EII). P indicates phosphorylation of the various
enzymes and the sugar molecules. Modified from (Postma et al. 1996).

Glucose is transported over the cytoplasmic membrane mainly through the glucose
and mannose specific PTS. GalP and the Mgl-system, proteins that are normally
involved in the transport of galactose, may however also transport glucose into the
cytoplasm. These proteins can be induced and used for glucose transport during
glucose limiting conditions. Glucose that is transported into the cytoplasm by the
Mgl-system or GalP is phosphorylated by glucokinase, encoded by the gene glk
(Gosset, 2005).
Glucose uptake is also controlled by the repressor protein Mlc. The phoshorylated
form of enzyme IICBGlc dominates in the absence of glucose. In this situation Mlc
binds upstream of the ptsG promoter and works as a repressor. Addition of glucose to
the medium results in dephosphorylation of enzyme IICBGlc. Mlc binds to the
dephoshorylated enzyme IICBGlc and is thereby sequestered away from the operator
making transcription of ptsG possible. Mlc is, as described above, thus capable of
binding both DNA and proteins (Plumbridge, 2002).
The PTS system also has an important role in catabolite repression, which results in
inhibition of gene expression and/or protein activity by the presence of a rapidly
metabolisable carbon source in the medium. Glucose is the preferred carbon source
for E. coli and decreased concentration of glucose in the medium results in
accumulation of the phoshorylated form of enzyme IIAGlc and IICBGlc. The
phosphorylated form of enzyme IIAGlc activates adenylate cyclase (AC), the enzyme
that converts ATP to cAMP, resulting in increased levels of cAMP. The cAMP binds
the product of the crp locus, termed the cAMP receptor protein (CRP). The cAMPCRP complex causes the induction of catabolite-repressed genes allowing uptake of
other sugars. The uptake of substrates is also regulated by another mechanism. The
unphosphorylated form of IIAGlc dominates when the glucose concentration in the
medium is high. When enzyme IIAGlc is in the unphosphorylated form, it can
inactivate several transport systems for non-PTS carbon sources, by binding to the
transporters. This mechanism is called inducer exclusion (Lengeler and Postma,
1999).

1.3.3 Acetic acid formation a result of high glucose uptake rate

Aerobic growth of E. coli on excess of glucose leads to the excretion of partially
oxidised metabolites, mostly in the form of acetic acid. This phenomenon is called
overflow metabolism and occurs if the glucose concentration exceeds a critical value.
This value is approximately 20-30 mg l-1 glucose and corresponds to a specific growth
rate of approximately 0.30 h-1 in minimal medium (Enfors and Hggstrm, 2000;
Meyer, et al., 1984). The reason for the overflow metabolism is not clear but it may be
a result of an imbalance between the glycolysis and the TCA-cycle or of saturation of
the TCA-cycle or the electron transport chain (Lee, 1996).
The main route for acetate production is from Acetyl-CoA through the enzymes
phosphotransacetylase (Pta) and acetatekinase (Ack) (Fig 4). This route generates
ATP. The other route for acetate production is directly from pyruvate by pyruvate
oxidase B (PoxB), but it plays a minor role (Wolfe, 2005).
Glucose!

PEP!
Pyruvate!
CO2!

pox

B!

CO2!
NADH!
Acetyl-CoA!

pta!

Acetyl-P!

Acetate!

ack!
ATP!

Figure 4. Simplified view of acetate formation in E. coli. Acetate is formed

either by the Pta-Ack route or by the PoxB route.

Most E. coli strains have the ability to assimilate acetate. This is primarily done by the
enzyme AMP-forming acetyl CoA-synthetase (AMP-ACS) (Wolfe, 2005). This route
is utilized when acetate is the carbon source or when there is a need to reabsorb
acetate that has been excreted as a consequence of overflow metabolism (Wolfe,
2005).

10

Acetate production is undesirable in recombinant protein production since it reduces

bacterial growth even at concentrations as low as 0.5 g l-1 (Nakano, et al., 1997).
Further, acetate formation reduces the yield of biomass and the production of
recombinant proteins has also been shown to be negatively affected (Jensen and
Carlsen, 1990; Turner, et al., 1994).
Acetate production depends on the bacterial strain, and it is known that E. coli Bstrains do not accumulate as much acetic acid as E. coli K12 strains. It has been
suggested that B-strains have a more active glyoxylate shunt than K12-strains, which
results in the direction of the pyruvate into biosynthetic precursors, e.g., succinate and
in the reduction of acetic acid (Phue and Shiloach, 2004; van de Walle and Shiloach,
1998). The lower accumulation of acetic acid in B-strains has also been explained by
differences in the transcription level of the acetate production genes (Phue and
Shiloach, 2004).

1.3.4 Reduction of acetate formation

The most commonly used approach in order to decrease the acetate production is the
fed-batch technique. Another strategy is to use genetic modification. The targets
genes are then i) genes that code for proteins involved in the uptake of glucose or ii)
genes that code for enzymes that are active in pathways resulting in acetic acid.
Using an E. coli strain with a mutation in ptsG, the gene coding for EIIGlc, resulted in
20-40% decrease in the specific acetate yield in comparison to the wild type. Other
consequences of the mutation were an increased biomass, from 13 to 19 g l-1 and an
increase in recombinant protein concentration exceeding 50% (Chou, et al., 1994).
Mlc overproducing strains were constructed by mutations in the promoter region of
the chromosomal mlc gene. This strain showed a reduced acetate accumulation, and as
a consequence of this, the pH of the media was higher in comparison to the wild type.
The effect of the mutation was also observed as an increase in the OD600 from 5 to 8 in
comparison to the wild type (Cho, et al., 2005).

11

Another approach that has been used in order to decrease the accumulation of acetic
acid is to replace the PTS system by the galactose permease. The galP gene is
normally repressed when E. coli grows on glucose. Its transcription is however
increased in PTS mutant strains. In this study, the chromosomal galP promoter was
replaced by the trc promoter, making induction with IPTG (isopropyl -Dthiogalactopyranoside) possible. The acetate concentration was decreased from 2,8 to
0,39 g l -1 and the GFP formation was increased four times in comparison to the wild
type (De Anda, et al., 2006).
Mutant strains, which lack the enzymes that form acetic acid, have also been
constructed and the excretion of acetate in these strains has been reduced (Bauer, et
al., 1990). Recently a triple BL21 mutant lacking all known acetate production genes
(ackA-pta-poxB) was constructed. Interestingly, this strain still accumulates acetic
acid, which indicates that there are other alternative acetate production pathways in
the cell (Phue, et al., 2010).

1.4 Limiting factors in recombinant protein production

The maintenance of a plasmid and high-level expression of the target protein
represents a metabolic burden to the cell. Production of a recombinant protein can
trigger stress responses in the cell that often resembles the cellular responses to
environmental stress such as heat shock and stringent response (Srensen and
Mortensen, 2005). The extent of the stress responses is determined by the rates of
transcription and translation, but stress can also emerge from the specific properties of
the recombinant protein, i.e. misfolding, which results in the degradation of the
recombinant product and in inclusion body formation (Hoffmann and Rinas, 2004).
Production of recombinant proteins can also lead to ribosome destruction (Dong, et
al., 1995) and in severe cases to cell-death (Miroux and Walker, 1996).
Generally, the goal in recombinant protein production is not only to maximise the
amount of recombinant protein but also to achieve a protein with a high quality, i.e. a
protein in an active, soluble and pure form. However, some proteins cannot be
produced as active, soluble entities in the cytoplasm and must therefore be directed to
other compartments of the cell. The advantages and disadvantages associated with
12

expression in each compartment are summarised in table 1. Overexpression in the

outer membrane has the purpose of producing whole cells with recombinant proteins
expressed at the surface, and differs in that context from the production in the other
compartments, where the goal is to produce recombinant proteins for isolation and
purification.

Table 1. Advantages and disadvantages with production of recombinant proteins in different

compartments of the cell.
Compartment

Advantages

Disadvantages

Cytosol (soluble protein)

Higher protein yield

No S-S bond formation

More complex purification
Many proteases

Cytosol (inclusion
bodies)

High protein yield

Protection from proteases
Reduced toxicity
Inclusion bodies are easy to isolate

Solubilisation and refolding needed

Lower yield
Higher cost
Refolding not always possible

Inner membrane (limited

to production of
membrane proteins)

The product can be purified after

detergent extraction

Low yield
Limited space in the membrane
Accumulation of product may be toxic to
the cell

Periplasm

Fewer proteases
Formation of S-S bonds
N-terminus authenticity
Simpler purification if selective release
of product possible

Inclusion bodies may form

Inefficient transport

Outer membrane (limited

to surface expression of
proteins)

On-cell protein characterisation possible

No purification of protein is needed
Increased stability of product
Whole cells can simply be removed

Limited space in the membrane

Transport not always possible

Extracellular

Less extensive proteolysis

Easier purification
N-terminus authenticity
Reduced toxicity

Transport not always possible

Diluted product

13

1.4.1 Cytoplasmic production

The goal for recombinant protein production in the cytoplasm is either to obtain
soluble proteins or to direct the product into inclusion bodies. The advantage with the
cytoplasmic production is the high yield of product. The cytoplasm is a reducing
environment, inhibiting the formation of disulphide bridges in the protein, a problem
that might be circumvented by secretion to the periplasm. Other disadvantages
associated with cytoplasmic production is the need for cell disruption, the complex
purification from a mixture of endogenous proteins and the higher amounts of
proteases in comparison to the periplasm. In some cases, cytoplasmic overexpression
of a gene results in the presence of one extra N-terminal methionine in the product
(Adams, 1968; Moerschell, et al., 1990), which may have negative impact on the
stability and solubility of the product (Chaudhiri, et al., 1999). That the product ends
up as inclusion bodies may for proteins that are easy to refold actually be beneficial.
Proteins in inclusion bodies are easy to separate form the cell debris after cell
disruption, they are mostly inactive and therefore not toxic to the cell, and they are
generally protected from degradation by proteases and are relatively pure (Jrgen, et
al., 2010; Makrides, 1996). However, when using this strategy, in vitro refolding is
needed to achieve a correctly folded product. There is however no guarantee that
proteins in inclusion bodies will regain activity or that the refolding will result in a
high yield. Refolding and purification from inclusion bodies is usually more
expensive and time consuming than purification of soluble proteins (Sorensen and
Mortensen, 2005). The renaturation conditions, such as temperature, buffer, pH and
ionic strength must be optimized for every protein (Hauke, et al., 1998) and using the
refolding strategy in HTP applications is therefore not an option.
Folding
The newly synthesized protein either folds independently or needs to be assisted by
molecular chaperones to attain a correct and biologically active structure. The
molecular chaperones are proteins that help the folding of other proteins, usually
through cycles of binding and release, without forming part of their final structure
(Young, et al., 2004). The chaperones favor on-pathway folding by shielding
interactive surfaces from each other as well as from the solvent, and by accelerating
rate-limiting steps. Chaperones are also invaluable in protein secretion and
14

translocation where their function is to prevent folding of the target protein, thereby
keeping them in a translocation-competent state. Chaperones are also involved in the
process of disaggregation of already formed aggregates (Baneyx and Mujacic, 2004).

Figure 5. Chaperone-assisted folding in the cytoplasm. The nascent polypeptide first encounters TF or
DnaK/DnaJ as it exits the ribosome. After release, the folding intermediate either reaches its native
conformation or is transferred to GroEL/GroES for further folding assistance. Partially folded proteins
may also be stabilized by the holding chaperones IbpA/B, Hsp31 and Hsp33 until folding chaperones
are available. The disaggregating chaperone ClpB promotes disaggregation of unfolded proteins and
cooperates with the folding chaperones to reactive them. 1

The molecular chaperones can be divided into three different groups based on their
function, the folding (e.g., DnaK and GroEL), the holding (e.g., IbpB) and the
disaggregating (e.g., ClpB) chaperones (Fig 5). Foldases constitute another important
group of chaperones and their role is to accelerate rate-limiting steps in the folding
1 Reprinted by permission from Macmillan Publishers Ltd: [Nature Biotechnology],
(Francois Baneyx and Mirna Mujacic, 2004, Recombinant protein folding and
misfolding in Escherichia coli, Nature Biotechnology, Vol. 22, No 11, p 1399-1408),
Copyright (2004)

15

pathways. The peptidyl-prolyl isomerases (PPIases) increases the rate of cis-trans

isomerization of peptide bonds involving proline residues and the thiol disulfide
oxidoreductases, known as the Dsb proteins, catalyse the formation and shuffling of
disulphide bridges in the periplasm (Baneyx and Mujacic, 2004).
The heat shock response in E. coli is regulated by 32 and it is induced by a large
variety of stress factors including temperature shift, metabolic harmful substances and
protein misfolding and aggregation. Major heat shock proteins are proteases and
molecular chaperones including DnaK/DnaJ/GrpE and GroEL/GroES (Arsne, et al.,
2000).
Proteolysis
Proteolysis and folding are tightly connected processes in the cell. The degradation of
misfolded proteins guarantees that abnormal polypeptides do not accumulate within
the cell and conserves the cellular resources by the recycling of amino acids. The
ATP-dependent cytoplasmic protease Lon and the Clp family of proteases are induced
by heat shock (Gross, 1996).
One strategy that can be used in order to avoid degradation of the recombinant
product is utilizing protease deficient mutants. Using such mutants is however often
associated with reductions of the growth rate and it is not known if the cell
compensates for the loss of one protease by increasing the concentration of another
(Martnez-Alonso, et al., 2010). One exception is however the strain BL21 that is
deficient of both Lon and OmpT but still exhibits good growth characteristics
(Srensen and Mortensen, 2005). However, in strains devoid of the Lon protease, the
aggregation of over-produced proteins has shown to be enhanced (Corchero, et al.,
1996). For proteins that are easy to refold, the deposition of them into inclusion
bodies can be an alternative superior strategy for avoiding degradation from proteases
(Hauke, et al., 1998).

16

Recovery and degradation of aggregated proteins

Protein aggregates accumulate when the cells capacity for folding, unfolding and
degradation is exceeded, which is usually a consequence of stress or of
overexpression of recombinant proteins (Srensen and Mortensen, 2005). The
molecular chaperones DnaK and GroEL not only mediate proper de novo folding of
proteins but are also involved in degradation of abnormal polypeptides by targeting
them to proteases such as Lon and ClpP (Martnez-Alonso, et al., 2010). The holding
chaperones IbpA and IbpB stabilize partly unfolded proteins without actively
promoting their refolding and are often found tightly associated to the target proteins
in inclusion bodies (Allen, et al., 1992). The disaggregating chaperone ClpB
cooperates with DnaK and GroEL in reversing protein aggregation (Weibezahn, et al.,
2004).
Strategies to improve specific productivity and solubility
The goal in recombinant protein production is to obtain as much as possible of high
quality product. A high total productivity can be achieved either by scaling up the
cultivation volume, by increasing the biomass or by increasing the specific
productivity (the productivity per cell). Promoter strength, inducer concentration, the
plasmid copy number and the specific growth rate of the producing cells are some
factors that have been shown to influence the specific productivity, but also the
solubility of the product (Sandn, et al., 2005; Sandn, et al., 2002; Swartz, 2001;
Tegel, et al., 2011; Terpe, 2006). When aiming at producing soluble proteins, the
strategy is in general to slow down the synthesis rate of the protein. Examples of such
strategies include using weaker promoters (Tegel, et al., 2011), using lower inducer
concentrations (Sandn, et al., 2005) and to lower the specific growth rate of the
producing cells (Sandn, et al., 2005). However, the opposite strategy, to use high
synthesis rates during the production, increases the specific productivity (Ryan, et al.,
1996; Sandn, et al., 2002). To find the best production conditions in order to obtain a
large amount of soluble protein therefore relies on finding the correct balance between
a high specific productivity and a high solubility.

17

The strength of a promoter is determined by the relative frequency of transcription

initiation. This is affected by the affinity of the promoter sequence for the RNApolymerase but also by the transcription rate of the RNA-polymerase. The T7
promoter, which is a very common promoter, requires host strains coding for the T7
RNA polymerase (Terpe, 2006). The E. coli BL21(DE3) strain and its derivatives,
contain a lambda lysogen DE3 with the T7 RNA polymerase under the control of the
IPTG inducible lacUV5 promoter (Terpe, 2006). The T7RNA polymerase transcribes
RNA five times faster than the E. coli RNA-polymerase (Golomb and Chamberlin,
1974). In many cases the use of the T7 leads to high product accumulation, in some
cases as high as 40-50% of the total cell protein (Studier and Moffatt, 1986).
Although the high product accumulation is desirable, the use of strong promoters such
as T7, has its drawbacks. High-level overexpression of genes can cause ribosome
destruction (Dong, et al., 1995) and cell death (Miroux and Walker, 1996) and the use
of strong promoter systems often results in the inability of the cell to fold the target
protein properly (Baneyx, 1999). Further, production of mRNA is energy demanding
and increases the metabolic burden on the cell.
Tegel and co-workers (2010) studied the effect of promoter strength on
overexpression of proteins during batch cultivation in shake flasks. The total
production as well as the soluble fraction of protein epitope signature tags (PrESTs),
short regions of human proteins, was measured. The expression from the T7, the
lacUV5 and the trc promoter was compared. In general, production under the control
of the T7 promoter resulted in the largest total amount of target protein, whereas the
use of the lacUV5 promoter, the weakest promoter in the study, resulted in the lowest
amount of total protein. The weakest promoter generated the largest fraction of
soluble protein and vice versa, and generally, the fraction of soluble protein was small
using both T7 and trc. However, the amount of soluble protein was highest using T7
even though the soluble fraction was the lowest. This is explained by the much higher
total production using this promoter (Tegel, et al., 2011).

18

The production of recombinant -galactosidase was studied with respect to the

specific growth rate at the time-point of induction (Sandn, et al., 2002). Induction
was performed at specific growth rates of 0.5 h-1 and 0.1 h-1, respectively. It was
shown that induction at the higher growth rate resulted in an almost 100 % higher
specific productivity. The amount of mRNA formed was the same at both occasions
and was therefore not the limiting factor. The ribosomal content, represented by the
rRNA amount, was five times lower at the low specific growth rate. At the high
specific growth rate, degradation of the ribosomes after induction, as a consequence
of the high product formation rate, was also observed. In the study, the conclusion
was drawn that translation, and not transcription, was the limiting factor in the protein
synthesis capacity. Ryan et al (1996) also showed that cells growing at a high specific
growth rate at the time of induction were more efficient in synthesizing the
recombinant protein. The synthesis rate also remained higher for fast growing cells
(Ryan, et al., 1996).
The concentration of IPTG and the feed rate of glucose during fed-batch cultivation
was shown to influence the quality of the recombinant product when production was
controlled from the lacUV5 promoter (Sandn, et al., 2005). The model proteins in
the study were maltose binding protein (MBP) and a mutated form of this protein that
was less stable and more prone to aggregation. Two different exponential feed
profiles were used which resulted in constant specific growth rates of 0.2 h-1 (low feed
rate) and 0.5 h-1 (high feed rate), respectively. Both the soluble and the insoluble
fractions of the proteins increased with the inducer concentration. Using the highest
concentration of inducer did not result in more soluble protein but in increased
formation of inclusion bodies. The lowest amount of soluble protein was achieved by
a high feed rate with the mutated, unstable form of the protein, in combination with a
high inducer concentration. A high feed rate in combination with a high inducer
concentration leads to a high syntheses rate of the protein. In this situation, the folding
machinery of the cell might get overloaded and is unable to stabilise and fold the
protein properly, which leads to increased proteolysis and aggregation.
Another factor that influences the efficiency of translation is the translation initiation
region (TIR) of the mRNA. The main elements of translation initiation in E. coli are

19

the Shine-Dalgarno sequence, the initiation codon that in E. coli is mostly AUG, and a
downstream region (DR). The SD is located 5-9 bases upstream of the initiation
codon and is important for the binding of the mRNA to 30S ribosomal subunit. The
sequence of the SD, in comparison to consensus sequence in E. coli, and its distance
to the initiation codon are factors that determines the translational efficiency. The
down stream region (DR) located immediately after the initiation codon also
influences the gene expression level (Stenstrm and Isaksson, 2002). Engineering the
translation initiation region of the mRNA is a promoter independent strategy of
influencing gene expression. However, manipulations in the DR will in many cases
change the amino acid sequence of the product (Baneyx, 1999).
A possible strategy for increasing the solubility of the recombinant proteins is by coexpression of chaperones. There are several chaperones or chaperone sets that have
been selected for over production along with the recombinant target protein. The
beneficial effect of co-expression of chaperones is however unpredictable and is a
matter of trial and error, and there is no guarantee that the overexpression of the
chaperones improves the solubility of the recombinant product (Sorensen and
Mortensen, 2005). Co-expression of the chaperones DnaK and GroEL/ES might
instead trigger proteolytic activities in the cell, which results in reduced yield, stability
and quality of the recombinant protein (Martnez-Alonso, et al., 2010). The dual role
of the chaperones, acting both as folding modulators and as proteolytic enhancers
might partly explain the diverse results reported from co-expression studies
(Martnez-Alonso, et al., 2010). Moreover, overproduction of chaperones contributes
to the metabolic burden of the cell, which might explain the reduced yield of the
recombinant protein.
Examples of other strategies that can be used in order to increase the solubility
include cultivation at low temperature and the use of fusion tags (Srensen and
Mortensen, 2005).

20

1.4.2 Periplasmic production

The transport to the periplasm
The main protein-translocation machinery in the inner membrane of E. coli is the Sectranslocase, which transport proteins across or insert proteins in the inner membrane.
The substrates for the Sec-translocase all contain hydrophobic N-terminal regions.
Proteins that are destined for secretion have N-terminal signal sequences that are
processed by signal peptidase that removes the signal sequence after the translocation
and allows release and folding of the protein in the periplasm. Inner membrane
proteins have membrane-anchoring signals in their N-termini that most often remain
associated with the inserted protein. The Sec-translocon can facilitate transport across
or integration of proteins into the membrane in a co-translational or post-translational
manner (Fig 6). The co-translational pathway is mainly employed for inner membrane
proteins, while the secretory proteins mainly utilize the post-translational pathway.
The selection of pathway lies at the early stage of translation when the nascent peptide
emerges from the ribosomal exit tunnel. Very hydrophobic signals that emerge from
the tunnel are bound by the signal recognition particle (SRP), which takes the
complex of the ribosome and the nascent chain to the membrane-associated SRPreceptor FtsY. Elongation of the chain and insertion of the protein into the membrane
via the translocon occurs simultaneously. The membrane protein YidC (not included
in Fig 6) has also been shown to play a role in the insertion of a subset of membrane
proteins via the translocon. If the signal sequence that emerges from the ribosomal
exit tunnel does not display a high level of hydrophobicity, it is bound by the trigger
factor (TF), which shields the nascent polypeptide from binding SRP. The newly
synthesised protein is maintained in an unfolded state by the cytoplasmic chaperone
SecB, which also targets the protein to the membrane bound ATPase SecA. Protein
translocation across the membrane occurs at the translocon (du Plessis, et al., 2011).

21

Figure 6. Schematic and simplified representation of protein targeting to the Sec-translocase. i) The
post-translational pathway: SecB binds to preproteins with less hydrophobic signal sequences and
targets the preprotein to SecA which is associated to the translocase SecYEG. The signal sequence is
cleaved off at the periplasmic side by the signal peptidase. ii) The co-translational pathway: More
hydrophobic signals are bound by the signal recognition particle (SRP) as they exit the ribosomal
tunnel. SRP takes the complex of the ribosome and the nascent chain to the membrane-associated
SRP-receptor FtsY. Elongation of the chain and insertion of the protein into the membrane via the
translocon occurs simultaneously.

Proteins may also be transported through the inner membrane by the twin arginine
translocation (TAT) system. This system transports proteins having specific twin
arginine motifs in their signal peptides and the proteins are transported in a folded
state (Berks, et al., 2005).
Targeting recombinant proteins to the periplasm
Recombinant proteins can be targeted to the periplasm by fusing natural signal
sequences to their N-termini. Periplasmic expression is associated with several
advantages in comparison with cytoplasmic production (Table 1). These include; (i)

22

authentic N-termini of the proteins are obtained after removal of the signal peptide by
the leader peptidase, (ii) possible formation of disulphides in the periplasm due to the
presence of the Dsb machinery (iii) lower amount of proteases in the periplasm
compared to the cytoplasm (iv) simplified purification of the target protein if the
periplasmic proteins are selectively released by osmotic chock or other strategies v)
higher solubility of the product (Baneyx and Mujacic, 2004; Mergulho, et al., 2005).
One difficulty with secretion is the inefficient export of the proteins across the inner
membrane. This may result in degradation or in inclusion body formation or in the
jamming of the membrane (Baneyx and Mujacic, 2004). Using signal sequences with
increased hydrophobicity may direct the preprotein to SRP-pathway and thus
eliminate toxic effects that might come from membrane jamming since the SRPpathway has a tighter coupling between translation and translocation than the SecBpathway (Wilson Bowers, et al., 2003).
The export capacity of the Sec-translocase was also studied by Mergulho and
Monteiro (2004). Different expression levels of two human proinsulin fusion proteins
were accomplished by using different promoters and copy number plasmids. The
periplasmic amount of product was almost the same although a 7 to 11-fold difference
in the total expression level was obtained. The study shows that the translation level
does not affect the maximum translocation efficiency and that a high synthesis rate of
the product is only wasting the resources of the cell (Mergulho and Monteiro, 2004).
Folding and degradation in the periplasm
The periplasmic chaperones are involved in the folding of the periplasmic proteins
and in the incorporation of proteins in the outer membrane. The Dsb proteins enable
the formation and reshuffling of disulphide bridges. SurA and FkpA are examples of
peptidyl-prolyl isomerases that are present in the periplasm (Moat, et al., 2002). Skp
is and example of a general periplasmic chaperone that binds its substrate in a cavity,
thereby protecting it from degradation (Walton, et al., 2009).
There are fewer proteases in the periplasm than in the cytoplasm. One example of a
periplasmic protease is DegP. DegP function both as a chaperone and as a protease

23

and is essential for the removal of misfolded and aggregated inner membrane and
periplasmic proteins. Synthesis of DegP is controlled by the E regulon, which is
activated in response to unfolded proteins in the periplasm (Missiakas, et al., 1996).
In order to increase the solubility of the product, the co-expression of chaperones may
be efficient. In a study by Sonoda and co-workers (Sonoda, et al., 2011) this effect
was studied on production of a single-chain Fv antibody secreted to the periplasm. It
was found that the overexpression of the periplasmic chaperones Skp and of FkpA
greatly increased the solubility of the product. However, co-expression of both FkpA
and Skp had no synergistic effect. Further, co-expression of the cytoplasmic
chaperones also affected the binding activity of the antibody fragment in the
periplasm, especially the co-expression of DnaKJE.
Bostrm and co-workers (Bostrm, et al., 2005) studied the effect of the feed rate on
recombinant protein secretion and degradation. Secretion of a protein to the periplasm
resulted in less degradation and in the avoidance of the stringent response. It was also
found that accumulation of acetic acid was ten times lower at a high specific growth
rate when the product was secreted to the periplasm.
Leakage to the medium
Periplasmic products show a tendency to leak to the culture medium, which is not
desirable in most periplasmic processes. The tendency of leakage can however be
utilized in processes where the recombinant proteins are targeted to the extracellular
medium. There are a number of hypotheses in the literature concerning the E. coli
cells inability to retain periplasmic products/leak periplasmic products to the medium
(Shokri, et al., 2003). A common basis is that the structural integrity of the membrane,
such as protein and lipid composition, is crucial for determining the retention. An
altered composition of the membrane may result from genetic changes or from
environmental changes such as medium composition or the feed rate of glucose
(Shokri, et al., 2002).

24

Some E. coli mutants leak periplasmic proteins to the medium in a large extent. These
mutants generally lack structural elements of the membranes or the cell wall, for
example, LPS (Tamaki, et al., 1971) and murein lipoprotein (Lpp) (Nikaido, et al.,
1977). Lazzaroni et al (1981) showed that mutants with a changed composition of
outer membrane proteins (OMP), in this case a lower amount of OmpF, released more
periplasmic protein to the medium (Lazzaroni and Portalier, 1981).
Secretion of overexpressed proteins to the periplasm also influences leakage. Cells
that were secreting -lactamase were more sensitive to detergents and they also had a
higher non-specific release of periplasmic proteins to the medium. Analysis of the
outer membrane protein composition showed that the amount of OmpA and OmpC
was lower (40-60%) in secreting cells (Georgiou and Shuler, 1988).
Some signal peptides seem, for unknown reasons, to enhance export of periplasmic
proteins to the medium. This was for example shown for insulin-like growth factor I
that was secreted by the use of the signal peptide from staphylococcal protein A. This
signal peptide has an extension in the N-terminus when compared to other signal
peptides (Abrahmsn, et al., 1986).

1.4.3 Production in the inner membrane

Many membrane proteins can be overexpressed in inclusion bodies in the cytoplasm
of E. coli, but their refolding into actively and functional proteins is difficult (Kiefer,
2003). The production thus relies on the expression in the cytoplasmic membrane
from where the protein can be purified after detergent extraction (Wagner, et al.,
2006). The accumulation of overexpressed proteins in the membrane is often toxic to
the cell, which results in low yield and low biomass formation (Wagner, et al., 2006).
The limited space in the cytoplasmic membrane, where the product can accumulate, is
one potential bottleneck in membrane protein overexpression. The formation of a high
biomass is thus of outermost importance for achieving high product yields (Wagner,
et al., 2008).

25

Membrane proteins are usually targeted via the Sec-pathway to the Sec-translocon
where they are inserted co-translationally into the membrane. Another potential
bottleneck in membrane protein overexpression is the saturation of Sec-translocon
(Essen, 2002). In a study by Wagner and co-workers (Wagner, et al., 2007), it was
shown that membrane overexpression resulted in cytoplasmic aggregates containing
the overexpressed proteins, chaperones and proteases. The aggregates also contained
precursors of periplasmic and outer membrane origin, which indicates jamming of the
Sec-translocon.
Co-expression of chaperones and cultivation at lower temperatures are common
strategies that are used in order to facilitate folding and to reduce the level of
inclusion body formation in membrane protein production. The CorA, the major
magnesium transporter form the bacterial inner membrane, was used as a model
protein (Chen, et al., 2003). In this study it was shown that lowering the cultivation
temperature below 37C reduced the levels of inclusion bodies formed and that a
higher fraction of the CorA was incorporated in the membrane. The co-expression of
the cytoplasmic chaperones DnaK/DnaJ and GroEL/GroES also increased the
incorporation of CorA in the membrane. A conclusion from this study is that
aggregation, and thus the formation of inclusion bodies, can be prevented by either
reducing the protein synthesis rate or by increasing the cytosolic concentrations of
DnaK/J and GroEL/ES.
The engineering or selection of host strains that are well suited for membrane protein
overexpression is another possible approach for improving membrane protein
overexpression. Two derivatives of BL21(DE3), named C41(DE3) and C43(DE3),
were isolated for their ability to produce eukaryotic membrane proteins at elevated
levels without toxic effects (Miroux and Walker, 1996). Through subsequent genomic
and proteomic studies (Wagner, et al., 2008), it was shown that the original lacUV5
promoter of these strains, which is generally stronger than the original lac variant, had
reverted to the original wild type promoter. It was shown that the lower strength of the
lac promoter led to a lower transcription rate of the T7 RNA polymerase, and that this
had a positive effect on the cellular viability leading to higher total production levels.

26

1.4.4 Surface display of proteins in E. coli

Proteins that are displayed at the surface of the E. coli cell have to be translocated
through the inner membrane and pass the periplasm before they are inserted in the
outer membrane. Before discussing surface display systems in E. coli, a short
description of the outer membrane proteome and the insertion process of proteins in
the outer membrane will therefore be given.
The outer membrane proteome
Murein lipoprotein exists in a large number of copies in each cell and anchors the
peptidoglycan layer to the outer membrane. OmpA, a monomeric protein that spans
the outer membrane, is also important for the stability of the cells since it is crosslinked to the peptidoglycan layer (Moat, et al., 2002). The outer membrane contains
many porins, trimeric proteins forming transmembrane -barrel pores in the outer
membrane that allow relatively unspecific passage of small hydrophilic molecules.
OmpF and OmpC are some examples of porins. The porins can represent as much as 2
% of the total protein content in E. coli, making porins some of the most abundant
proteins in terms of mass (Nikaido, 1996).
Maltoporin B (LamB) forms a channel in the outer membrane that specifically allows
passage of maltose and maltodextrins (Nikaido, 1996). LamB also contributes to the
ability of the bacteria to take up other sugars from the surroundings during glucose
limiting conditions (Death, et al., 1993).
OmpT is a protease present in the outer membrane. It cleaves peptides between two
consecutive basic amino acids (Kramer, et al., 2000). OmpT has been associated with
the degradation of many recombinant proteins (Baneyx and Georgiou, 1990). It is
stable under denaturating conditions and hence acts during purification and refolding
processes (White, et al., 1995).

27

Insertion of proteins in the outer membrane

A network of proteins is involved in the folding and insertion process of outer
membrane proteins. The -barrel assembly machinery (BAM) complex and a number
of periplasmic chaperones, especially SurA, Skp and Deg P, play important roles in
this process (Fig 7). The insertion process is ATP-independent due to the lack of a
periplasmic ATP-pool (Knowles, et al., 2009).
SurA function both as a peptidyl-prolyl isomerase and as general chaperone. The
main role of SurA in OMP maturation is that of a general chaperone (Sklar, et al.,
2007). SurA has a high specificity for outer membrane proteins due to a specific
pattern of aromatic residues that is found frequently amongst this group of proteins.
SurA deficient mutants have a reduced amount of outer membrane proteins
(Hennecke, et al., 2005).
The chaperone Skp has a selectivity for denatured OMPs (Chen and Henning, 1996).
Skp deficient mutants have shown decreased levels of outer membranes proteins, for
example decreased amounts of LamB, OmpA and OmpF. The skp gene is located
downstream of BamA (one of the genes in the BAM complex) and both of these genes
are regulated by the E stress response that, for example, is activated in response to
unfolded OMPs (Sklar, et al., 2007). Skp mediates transport of OmpA in an unfolded
state across the periplasm (Walton, et al., 2009).
The last periplasmic chaperone that has a large impact on OMP biogenesis is DegP.
This chaperone has both protease and general chaperone activity, and it was suggested
that there are two different pathways for periplasmic chaperone activity in OMP
maturation (Sklar, et al., 2007). SurA is the main chaperone during normal conditions
whereas Skp/DegP have a primary role of rescuing OMPs that have fallen off the
normal assembly pathway. However, under stress, or in the absence of SurA, the
importance of Skp/DegP is increased.

28

Figure 7. A model for the periplasmic chaperone-mediated biogenesis of OMPs in E. coli. The OMPs
are transported to the periplasm by the Sec-pathway. Most OMPs are escorted through the periplasm by
SurA while off-pathway OMPs are rescued by the DegP/Skp complex. The DegP/Skp complex can
deliver OMPs back to SurA, directly to the BAM-complex or use the protease activity of DegP for
degradation. Modified from (Sklar et al. 2007).

-barrel assembly machinery (BAM) complex

The E. coli BAM-complex is composed of five domains; BamA, BamB, BamC, Bam
D and BamE (Fig 7). The names of these proteins have recently changed and the
former names were: BamA (YaeT, also called Omp85), BamB (YfgL), BamC (NlpB),
BamD (YifO) and BamE (SmpA). BamA is an integral membrane protein and the
other four proteins are lipoproteins that are localized to the inner leaflet of the outer
membrane. All of the five BAM-proteins are involved in the OMP biogenesis but
their specific roles in the process are not clear (Knowles, et al., 2009).
Surface display systems in E. coli
The display of recombinant proteins at the surface of the bacteria is useful in many
different biotechnological applications. It may be applied in the field of live-vaccine
development,

peptide

library

screening,

whole-cell

biocatalysis,

biosensor

development and bioadsorbents (Jose and Meyer, 2007). Surface expression of

proteins in bacteria commonly relies on a fusion between the target protein (the
passenger) and a carrier protein that usually is a cell surface protein naturally present
in the host. For examples of surface display systems that have been used in E. coli,

29

see table 2. A successful carrier, (i) has an efficient signal peptide that transports the
fusion protein through the inner membrane, (ii) has a strong anchoring structure so
that the targets stays attached to the surface, (iii) is compatible with the target protein,
meaning that stability of the carrier is not influenced by the target, and (iv) is resistant
against degradation by proteases. The passenger protein itself also affects the
translocation process. The folding structure of the passenger, such as the presence of
disulphide bridges, is one factor that influences the success of the surface display. The
size of the passenger, as well as the presence of certain amino acids in the passenger,
are also known to influence the process (Lee, et al., 2003).

Table 2. Selected examples of surface display systems in E. coli. Advantages and disadvantages
associated with each display system.

Display system

Advantages

Disadvantages

OMP

Limited to transport of small

passengers

Lpp-OmpA

Transport of large passengers

Intermediate number of passenger
copies per cell

Difficult to transport proteins with S-S

bridges

Surface organelles

High number of passenger copies per

cell

Limited to transport of small

passengers
Difficult to transport proteins with S-S
bridges

Autotransporters

Transport of large passengers

High number of passenger copies per
cell
Dimerisation at the surface

In some cases difficult to transport

proteins with S-S bridges

Fusions to OMPs and lipoprotein

The first examples of recombinant surface display in E. coli was reported more than
two decades ago and relies on fusions between outer membrane proteins such as
OmpA, Lam B and OmpC and the passenger proteins. This type of display systems
has been used for display of different peptides but it is not suitable for display of large
proteins as the upper limit seems to lie between 60 and 70 amino acids (Charbit, et al.,
1988).

30

The LppOmpA hybrid display system is based on a fusion between the signal peptide
and the first nine amino acids of the E. coli lipoprotein (Lpp) and three or five
membrane spanning loops from the E. coli outer membrane protein A (OmpA). This
system is not that sensitive to the sizes of the passengers, but appears instead to be
sensitive to the presence of disulphide bridges in the passengers (Stathopoulos, et al.,
1996). Intermediate numbers, approximately 104 recombinant passengers, has been
reported to be expressed on the surface of the cell (Francisco, et al., 1993).

Fimbriae and other polymeric surface organelles

Surface structures like fimbriae and flagella are present at high numbers at the surface
of gram negative bacteria and are therefore attractive for display purposes. A large
variety of fimbriae proteins have been used for surface display of different peptides.
One drawback associated with this system is that there seems to be a restriction with
regard to passenger size and composition. The successfully transported peptides are
not larger than 10-30 amino acids and they are relatively hydrophilic. The formation
of disulphide bridges in the passengers also seems to be a critical factor for transport
(Klemm and Schembri, 2000).

Autotransporters
Pathogenic gram-negative bacteria transport various kinds of virulence factors to the
surface, or to the exterior of the cell, by the autotransporter pathway. The
autotransporter protein family represents a large and highly diverse group of proteins
that can be used as carriers for recombinant proteins. The passenger proteins that are
naturally transported vary in length from 20 to 400 kDa and are also highly variable in
sequence (Dautin and Bernstein, 2007). The autotransporters are synthesised as
precursor proteins with a cleavable signal peptide, an N-terminal passenger domain
and a C-terminal translocator domain composed of a -helical linker region and a
pore-forming -barrel part (Fig 8). The signal peptide directs the preprotein to the Sec
translocon and initiates the translocation process of the precursor protein across the
inner membrane. The signal peptide is cleaved off and the -domain becomes
integrated in the OM. The -helical linker is thought to form a hairpin structure in the
pore and pull the passenger domain towards the surface (Jose, 2006). The
autotransporters initially got their name because it was thought that they contained

31

everything that was needed for the transport and incorporation into the outer
membrane in their own sequence. Today, there are several co-existing models
describing how the passengers are actually transported to the surface of the cell. The
model described in figure 8 is the hairpin model, which is the classical model. One
of the newer models suggests the involvement of the BAM-complex in the
incorporation process (Dautin and Bernstein, 2007).
There are several examples in the literature where different autotransporters have
been used for surface expression of recombinant proteins in E. coli. The AIDA-I
(adhesin involved in diffuse adherence) autotransporter from E. coli and the IgA
protease from Neisseria gonorrhoeae are two of the most frequently applied
autotransporters for this purpose (Jose, et al., 2002; Jose, et al., 2009; Jose and Meyer,
2007; Jose and von Schwichow, 2004; Klauser, et al., 1990; Li, et al., 2008; Maurer,
et al., 1997).
A
medium!

MECHANISM FOR AUTOTRANSPORT!

OM!
periplasm!

IM!
cytoplasm!

B
Passenger!

Translocation unit (TU)!

SP!
C!

N!

#-barrel pore!
"-helical linker region!

Figure 8. The autotransporter system. A) Suggested mechanism for autotransport. The signal peptide is
cleaved off following translocation across the inner membrane through the Sec-translocon. The poreforming part of the translocation unit forms a pore in the outer membrane and pulls the passenger
towards the surface of the cell. B) Structure of an autotransporter vector. The vector includes a signal
peptide (SP), a passenger protein and a translocation unit that is composed of a linker region and a
pore-forming part.

32

1.4.5 Extracellular production

Recombinant proteins can also be targeted to the medium, which offers the benefits of
low levels of proteolysis, simple detection and purification, a better folding
environment and the avoidance of the N-terminal methionine extension. Extracellular
production of toxic proteins minimizes their impact on the host and may represent the
only possible way for their production (Hannig and Makrides, 1998; Ni and Chen,
2009). The high dilution of the product may however be disadvantageous. One
strategy is to utilize leaky mutants that release periplasmic proteins to the medium.
Another strategy relies on import of secretion mechanisms, either lysis or whole
transport mechanisms, from pathogenic E. coli (Shokri, et al., 2003).
The haemolysin transport system is one candidate for transport of the recombinant
products to the medium. The proteins are translocated directly to the medium in a
protein channel that spans both of the membranes. The channel is composed of
HlyB/HlyD and TolC and the signal peptide of HlyA is fused to the product and
directs it to the protein channel. The proteins that form the channel also need to be coexpressed which is one disadvantage with the system. However, transport of the
product by the haemolysin system does not compete with transport of endogenous
proteins, which is advantageous (Blight and Holland, 1994).
Periplasmic products can also be released to the medium by import of lysis
mechanisms. This strategy is based upon the action of colicin lysis proteins where the
colicin E1 lysis protein (kil) has been mostly used. The induction of the lysis protein
results in permeabilisation of the outer membrane and the subsequent release of
products (Miksch, et al., 1997).
The autotransporters can also be utilized for extracellular transport of recombinant
proteins since some autotransporters release their passengers to the medium (Jose and
Meyer, 2007).

33

2 PRESENT INVESTIGATION
With the completion of the human genome project (HUGO) during recent years, there
is today a large need for production of proteins, not only in order to determine their
structures and functions, but also as they may constitute proteins of large value for the
biopharmaceutical industry. However, since proteins are very diverse and different
from each other, it is virtually impossible to know how to produce a certain protein in
the most suitable way just by looking at the amino acid sequence. Thus, in order to
increase the probability of finding production conditions appropriate for a particular
protein, there is a need for an increased understanding of production strategies already
available as well as the establishment of new and better production methods.
Proteins that are used for research purposes are preferably produced in an active,
soluble form. Typically, the proteins are produced in small-scale cultivations using
batch technology. Unfortunately, this technology suffers from drawbacks related to
e.g., oxygen limitation, acetic acid production and low biomass, but is in spite of that
a frequently used method since it is so easy to perform. However, cells grow at their
maximal specific growth rate in a batch culture, which in many cases is not optimal
when aiming at producing active soluble proteins, as described earlier.
In the biopharmaceutical industry, the fed-batch technology is commonly used for
control of the glucose uptake rate. The glucose uptake rate, or the feed-profile, has in
the literature been described to influence parameters such as specific productivity,
solubility and proteolysis (Bostrm, et al., 2005; Ryan, et al., 1996; Sandn, et al.,
2005; Sandn, et al., 2002). Some proteins cannot be produced in an active form in
the cytoplasm and are therefore instead secreted to the periplasm, but this production
strategy is usually associated with low yields. Other difficult-to-express proteins,
e.g., proteins that are toxic to the cell, are also preferably transported to parts of the
cell other than the cytoplasm. This will not only provide another possibility for
successful protein production but also simplified purification as the number of
endogenous proteins are significantly less in the periplasm or outside the cell than in
the cytoplasm.

34

A compartment that has been neglected for long from the perspective of large-scale
protein production is the cell surface. Despite this, bacterial surface display systems
has during the last 20 years emerged as an important research tool, e.g., display of
peptide/protein libraries in various protein engineering efforts, display of protein
immunogens for the construction of live vaccines, for biocatalysis and
bioremediation, recently reviewed in (Daugherty, 2007; van Bloois, et al., 2011).
Potentially, bacterial display could be an attractive alternative to more established
large-scale protein production strategies, as it offers a simple purification scheme and
possibilities for on-cell protein characterisation. However, the utility of surface
display in this context (large scale protein production) has to be proven by further
investigations.
As an alternative to traditional fed-batch technique, we here describe an approach for
control of the glucose uptake based on mutant bacterial strains deficient in the
phosphotransferase system (PTS) (Paper I), and its impact on cell density, specific
productivity, acetic acid formation and oxygen consumption (Paper I & III),
Historically, there have been very few reports on the impact of the feed-rate on
processes with product localisations other than the cytoplasm. Thus, we also
investigated how the glucose feed-rate affects recombinant protein production in the
periplasm (Paper II) and when displayed on the bacterial surface (Paper IV).

2.1 A cellular alternative to fed-batch cultures (I, III)

2.1.1 Strain evaluation (I)
The aim of this part of the study was to find a method to control the glucose uptake
rate in small-scale batch cultivations where the traditional fed-batch technique is not
applicable. The strategy was to use strains with mutations in the phosphotransferase
system (PTS) that is involved in the glucose uptake. We reasoned that the mutants
would have lower growth rates due to the mutations and as a result of that also lower
oxygen consumption and decreased formation of acetic acid. Hopefully, this would
result in the establishment of higher cell densities and improved product yield, and the
strains would thus represent a novel, simple alternative to fed-batch cultures.

35

The strain AF1000, that originates from the E. coli K12 strain MC4100 (Casadaban,
1976; Sandn, et al., 2002) was used in this study and is referred to as wild type (WT)
strain. This strain lacks all constituents of the lactose operon and it grows on minimal
salt medium without any additional supplements. The three variants of this strain,
with mutations in genes coding for proteins belonging to the PTS system (Picon, et
al., 2005), that where used are presented in table 3.

Table 3. Strains with mutations in PTS. The mutations in the different strains are
shown as well as the absent enzymes.

Original name
of strains

Name of the strain

in this work

Mutation in
gene cluster

Absent enzyme

PPA 668

PTSMan

manX

Enzyme IIABMan

PPA 652

PTSGlc

ptsG

Enzyme IICBGlc

PPA 689

PTSGlcMan

manX and
ptsG

Enzyme IIABMan
Enzyme IICBGlc

The E. coli homologous enzyme -galactosidase, which hydrolyses lactose into

monosaccharides, was used as a model protein since it is a stable protein and has a
high solubility even at high concentrations. Another advantage with the model protein
is the straightforward analysis for enzyme activity. The lacUV5 promoter, which is
theoretically not subjected to catabolite repression (Arditti, et al., 1973), controlled
the expression of the recombinant protein from a low copy number plasmid
(pACYC). AF1000 and its derivatives are lac negative and production from the
lacUV5 is thus constitutive. A system that could be induced by IPTG addition was
also constructed by insertion of the F-factor (lacIq, lacY, lacA).

Cell-growth
First, we studied impact of the mutations on the growth rate of the mutant strains. All
strains were therefore grown in batch cultivations with unlimited access to glucose.
The cell density increased exponentially and the resulting growth rates were 0.78 h-1,
0.38 h-1 and 0.25 h-1 for PTSMan, PTSGlc and PTSGlcMan, respectively (Fig 9). This is to
be compared to the wild type, which had a specific growth rate of 0.72 h-1. This
growth rate can however not be considered as deviating from the one obtained for

36

PTSMan. This result was encouraging as mutations in the PTS system in a previous
publication had been shown to reduce the glucose uptake rate (Picon, et al., 2005).

6
! = 0.72
! = 0.78

Cell dry weight (g l-1)

! = 0.38
4

3
! = 0.25
2

10

12

14

16

Cultivation time (h)

Figure 9. Biomass accumulation as a function of cultivation time.

Comparison of the wild type cell (WT) and the mutants grown in batch
cultivation. Circles:WT, squares: PTSMan, diamonds: PTSGlc, triangles:
PTSGlcMan.

Acetic acid and specific oxygen consumption rate

The next variable studied was the acetic acid formation. The wild type and PTSMan
produced acetic acid in proportion to the growth rate (Fig 10). The PTSGlc and
PTSGlcMan produced small amounts of acetic acid. The lower production of acetic acid
in PTS-mutants has been observed previously (Chou, et al., 1994).
A further step was to compare the mutants grown in batch with the wild type grown in
fed-batch. Exponential feed rates were therefore designed to theoretically give the
same growth rates for the wild type in fed-batch as the mutants had in batch. The
PTSMan had a specific growth rate approximately equal to that of the wild type,
making this type of comparison irrelevant to perform for this mutant.
The mutants had a specific oxygen consumption rate that was proportional to the
growth rate (data not shown). The oxygen consumption rate for the PTSGlcMan
cultivated in batch was almost identical to the WT cultivated in fed-batch. This
comparison was important to make in order to exclude the possibility that the

37

mutations resulted in increased respiration, since this would have had negative
impacts on the establishment of high cell densities.
The acetic acid accumulation was close to zero in the fed-batch cultivations with the
wild type as was it for the mutants in batch (data not shown). Batch cultivated mutants
are thus equal to fed-batch cultivated WT with respect to acetic acid formation and
oxygen consumption.

1200

1000

HAc (mg L-1)

800

600

400

200

6
8
10
Cultivation time (h)

12

14

16

Figure 10. Accumulation of acetic acid as a function of cultivation time.

Comparison of the wild type cell (WT) and the mutants grown in batch
cultivation. Circles: WT, squares: PTSMan, diamonds: PTSGlc, triangles:
PTSGlcMan.

Product formation
The next parameter to evaluate was the specific product formation rate. The
constitutive system was initially used for production of the model protein galactosidase. Figure 11 shows data from batch cultivations with the wild type and the
mutants. The PTSMan strain had the highest and most stable specific production rate.
The wild type also had a relatively high specific production rate in the beginning of
the cultivation, but it dropped to around 50% at the time of harvest. PTSGlc had an
initial low specific production rate that dropped even more as a function of time. The
PTSGlcMan had an even lower specific productivity (data not shown). By using cells
containing an F-factor with lacIq, the production could be induced by addition of
38

IPTG. The production was increased in the PTSGlc (as can be seen in figure 12) but
remained low in PTSGlcMan. The reason to why the production is so low in the double
mutant, PTSGlcMan, is not known. The low production might be a consequence of the
low synthesis rate of proteins in this mutant. It is also possible that the absence of the
two PTS enzymes, which are central for the glucose uptake in the cell, somehow
affects the transcription from the substrate-induced promoter lacUV5.

70

60

q (U mg-1 h-1)

50

40

30

20

10

10

11

12

13

Cultivation time (h)

Figure 11. Specific production rate of -galactosidase during batch

cultivation of the WT strain and the mutants as a function of the
cultivation time. Circles: WT, squares: PTSMan, diamonds: PTSGlc.

The last step of this evaluation was to compare the specific production rate of the
PTSGlc cultivated in batch with the WT cultivated in fed-batch (Fig 12). The
production was induced by addition of IPTG when the cells had a specific growth rate
of approximately 0.4 h-1. The specific production rate was approximately 50 U mg-1
h-1 after the induction in both strains. A reduction in the specific productivity was seen
at an earlier time-point for the PTSGlc than for the wild type. The total -galactosidase
accumulation at the end of the cultivation was estimated to be 33% and 13% of the
total protein, for the wild type and PTSGlc, respectively.

39

30

60
50

25

30
15

-1

20

-1
qp (U mg h )

Cell dry weight (g l-1)

40
20

10
10
5

-4

-2

10

12

-10

Time from induction (h)

Figure 12. Dry weight and specific production rate of -galactosidase of the
PTSGlc mutant grown in batch and the WT strain grown in fed-batch at the
corresponding growth rate. Circles: WT (fed-batch), diamonds: PTSGlc
(batch). Closed symbols: cell dry weight, open symbols: specific production
rate.

High cell density cultivation

As a reduction of the growth rate, by the use of the mutants, potentially would result
in higher cell densities in batch cultivations, we studied this. Therefore, the WT and
the PTSGlcMan were grown in a repetitive batch mode where new batches of glucose
were added as the glucose in the medium was consumed. This glucose addition
strategy was chosen in order to avoid negative effects from high glucose
concentrations (Lara, et al., 2008; Shiloach and Fass, 2005). The final cell mass
reached was 27 g l-1 for the wild type and 34 g l-1 for PTSGlcMan respectively (Fig 13).
The WT produced ten times more acetic acid during the cultivation than the mutant,
9000 mg l-1 compared to 900 mg l-1. It was noted that already at acetate levels of 500
mg l-1, the growth rate of the WT was affected, and at this time the cell concentration
was only 3 g l-1. The shaded area in figure 13A shows the part of the cultivation that is
principally not operational due to the high concentration of acetic acid. The PTSGlcMan
(Fig 13 B) has constant but lower growth rate, a much longer period. At the end of the
cultivation an increase in the acetic acid accumulation is observed. This acetic acid
probably originates from mixed acid fermentation, which is a result of insufficient
supply of oxygen to the fermentor.
40

0,4

B
8000
0,3

-1 -1

(g g h )
O2

-1

my (h ), q

-1

O2

-1

my (h ), q

0,1

4000
O2

O2

2000

0,2

2000

HAc
0

-0,1

0
0

10

12

0
0

10

15

20

25

Cultivation time (h)

Cultivation time (h)

Figure 13. High cell density cultivations of A wild type (AF1000) and B PTSGlcMan mutant strain.
Closed circles: specific growth rate which is also approximated by a solid straight line, open circles:
acetic acid concentration, solid line: specific oxygen consumption rate, qO2. The shaded area in
figure A indicates the interval of cultivation that is preferably avoided.

2.1.2 Production of integral membrane proteins by the PTS-mutants (III)

A following logical step was to explore the possibility of using the mutants for
production of other proteins, especially proteins that are known to be difficult to
produce. Integral membrane proteins were chosen as model proteins since their
production is usually associated with low yield, toxicity and inclusion body formation
(Essen, 2002). A set of five different E. coli integral membrane-spanning proteins
(IMPs) with human homologues were selected as model proteins (Table 4) and were
produced with N-terminal His6 tags to facilitate purification. These proteins have
earlier been expressed using the T7 promoter with varying success in different strains
(Eshagi, et al., 2005), but in the present study the expression was controlled from the
araBAD (PBAD) promoter. The B-strain, BL21(DE3)(Novagen) that is commonly used
for production of membrane proteins was also included in the study. This strain has a
specific growth rate of 0,76 h-1 in minimal medium, which is approximately equal to
the specific growth rate of AF1000 (WT).

41

-1

-1

4000
0,4

6000
0,2

HAc (mg l )

0,6

-1

6000

HAc

HAc (mg l )

(g g h )

0,8

8000

Table 4. Membrane protein characteristics. The integral membrane proteins

used in the study.

Target

Function/Predicted function

EM03

Ammonium transporter

EM09

Predicted transporter

EM16

Chloride transporter

EM20

Multi drug efflux system protein

EM29

Intramembrane serine protease

Small-scale production
All strains that were included in the study were grown in minimal medium in micro
titre plates, and expression of the IMPs was induced at OD600 1 by addition of Larabinose. All cells were harvested after the same time of expression. A dot blot (Fig
14) was used to show the expression of the targets. PTSGlcMan and PTSGlc produced
three out of the five proteins, as did the BL21(DE3) strain. The amounts of cells at the
time of harvest varied since the strains have different growth rates. A quantitative
comparison of the expression levels in the different strains is not therefore possible
from this dot blot.

!"#$ %!"#& %!"'( %!")# %!")&%

*+'###%
,-."/0%
,-.123%
,-.123"/0%
45)'67!$8%

Figure 14. Expression of membrane proteins. Dot blot of

purified His-tagged proteins overexpressed in the following
strains: AF1000, PTSMan, PTSGlc, PTSGlcMan and BL21 (DE3).

42

Medium scale production and quantification

In order to be able to compare the relative amounts of IMPs and to assess the degree
of homogeneity of the target protein, the production of the EM03 target was scaled up
into shake flasks. The membrane fraction was subjected to IMAC resin in order to
purify the His6-tagged product. Expression of the target protein was verified by
Comassie-stained SDS-PAGE (Fig 15).

+,#
-./01#
!""#

!#####'#####(#####%######*#####&######

$"#
&'#
%$#
("#
'"#
!)#

Figure 15. Verification of expression of EM03. SDS-PAGE analysis of IMACpurified target EM03. 1 marker, 2 AF1000, 3 PTSMan, 4 PTSGlc, 5 PTSGlcMan, 6
BL21 (DE3).

The samples were further subjected to analytical gel filtration to allow a quantitative
comparison of the expressed levels in the different strains. The gel filtration
chromatogram is shown in figure 16A where values for the same amounts of cells are
shown normalized so that the top peak (maximum) value equals 1. The chromatogram
shows that EM03 could be purified as a non-aggregate.

43

Figure 16. Purification and quantification of the selected membrane protein. (A) Analytical gel
filtration from medium-scale shake flask cultivations using the target protein, EM03. The
chromatogram shows values for an equal OD6oo -value of cells in the different samples, normalized
with respect to the highest top peak absorbance set equal to one. (B) Estimated peak areas of
analytical gel filtration chromatograms from the production of the target EM03. The values are
shown as calculated peak areas for an equal OD600 -value of cells in the different samples,
normalized with respect to the largest top peak area set equal to one.

The amounts of EM03 that were produced in the different strains were calculated
from the areas under the curves and the results are plotted in figure 16B. The
comparison shows that PTSGlc produced approximately twice as much protein as
PPA689 PTSGlcMan and BL21(DE3). AF1000 and PTSMan produced very small

44

quantities of EM03 also in shake flasks, so that it was not possible to determine the
areas in a statistically relevant fashion.
It is obvious that the glucose uptake rate influences the amount of protein that can be
extracted in a soluble form from the membrane, but this is apparently not the only
factor since AF1000 and BL21 have the same growth rates but very different
production levels.
Acetic acid
As stated before, the by-product acetate has a negative impact on both the growth rate
and on the production of recombinant proteins (Eiterman and Altman, 2006; Jensen
and Carlsen, 1990; Koh, et al., 1992). It is difficult to separate the effect of the acetic
acid from the effect of the growth rate on the production, since a growth rate above a
certain threshold value leads to the excretion of acetic acid. In some studies, acetic
acid has therefore been added to the cultivation and its effect on growth rate and on
production of recombinant proteins has been studied. Jensen and Carlsen (1990)
showed that acetic acid concentrations of above 6.1 g l-1 resulted in decreased growth
rate but in another study (Nakano, et al., 1997) this effect was seen already at
concentrations of 500 mg l-1. In our study (I) AF1000 showed a reduction in growth
rate at concentrations of 500 mg l-1 and at this time the cell concentration was
approximately 3 g l-1.

Table 5. Yield of acetic acid per cell for the strains used in the
study.
Strain

HAc (mg/L) per OD600-unit

AF1000

158

PTSMan

147

PTSGlc

PTSGlcMan

BL21(DE3)

58

45

The BL21(DE3) strain has a high specific growth rate (approximately 0,76 h-1) which
is comparable to the specific growth rate of AF1000, but has a comparably low
production of acetic acid (Table 5). The production of the membrane protein target
EM03 is twice as high in PTSGlc as in PTSGlcMan and BL21(DE3) and very low in
AF1000 and PTSMan. The differences in the accumulation of acetic acid between the
strains might be one possible explanation for the different production levels.
However, this in not the only explanation since the PTSGlc and the PTSGlcMan has the
same production of acetic acid, but different levels of production of EM03. The
production level is probably a combination of the glucose uptake rate and the acetic
acid accumulation level. The BL21(DE3) strain is deficient in the proteases Lon and
OmpT, which might also explain the relatively high production level in this strain.

2.2 Impact of feed-rate on processes with other product localisations

than the cytoplasm (II, IV)

2.2.1 Leakage of periplasmic products in relation to the glucose uptake
rate (II)
Some proteins need to be transported to the periplasm in order to fold properly.
Leakage of periplasmic proteins to the medium is a problem associated with
periplasmic production provided that the product ending up in the medium is not
collected. The aim of the present study was to investigate how the glucose uptake rate
is correlated to the leakage/periplasmic retention of secreted products. The growth
rate was varied and its effect on leakage of two periplasmic products to the medium
was studied. The focus was also on how the amount and specificity of the outer
membrane proteins was affected by the growth rate and by recombinant protein
production. Could the amounts of these proteins be connected to leakage of
periplasmic products to the medium?
Cutinase, a lipolytic enzyme, from Fusarium solani pisi was used as one of the model
proteins (Mannesse, et al., 1995). Two Z domains, i.e., engineered forms of the B
domain of staphylococcal protein A, which is present in the cell wall of
Staphylococcus aureus, were fused to the N-terminus of the cutinase (Bandmann, et
46

al., 2000). The expression of the product was controlled by the constitutive
staphylococcal protein A promoter (Lfdahl, et al., 1983), and the signal peptide from
the same protein was used to direct the recombinant product to the periplasm. An
antibody fragment (Fab) was used as a second model protein, which was directed to
the periplasm by an OmpA signal sequence. The production of Fab was under control
of the tac promoter. ZZ-cutinase was expressed in the K12 strain 0:17 (Olsson and
Isaksson, 1979) and Fab in W3110 (ATCC 27325).

Specific productivity and leakage

The specific productivity of the model proteins followed the growth rate of the cells
and was higher at higher growth rates. This trend was also observed for the
production of -galactosidase (I) and is in accordance with earlier findings (Sandn,
et al., 2002). Figure 17 shows the leakage of ZZ-cutinase, the quotient between
product activity in the medium and product activity in total, as a function of the
growth rate. A higher specific growth rate resulted in a higher leakage of periplasmic
product to the medium. The same trend was observed for the production of Fab (data
not shown). Leakage is defined as the selective passage of proteins through the outer
membrane, whilst maintaining a functional cell. This is distinguished from lysis,
which leads to disruption and cell-death, but that also results in release of periplasmic
products to the medium. The presence of protein in the medium corresponding to the
normal endogenous composition of E. coli proteins (here referred to as total protein)
is usually considered as an indication of lysis. Product activity and total protein in the
medium was measured in order to exclude that the product found in the medium was a
result of lysis. Not more than 4% of the total protein was found in the medium in the
batch cultivation with recombinant product formation and not more than 3% in all
other cultivations (data not shown). The leakage of the product to the medium was as
high as 20% in the batch cultivation, and 9 and 6% respectively in the fed-batch
cultivations. The main part of the ZZ-cutinase activity found in the medium was thus
a result of leakage and not of lysis.

47

Cutinase activity (medium/total)

0.20

0.15

=0.3 h-1 (batch)

0.10

=0.2 h-1 (exp. fedbatch)

0.05
=0.1 h-1 (exp. fedbatch)

0
0

10

15

20

DW (g l-1)

Figure 17. Leakage of ZZ-cutinase to the medium in percent of the total

production. The specific growth rates (h-1) are indicated in the figure.

Effects of glucose uptake rate and recombinant protein production on outer

membrane composition
In order to investigate the relationship between the content of outer membrane
proteins and leakage, the first step was to identify which proteins that were present in
the outer membranes during the cultivations. The outer membrane proteins were
therefore isolated from cultivations with different growth rates (both with and without
production of ZZ-cutinase). The most abundant proteins were identified as OmpA,
OmpF and LamB by N-terminal sequencing (Fig 18). The expression of the outer
membrane proteins involved in uptake of nutrients, LamB and OmpF, was dependent
on the specific growth rate. The titre of these proteins decreased as the growth rate
increased for both ZZ-cutinase and non-producing cells. However, the reduction was
more noticeable for the producing cells. No LamB was detected in the batch
cultivation, which was to some degree expected since Death and co-workers (Death,
et al., 1993) showed that the LamB protein is upregulated during glucose limitation.
The OmpA protein, which is thought to have a role in stabilizing the cell, seemed to
be more or less constant regardless of the growth rate. There was however a slight
tendency that the amount of the protein was lower in producing cells than in nonproducing cells at comparable specific growth rates.

48

600

OmpF (mg l

-1

500

400

300
200
100

0
200

180
160

LamB (mg l

-1

140
120
100
80
60
40
not
det

20
0

not
det

400

350

OmpA (mg l

-1

300
250
200
150
100
50
0
1000

900

Figure 18. Outer membrane proteins

isolated from cultivations with different
growth rates (exponential feed profiles)
without production (black bars) and during
ZZ-cutinase production (white bars). Lam B
was not detected in all cultivations, this is
indicated by not det in the figure.
A. OmpF (mg l-1)
B. LamB (mg l-1 )
C. OmpA (mg l-1)
D. Sum of OmpF, LamB and OmpA (mg l-1)

800

OMP (mg l

-1

700
600
500
400
300
200
100
0
0,1

0,2

0,3

0,45

0,6

-1

Specific growth rate (h )

!
49

Figure 18D summarises the combined titre of proteins investigated. The combined
titre of outer membrane protein decreased as the specific growth rate increased for
both producing and non-producing cells. The overall titre of outer membrane proteins
was lower if a recombinant protein was produced. The total reduction was almost
60% with a high product formation rate at = 0.3 h-1.
It is reasonable to hypothesise that outer membrane protein accumulation influences
product retention. When studying the total titre of outer membrane proteins it was
clear that the titre was inversely proportional to the growth rate and that a lower titre
was found in producing cells. Secretion of a recombinant product results in an
increased transport of proteins through the inner membrane. Our model system
showed a specific productivity that was proportional to the specific growth rate,
allowing different levels of transport through the Sec translocase to be studied.
Depletion of outer membrane proteins, due to jamming and overloading of the sec
system, has earlier been proposed as possible reasons for periplasmic leakage
(Baneyx, 1999). In the present study, a higher growth rate, which is connected to a
higher transport through the Sec translocon, resulted in a higher leakage and in a
lower amount of outer membrane proteins. Inhibited transport of outer membrane
proteins due to the overloading of the Sec translocon is therefore a possible
explanation to our results.
Another approach that can be used when studying consequences of limited transport
through the Sec translocon is by altering the levels of the components of the Sec
translocon itself. Baars and co-workers (2008) used this approach and compared the
outer membrane proteome of cells depleted of SecE with the outer membrane
proteome of cells expressing normal levels of this protein. It was shown that the
amount of most outer membrane proteins was reduced upon SecE depletion.
However, the amount of OmpA was however unaffected, which is in accordance with
our findings (Baars, et al., 2008).
In a previous study (Shokri, et al., 2002), it was shown that the growth rate influenced
the composition of phospholipids and fatty acids in the membranes. The leakage of
the periplasmic product, in that study -lactamase, showed a peak at a dilution rate

50

corresponding to a specific growth rate of 0.3 h-1. At this specific growth rate, there
was an increased amount of phosphatidyl glycerol and unsaturated fatty acids in the
membrane. The expression level of the product in that study was low and maybe not
enough for influencing the outer membrane composition. The growth rate intervals
investigated in these two studies were different and therefore a direct comparison was
difficult to do.
In the present study, we investigated how the composition of the outer membrane
proteins was connected to leakage of recombinant proteins. Our results suggest that
the permeability of the outer membrane is a function of both the lipid composition and
its protein content.
300

Cutinase activity in cell pellet (U ml-1)

250

200

150

100

50

0
0

10

15

20

-1

DW (g l )

Figure 19. Periplasmic retention of ZZ-cutinase (cell pellet) as a function of dry weight (DW). The
specific growth rates are indicated in the figure as follows: a specific growth rate of 0.3 h-1 (filled
circles), a specific growth rate of 0.2 h-1 (filled triangles) and a specific growth rate of 0.1 h-1 (open
squares), respectively.

Figure 19 shows the retention of ZZ-cutinase, i.e. the amount of product that can be
harvested from the cell paste, as a function of cell accumulation. Interestingly, the
amount of product that can be retained within the cell is always the same and not a
function of the growth rate. This means that a higher specific growth rate, which
results in higher synthesis rate, will not result in more product in the periplasm of
each cell due to the elevated leakage.
51

2.2.2 Optimisation of surface expression using the AIDA autotransporter

(IV)
Although bacterial surface display has been used extensively in various research
applications, there are few studies focusing on its use as a large-scale protein
production tool. If surface-display technology is to be used in the context of largescale production, methods for cultivation of large quantities of cells whilst
maintaining a high surface expression is needed. Here, we investigated the influence
of different cultivation conditions and techniques, batch and fed-batch, as well as the
impact of two different cultivation media on the production of cell surface-displayed
recombinant proteins.
As a model protein, we used the staphylococcal protein A-derivative Z, which was
chosen because it is a small protein without disulphide bridges, and should
theoretically not be difficult to transport to the surface. Further, Z binds to the Fc
region of IgG, thus enabling flow cytometry-based verification and quantification of
surface expression of Z by using fluorescent IgGs. The vector that was used for the
surface display is based on the AIDA autotransporter with its own wild type promoter
aidA (Fig 20).

PaidA

SP

AIDAC

Figure 20. The AIDA-vector containing the aidA promoter, the signal peptide
(SP), Z (the passenger protein), a linker (L) and the -barrel pore (AIDAc).

Impact of OmpT on surface expression level of Z

Z contains a potential cleavage site for the outer membrane protease OmpT. A first
step of this work was to assess if the potential cleavage site was accessible for OmpT.
Analysis of the AIDA-Z fusion protein expressed in strains with and without OmpT
(0:17 (Olsson and Isaksson, 1979) and 0:17OmpT, respectively) revealed that both
strains expressed the fusion but only the OmpT negative strain yielded the full-length

52

protein (Fig21). The degradation product in 0:17 has approximately the same size as
AIDAc, a size that is expected if OmpT cleaves within the Z-domain.

Figure 21. Comparison of surface expression of Z in OmpT positive and OmpT negative E. coli 0:17.
(A) Western blot of the isolated outer membrane fraction, using antiserum against AIDAc. Lane 1:
Marker, 2: 0:17, 3: 0:17OmpT, 4: 0:17 with empty vector, 5: 0:17OmpT with empty vector, 6: 0:17
with vector containing Z, 7: 0:17OmpT with vector containing Z. (B). Cells analysed by flow
cytometry using human IgG linked to AlexaFluor488. Red: 0:17 with vector without passenger, Blue:
0:17 with vector containing Z, Green: 0:17OmpT with vector containing Z.

Flow cytometry analysis of the different clones confirmed that Z was accessible and
functional at the cell surface, since binding to fluorophore-labeled IgG was achieved.
The fluorescence intensity was, as expected from the western blot analysis, lower in
0:17 than in 0:17OmpT. The strain 0:17OmpT was thus chosen for the further
experiments. The effect of the temperature on the expression level was also
investigated and expression was found to be favoured by cultivation of the cells at
37C.
Effects of growth medium and regulation of the aidA promoter
In order to evaluate the effect of the growth medium on the surface expression of Z
the cells were grown either in minimal medium with glucose as carbon source or in
Luria Broth (LB) medium. As can be seen in figure 22, the expression level was
higher in the minimal medium than in LB, which is in accordance with earlier
findings (Benz, et al., 2010). Glucose was added to the LB medium in order to assess
53

if the lower expression level was a result of the absence of an easily available carbon
source. This was not the case since the addition of glucose to the LB medium resulted
in even lower expression levels (Fig 22). The concentration of amino acids in the
medium was followed during the cultivation (data not shown). Some amino acids
were depleted but their depletion did not result in increased expression levels of Z.
Another idea that we had was that glucose starvation and the subsequent entrance into
the stationary phase would affect the level of expression. The cells were thus grown in
minimal salt medium containing glucose and were allowed to grow until the glucose
was consumed. It was shown that entry into the stationary phase reduced the surface
expression level of Z (data not shown). These data together with those from the
cultivations in different media indicate that the aidA promoter is not induced by
stringent response.

Figure 22. Comparison of surface expression of Z in 0:17OmpT grown

in glucose minimal medium (grey), LB medium (white) and LB medium
with 10 g l-1 glucose (black), respectively.

Nevertheless, cultivation in different media does result in different growth rates.

Going from minimal medium to LB medium and finally to LB medium supplemented
with glucose result in progressively higher growth rates. Since it appeared likely that
lower growth rates resulted in enhanced surface expression, the next logical step was
to investigate if further reductions of the growth rate would be beneficial.

54

Effects of glucose uptake rate in minimal salt medium

Different fed-batch cultivations were performed in order to investigate the influence
of the glucose uptake rate/growth rate on expression. Three different growth rates
(=0.1, 0.2 and 0.4 h-1) were achieved by exponentially feeding glucose to the
reactors after an initial batch phase. Surface expression of Z was followed using flow
cytometry. There is a tendency that the expression level is higher at a higher growth
rate (Fig 23). The limited transport capacity of the Sec-translocon (Baars, et al., 2008;
Baneyx, 1999; Mergulho and Monteiro, 2004) is a potential bottleneck in production
systems where the recombinant protein is localised to parts of the cell other than the
cytoplasm. Likewise, the insertion of proteins in the outer membrane, mediated by the
BAM-complex, is a potential bottleneck in surface display systems. The outer
membrane protein fraction was isolated from the cultivations with different growth
rates (data not shown) and no clear difference in outer membrane protein content
could be seen. In (II) those differences appeared clear. The expression level of Z by
the aidA promoter is probably not high enough for influencing transport and insertion
of endogenous proteins and the pattern of protein expression therefore probably
reflects the synthesis rate of the protein.

Fig 23. Cell growth and antibody-binding activity of the surface displayed Z. OD600 values
(filled symbols) from the fed-batch cultivations with different growth profiles and mean
fluorescence per cell (open symbols) analysed by flow cytometry. Circles =0.1 h-1, squares
=0.2 h-1, triangles =0.4 h-1.

55

Optimisation of cultivation
The last step of this study was to develop an efficient process for cultivation of cells
with high-level expression of proteins at the surface. Expression was favoured by
cultivation in minimal medium and there was a trend that expression was higher at
higher growth rates (Fig 23). We therefore reasoned that a repeated batch strategy,
were the cells grow at the maximal specific growth rate and where new batches of
glucose are added as the glucose is consumed, would be suitable for obtaining cells
with high-level expression (Fig 24).

Figure 24. Cell growth, acetic acid formation and surface expression during repeated batch
cultivation. (A) Cell growth measured as optical density (filled squares), dry weight (open squares),
surface expression per cell measured by flow cytometry (circles, line). (B) Specific growth rate (,
diamonds), acetic acid concentration in the medium (triangles, line) and dissolved oxygen (DOT,
line).

A furhter advantage of the repeated batch strategy is seen when looking at the
fluorescence peaks from the flow cytometer analysis (Fig 25). The peaks obtained
during the extended batch culture are narrower than during the fed-batch cultivations,
indicating that the bacterial population is more homogenous. The peak width can be
considered as a quality parameter for cells expressing proteins at their surface. As
discussed before, acetic acid accumulation, due to overflow metabolism, is a problem
when using the batch cultivation strategy. The acetic acid accumulation in strain
0:17OmpT is however much lower than in AF1000, even though the strains are both

56

K-12 derivatives and have approximately the same growth rates, and this explains
why the repetitive batch strategy can be used. In the former study (I) the cell mass
was 3 g l-1 when an inhibiting concentration of acetic acid, 500 mg l-1, was reached. In
the present study the cell mass was 8 g l-1 when the acetic acid reached this
concentration but the cells continued to grow without severe effects on the growth
rate until the cell mass reached approximately 20 g l-1. The acetic acid concentration
does not reach inhibiting concentrations until the oxygen limitation of the fermentor is
reached. At this point, coinciding with the DOT approaching zero, a sharp increase in
the acetic acid accumulation is observed. This additional acetic acid probably
originates from mixed acid fermentation, which is a result of insufficient supply of
oxygen to the fermentor. The final dry weight reached, by using the repetitive batch
process, was approximately 30 g l-1, and the surface expression level remained high
up to a cell mass of approximately 20 g l-1 (Fig 24). For a further increase in cell
mass, the fed-batch technology is needed, even though this results in a less
homogenous cell population.

Figure 25. Flow cytometry analysis of antibody-binding activity of surface

displayed Z from two selected samples. Black (repeated batch) and grey (fedbatch, =0.4 h-1)

57

3 CONCLUDING REMARKS
The potential of E. coli as host for production of recombinant proteins has not yet
been fully exploited. There are several examples in the literature describing efforts to
overcome problems associated with heterologous protein production in this host. The
development of novel and better methods for production of recombinant proteins is
essential for the purpose of structural and functional studies, but also for the industrial
production of biopharmaceuticals. In this thesis, the impact of the glucose uptake rate
was studied with the objective to improve and optimise production of recombinant
proteins in E. coli. The glucose uptake rate was shown to influence the production of
the recombinant protein in all works included in this thesis. The impact of the glucose
uptake rate was however different depending on the product localisation and the
protein product in question.
As an alternative to traditional fed-batch technology, we here showed that mutant
strains deficient in the phosphotransferase system (PTS) could be used for control of
the glucose uptake rate (I). This allows for control of the specific growth rate at a
genetic level rather than by physical means (e.g., by feed rate, temperature). We
suggest that the mutants will be valuable tools in small-scale parallel recombinant
protein production where the fed-batch technology, for practical reasons, is not
applicable. The mutants were further applied for production of integral membrane
proteins (III) and were able to produce membrane proteins that were not possible to
produce by the corresponding wild type strain. Our results suggest that the mutants
could be useful not only for production of membrane proteins but also for other
difficult-to-express proteins, when production benefits from a lower synthesis rate
and when a high cell density is needed in order to obtain a sufficient amount of
protein.
Process development is quite often characterised by a screening stage performed
under batch conditions in small parallel scale, e.g., in microtitre plates or shake flasks.
Unfortunately, the level of control of the environmental conditions in such shaken
systems is low and the conditions also change rapidly as a consequence of the
exponential biomass-increase. In contrast, in the production scale, the environmental

58

conditions are strictly controlled, the fed-batch technology is usually applied and the
production takes place under lower growth rates in comparison to batch cultures.
Thus, the results obtained from small-scale cultivations do not necessarily apply in a
large-scale production format, which makes the scale up process more complex. By
using the PTS-mutants, screening can be performed under fed-batch-like conditions.
Potentially, this will limit the amount of work for the process development team and
hopefully increase the probability of selecting conditions that will work also in the
large-scale production process.
That there is a need for control of the glucose uptake rate in small-scale cultivation is
evidenced by the recent development of other innovative methods. One such method
is EnBase, or enzyme-based-substrate-delivery, which has been developed as a
complement to the fed-batch technology (Panula-Perl, et al., 2008; Siurkus, et al.,
2010). Glucose is released into the medium by enzymatic degradation of starch by the
enzyme glucoamylase and no external feed of glucose is required. The main benefit of
this method is that it results in significantly higher cell densities compared to
traditional shake-flasks cultures (Siurkus, et al., 2010). However, as EnBase is a kitbased method, it is expensive to use and since the ingredients of the different
components in the kit are not fully disclosed, using the EnBase technology as a tool
in the process development might be more complicated.
One problem associated with periplasmic processes is that the product tends to leak to
the medium, resulting in decreased yields if the product in the medium is not used in
the down stream processing. Leakage of recombinant products to the medium was
clearly affected by the feed rate of glucose (II) since leakage increased with the feed
rate. Although a higher feed rate resulted in an increased specific productivity, the
amount of product inside the cells was constant. In order to retain the product inside
the cells, the induction should preferably take place when the feed rate is low, which
would not only decrease leakage but also improve the product quality (Bostrm, et al.,
2005; Sandn, et al., 2005). In this study, we also showed that the feed rate influenced
the outer membrane composition. The total amount of outer membrane proteins
decreased as the feed rate increased and further reductions in outer membrane protein
accumulation was seen when a recombinant product was secreted to the periplasm.

59

Although bacterial surface display has been used extensively in various research
applications, there are few studies focusing on its use as a large-scale protein
production tool. Here we applied the AIDA autotransporter for surface display of a
recombinant protein and studied protein expression under control of the aidA
promoter (IV). We showed that cultivation in minimal salt medium resulted in higher
expression levels, as did higher feed rates of glucose during fed-batch cultivation.
Batch cultivation with repeated additions of glucose was distinguished as the best
method to improve the surface expression homogeneity in the bacterial population. In
the future, it would be interesting to change the aidA promoter to an inducible and
tunable promoter. By varying the inducer concentration it will then be possible to
investigate if the expression level can be further increased and also potential
limitations regarding Sec-translocation and the insertion process of proteins in the
outer membrane can be studied. The space in the outer membrane where the
transporters accumulate is limited and it will be interesting to find out (i) how many
transporters that can actually be inserted in the membrane and (ii) how many
transporters that can be inserted without affecting the stability of the membrane.
Extracellular production of recombinant proteins is desirable since it allows easy
purification, an environment free of cell-associated proteolytic degradation and
minimized impact on the host cell from toxic proteins. Some autotransporters release
their passenger to the medium and may therefore be exploited for extracellular
production of recombinant proteins. It would be interesting to investigate how
efficient such systems would be in terms of productivity and protein quality. The
number of transporters that can be inserted in the membrane is, as discussed above,
limited and furthermore, the cell has to produce one transporter for each passenger,
which is disadvantageous. However, for some proteins the extra cellular medium
represents the only possible method for their production, and for production of these
proteins, the autotransporter-based excretion strategy might be promising.
Considering what has been presented in this thesis, the glucose uptake rate is an
important parameter in recombinant protein production. It does not only effect the
biomass that can be reached in a certain process but influences also parameters such
as protein quality, specific productivity and leakage of recombinant proteins to the

60

medium. Therefore, it is important to be able to control the glucose uptake rate also in
small-scale cultivation, and as shown in this thesis, this becomes possible by using
PTS-mutants.

61

4 ABBREVIATIONS
AC
Ack
AMP-ACS
AIDA
AMP
ATP
BAM
cAMP
CCR
cDNA
CL
CRP
DNA
DOT
DR
DSB
DW
E. coli
EI
EII
F
Fab
HAc
His6
HPr
HTP
HTS
HUGO
IgG
IMAC
IMP
IPTG
kDa
LamB
LB
Lpp
LPS
MBP
mRNA
OD
OMP
PE
PEP
PG
PoxB
ppGpp

Adenylate cyclase
Acetate kinase
AMP-forming acetyl CoA-synthetase
Adhesin involved in diffuse adherence
Adenosine monophosphate
Adenosine triphosphate
-barrel assembly machinery
Cyclic adenosine monophosphate
Carbon catabolite repression
Complementary DNA
Cardiolipin
cAMP receptor protein
Deoxyribonucleic acid
Dissolved oxygen tension
Down stream region
Disulphide bond
Dry weight (g l-1)
Escherichia coli
Enzyme I
Enzyme II
Flow rate (L/h)
Antibody fragment
Acetic acid
Hexahistidine tag
Phosphohistidine carrier protein
Highthroughput production
High-throughput screening
The Human Genome Organisation
Immunoglobulin G
Immobilized metal affinity chromatography
Integral membrane proteins
Isopropyl--D-thiogalactopyranoside
kilo Dalton
Maltose outer membrane porin
Luria broth
Murein lipoprotein
Lipopolysaccaride
Maltose binding protein
Messenger ribonucleic acid
Optical density
Outer membrane protein
Phosphatidyletanolamine
Phosphoenolpyruvate
Phosphatidylglycerol
Pyruvate oxidase B
Guanosine tetraphoshate

62

PPIase
PrEST
Pta
PTS
qp
qs
RNA
rRNA
SD
SDS-PAGE
Si
SRP
TAT
TCA
TF
TIR
tRNA
V
wt
X

24 = E
32= H
38= s

Peptidyl-prolyl isomerase
Protein epitope signature tag
Phosphotransacetylase
Phosphotransferase system
Specific productivity (g g-1 h-1)
Specific substrate consumption rate (g g-1 h-1)
Ribonucleic acid
Ribosomal ribonucleic acid
Shine Dalgarno sequence
Sodium dodecylsulfate polyacrylamide gel electrophoresis
Substrate concentration (g l-1)
Signal recognition particle
Twin arginine translocation
Tricarboxylic acid cycle
Trigger factor
Translation initiation region
Transfer ribonucleic acid
Cultivation volume (l)
Wild type
Cell dry weight (g l-1)
Specific growth rate (h-1)
Extra-cytoplasmic stress sigma factor
Heat shock sigma factor
Starvation/stationary phase sigma factor

63

5 ACKNOWLEDGMENT
Allting har ett slut, s ven en doktorandtid, och jag skulle drfr vilja ta tillfllet i akt
att tacka alla Er som p ett eller annat stt stttat mig p vgen till examen. Ett
speciellt tack till:
Vinnova, Vetenskapsrdet och KTH fr finansiellt std.
Professor Gen Larsson, fr handledning och std genom ren, fr att du alltid delar
med dig av dina kunskaper och ider samt fr att du r s entusiastisk ver vr
forskning.
Professor Pr Nordlund och Marina Ignatushchenko och vriga medarbetare p MBB,
Karolinska Institutet. Tack fr att jag fick mjlighet att komma till Ert labb och
genomfra min studie. Det var bde lrorikt, roligt och trevligt att arbeta tillsammans
med er.
Dominic Reeks, Neil Weir and Leigh Bowering at Celltech for great cooperation.
Katrin fr ett ypperligt samarbete i brjan av doktorandtiden. Allt blir s mycket
lttare och roligare nr man r tv.
Martin fr att du fortsatte ytexpressionsprojektet nr jag labbade p KI, samt fr ett
finfint samarbete drefter. Tack ocks fr att du alltid stller upp och lser mina
datorrelaterade problem och fr att du ritar s fina bilder. Din hjlp nu i skrivartider
har verkligen varit vrdefull.
Patrik fr handledning i autotransportprojektet och fr ovrderlig hjlp med
avhandlingen.
Examensarbetarna Maria, Sofia och Cecilia. Det var givande att jobba tillsammans
med er och tack fr era bidrag till mina projekt.
Andres fr hjlp med proteinrening och fr att du alltid tar dig tid att diskutera
forskningsrelaterade frgor. Per-ke Nygren och Per-ke Lfdahl fr kloningstips,
John Lfblom fr hjlp med FACS-analys.
Alla nuvarande och fre detta kollegor och vnner p avdelningen (ingen nmnd,
ingen glmd) fr trevligt sllskap och roliga stunder under rens lopp, samt tack fr
all hjlp och goda rd.
Ett extra tack till familjen som alltid hjlper mig nr det behvs. Pappa och Ida fr att
ni lyssnar och frstr. Irene, Sren, Ida och Tobias fr att ni varit barnvakt s att jag
kunnat fokusera p att skriva. Jakob och Ines fr att ni sprider s mycket gldje
omkring er. Och sist men inte minst, ett stort tack till dig Lars fr ditt tlamod, fr att
du r en fantastisk hemma-pappa och fr att du alltid finns dr.

64

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